Download - Research Article Safety Evaluation of the Coagulase ...downloads.hindawi.com/journals/bmri/2015/483548.pdf · Research Article Safety Evaluation of the Coagulase-Negative Staphylococci

Transcript
Page 1: Research Article Safety Evaluation of the Coagulase ...downloads.hindawi.com/journals/bmri/2015/483548.pdf · Research Article Safety Evaluation of the Coagulase-Negative Staphylococci

Research ArticleSafety Evaluation of the Coagulase-NegativeStaphylococci Microbiota of Salami Superantigenic ToxinProduction and Antimicrobial Resistance

Raquel Soares Casaes Nunes Eduardo Mere Del Aguilaand Vacircnia Margaret Flosi Paschoalin

Instituto de Quımica Universidade Federal do Rio de Janeiro Avenida Athos da Silveira Ramos 149 Sala 545Cidade Universitaria 21949-909 Rio de Janeiro RJ Brazil

Correspondence should be addressed to Vania Margaret Flosi Paschoalin paschviqufrjbr

Received 26 May 2015 Accepted 20 October 2015

Academic Editor Rosaria Scudiero

Copyright copy 2015 Raquel Soares Casaes Nunes et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

The risks of contracting staphylococci food poisoning by the consumption of improperly manufactured salami and the possibilityof this food being reservoirs for antibiotic resistance were evaluated Nineteen coagulase-negative staphylococci (CNS) strainswere found in commercial and artisanal salami The species in commercial salami were S saprophyticus S sciuri S xylosusand S carnosus Artisanal salami showed S succinus S epidermidis and S hominis but no S carnosus Phylogenetic analysesgrouped the strains into three major staphylococcal species groups comprised of 4 refined clusters with similarities superiorto 90 Fifteen strains harbored multiple enterotoxin genes with high incidence of sebsec and sea 57 and 50 respectivelyintermediate incidence of sedsehselm and seiselntst-H 33 and 27 correspondingly and low incidence of seeseljselo andseg of respectively 13 and 1 Real time RT-PCR and enzyme-linked-immunosorbent assays confirmed the enterotoxigenicity ofthe strains which expressed and produced enterotoxins in vitro The CNS strains showed multiresistance to several antimicrobialsof therapeutic importance in both human and veterinarian medicine such as 120573-lactams vancomycin and linezolid The effectivecontrol of undue staphylococci in fermented meat products should be adopted to prevent or limit the risk of food poisoning andthe spread of antimicrobial-resistant strains

In memoriam of Professor Joab Trajano Silva PhD

1 Introduction

Staphylococcal food poisoning is an illness caused by theingestion of contaminated food containing enterotoxins pro-duced by bacteria belonging to this genus Enterotoxins thatexhibit superantigenic activities are heat stable proteins andmay not be destroyed even during cooking conditions

In Brazil according to data from the Ministerio daSaude (Ministry of Health) staphylococcal poisoning isthe second most common foodborne disease ranking onlyafter outbreaks involving Salmonella spp [1] Staphylococcusclassified as coagulase-positive are considered potential food

enterotoxin-producing strains [1] although recently theenterotoxigenic potential of coagulase-negative staphylococci(CNS) species in food poisoning has also been recognized [2]

Initially enterotoxin SEs family members were dividedinto five serological types (sea through see) based on theirantigenicity [3 4] In recent years however newly describedtypes of SEsmdashSEG SEH SEI SElJ SElK SElL SElMSElN SElO SElP SElQ SElR and SElUmdashwith amino acidsequences similar to the classical SEs were discoveredThesenewly described enterotoxins are designated as SE or SE-like (SEl) according to their emetic properties displayed ina primate model following oral administration [5]

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 483548 17 pageshttpdxdoiorg1011552015483548

2 BioMed Research International

The toxic shock syndrome toxin-1 (TSST-1) is also amember of the SE-related toxin family and has the ability tostimulate large populations of T cells containing a particularV120573 element in their T-cell receptors (TCR) Like othersuperantigenic toxins it bypasses normal antigen presenta-tion by binding to class II major histocompatibility complexmolecules on antigen-presenting cells and to specific variableregions on the beta-chain of the T-cell antigen receptorThrough this interaction a massive proliferation of T cells atorders ofmagnitude above antigen-specific activation occursresulting in a massive cytokine release that is believed to beresponsible for the most severe features of TSST [6]

Enterotoxin (SE) genes are encoded in mobile geneticelements such as plasmids prophages and Staphylococcuspathogenic islands (SaPIs) [7]

Salami is a kind of dry sausage obtained by the microbialfermentation of raw pork meat using Staphylococcus startercultures as technological accessories to ferment the productand give it its organoleptic characteristics In Brazil Italiantype salami similar to salami produced in Southern Europeis avidly consumed with a production trade of 13093 tonsbetween 2000 and 2014 [8]

S xylosus S equorum and S carnosus are part of thestarter culture microbiota that participate in the reactionsrequired for creating the flavor and aroma during the matur-ing period of fermented meat production [9] In additionother species such as S epidermidis S pasteuri S sciuri andS succinus may also occasionally be present in meaningfulamounts [10]

However even the combination of physical and chemicalbarriers cannot always guarantee the stability and microbialsafety of starter cultures Contamination of salami fermen-tation starter culture microbiota by pathogenic coagulase-negative staphylococci (CNS) strains is perhaps the mostharmful factor in the production of cured meat productssince these pathogens are able to produce heat-stable entero-toxins with superantigenic activities in food matrices [11 12]

Staphylococci species are not usually identified at thespecies level by routine laboratory testing and commercialkits since phenotypic discrimination cannot reliably identifythese species due to the variable expression of some phe-notypic traits [13] For this purpose molecular techniquesincluding nucleotide sequencing within the 16S rDNA hsp60tuf sodA and rpoB genes have been successfully used toidentify Staphylococcus species [14]

Depending on the conditions some species of coagulase-negative staphylococci can present health risks since theyhave shown resistance to several antibiotics of therapeuticimportance such as 120573-lactams [15]

The aim of the present study was to identify the membersof the CNS microbiota from salami We sampled the salamimarketed in Brazil comparing the CNS microbiota in salamiproduced by industrial companies and in artisanal salamimanufactured by small producers The CNS strains wereidentified by sequencing of a 16S rDNA region and thephylogenic relationships between the observed species wereestablished The presence of multiple genes encoding theclassical and newly described sesel and tstH1 toxins in theCNS genomes was investigated The risk of food poisoning

was assessed by evaluating the ability of the CNS strains intranscribing and expressing the classical and newly describedenterotoxins in vitro by using real time RT-PCR and enzyme-linked immunosorbent assay (ELISA) The resistance ofthe isolated strains to antimicrobial agents of therapeuticimportance in staphylococci infections was also evaluated

2 Materials and Methods

21 Isolation of Bacterial Strains Six samples of distinctbrands of salami 03 from themeat industry and 03 from smallartisanal producers were collected in the municipality of Riode Janeiro Brazil Twenty-five grams of salami was added to225mL of 01 peptone water The suspensions were trans-ferred to homogenizer bags (Interscience SaintNom France)and coupled to a Stomacher 400 circulator (Seward Wor-thing West Sussex UK) at 260 rpm for 1min The suspen-sions were serial-diluted from 10minus6 to 100 and 100 120583L of eachdilution was transferred onto 20120583L of Baird-Parker agar con-taining egg yolk tellurite emulsion (BPAthorn RPF bioMerieuxFrance) Eighty presumptive coagulase-negative staphylo-cocci colonies were tested by Gram-staining catalase coagu-lase and thermostable DNAse activities according to BergeyrsquosManual of Systematic Bacteriology Sixty presumptive CNSstrains were stored at minus80∘C in tryptone soy agar (TSAFranklin Lakes New Jersey USA) plus 45 vv glycerol

22 DNA Preparation The strains were cultured aerobicallyovernight in 10mL Brain Heart Infusion broth (BD BBL LePont de Claix France) at 37∘C for 24 h The suggestive CNScolonies were adjusted to 106UFCmL in a spectrophotome-ter and harvested by centrifugation at 5700times g for 1minThecell pellet was used for DNA extraction using the DNeasyblood and tissue kit (Qiagen Dusseldorf Germany) fol-lowing the manufacturerrsquos instructions Genomic DNA wasquantified using the Qubit fluorometer (Invitrogen GrandIsland New York USA) and Qubit assay kits

23 PCR Tests

231 Primer Sequences and Target Genes Primer sets flank-ing the sea seb sec sed see seg seh sei selk selm seln seloselq selr selu and tstH1 sequences are listed in Table 1

232 Uniplex- Duplex- and Multiplex-PCR Tests Uniplex-PCR tests targeting the tstH1 sequence and duplex-PCRtargeting the seaseb and secsed sequences were performedPCR mixtures contained 25120583L of 20mM MgCl

2 10x PCR

buffer (Invitrogen Grand Island New York USA) 100mMdNTPmix (FermentasThermoScientific Vilnius Lithuania)02mM of each primer (Table 1) 05U Taq DNA polymerase(Invitrogen Grand Island New York USA) and 100 ngof DNA templates Uniplex- and duplex-PCR assays wereperformed under the following conditions 94∘C for 5minfollowed by 35 cycles of 94∘C for 2min 53∘C for 2min and72∘C for 1min for extension ending with a final extensionat 72∘C after 7min [16] with modifications in the annealingtemperature using a thermal cycler (MyCycler Bio-Rad

BioMed Research International 3

Table 1 Primer set for V3 16S rDNA sequencing and PCRreal time RT-PCR tests targeting the classical and newly described staphylococcalenterotoxin genes

Primers set and sequences (51015840-31015840) Gene Amplicon (bp) ReferencesSEAf TTGGAAACGGTTAAAACGAA sea 120 [17]SEA

rGAACCTTCCCATCAAAAACA

SEBf TCGCATCAAACTGACAAACG seb 478 [17]SEBr GCAGGTACTCTATAAGTGCCSECf GACATAAAAGCTAGGAATTT sec 257 [17]SECr AAATCGGATTAACATTATCCSEDf CTAGTTTGGTAATATCTCCT sed 317 [17]SEDr TAATGCTATATCTTATAGGGSEEf TAGATAAAGTTAAAACAAGC see 170 [17]SEEr TAACTTACCGTGGACCCTTCSEGf TGCTATCGACACACTACAACC seg 704 [18]SEGr CCAGATTCAAATGCAGAACCSEHf CGAAAGCAGAAGATTTACACG seh 495 [18]SEHr GACCTTTACTTATTTCGCTGTCSEIf GACAACAAAACTGTCGAAACTG sei 630 [18]SEIr CCATATTCTTTGCCTTTACCAGSElJf CAGCGATAGCAAAAATGAAACA selj 426 [19]SElJr TCTAGCGGAACAACAGTTCTGASElMf CCAATTGAAGACCACCAAAG selm 517 [20]SElMr CTTGTCCTGTTCCAGTATCASElNf ATTGTTCTACATAGCTGCAA seln 682 [20]SElNr TTGAAAAAACTCTGCTCCCASElOf AGTCAAGTGTAGACCCTATT selo 534 [20]SElOr TATGCTCCGAATGAGAATGASElKf ATGAATCTTATGATTTAATTTCAGAATCAA selk 545 [21]SElKr ATTTATATCGTTTCTTTATAAGAAATATCGSElQf GGAAAATACACTTTATATTCACAGTTTCA selq 539 [21]SElQr ATTTATTCAGTTTTCTCATATGAAATCTCSElRf AATGGCTCTAAAATTGATGG selr 363 [22]SElRr TCTTGTACCGTAACCGTTTTSElUf AATGGCTCTAAAATTGATGG selu 215 [22]SElUr ATTTGATTTCCATCATGCTCTSST-1f ATGGCAGCATCAGCTTGATA tstH1 350 [17]TSST-1r TTTCCAATAACCACCCGTTT16S rDNAf ATA AGA CTG GGA TAA CTT CGG G 16SrDNA 500 [23]16S rDNAr CTT TGA GTT TCA ACC TTG CGG TCGf forward r reverse

Hercules CAUSA)The amplified fragments were visualizedon 10 agarose gels (Sigma) stained with GelRed (dilution1 1000) (BioAmerica Tel Aviv Israel) and documented on atransilluminator (MiniLumi Imaging Bio-Systems BioAmer-ica Tel Aviv Israel)

233 Multiplex-PCR Tests Multiplex-PCR assays were per-formed by the simultaneous amplification of the see seg sehsei selj selm seln selo selk selq selr and selu sequences usingthe primer sets listed in Table 1 Each reaction contained50120583L of a mix containing 05U Taq DNA polymerase 10xPCR buffer 100mMdNTP 02120583Mof each primer and 100 ng

of DNA template DNA amplification of see seg seh andsei was carried out as follows 95∘C for 5min 35 cycles of95∘C for 30 s 53∘C for 90s and 72∘C for 90 s and a finalextension at 72∘C for 10min The DNA amplifications of theselj selm seln and selo group and the selk selq selr andselu group were carried out in the same conditions [3] PCRproducts were visualized by electrophoresis on 12 agarosegels (Uniscience do Brasil Sao Paulo Brazil) in 1x TAE (Tris-boric acid-EDTA) buffer stained by 05 120583gmLminus1 of GelRed(BioAmerica Tel Aviv Israel) and documented on a transil-luminator (MiniLumi Imaging Bio-Systems BioAmerica TelAviv Israel)

4 BioMed Research International

DNA templates from the following reference strains wereused S aureusATCC 29231 (sea) S aureusATCC 14458 (sebtstH selk selq selr and selu) S aureus ATCC 19095 (sec segseh and sei) S aureus ATCC 13563 (sed) S aureus ATCC27664 (see) and S aureus ATCC 27154 (selj selm seln andselo) and S xylosus ATCC 29971

24 Enterotoxin ExpressionAssays Theobserved strainswerecultured aerobically overnight in 10mL Brain Heart InfusionBroth (BD BBL Le Pont de Claix France) at 37∘C for 72 hBacteria supernatants were collected by centrifugation at4000timesg for 10min and used for the detection of sea seb secsed and see by an ELISA assay using a commercial detectionkit (RIDASCREEN SET A B C D E Art number R4101 R-Biopharm AG Germany) The assay was performed accord-ing to the manufacturerrsquos recommendation and as describedelsewhere [16]Themean lower limit of detection of the assaywas 025 ngmLminus1 The threshold is defined as the average ODof two negative controls plus 015 a constant established bythe kit Samples containing SEs showed absorbance valuesequal to or greater than the threshold value All experimentswere performed in duplicate

25 Real Time RT-PCR Assays Total RNA was extracted byusing the QIAGEN RiboPure Bacteria kit (Life TechnologiesCarlsbad California USA) following the manufacturerrsquosinstructions and quantified using the Qubit fluorometer(Invitrogen Grand Island New York USA) and Qubit assaykits The cDNA synthesis was performed by using the HighCapacity cDNA Reverse Transcription Kit (Applied Biosys-tems California USA) and the ABI PRISM 7500 Fast RT-PCR system (Applied Biosystems California USA) Sampleswere plated in triplicate in 96-well plates as follows 12 120583Lof the SYBR Green PCR Master Mix 1 120583L of primer mix(sea seb sec sed and see) and 45 120583L of the cDNA ultrapurewater in each well Amplification was performed under thefollowing conditions 95∘C for 15min 40 cycles at 95∘Cfor 15 s 54∘C for 30s and 72∘C for 30 s The dissociationcurve was performed at 95∘C for 15 sec 54∘C for 30 sec and95∘C for 15 sec CT means the standard deviations and thecDNA semiquantification were calculated using the Graph-Pad Prism 5 software package Calibration curves based onfive points were constructed in triplicate corresponding toserial dilutions (1 1 10 1 100 1 1000 and 1 10000) from100 ng of a DNA template stock solution

26 16S rDNA Sequencing Amplification of the V5 regionof 16S rDNA fragment was performed using 50 ng of DNAtemplates from the 65 strains found in the salami samplesPCR was performed under the following conditions 95∘Cfor 10min followed by 30 cycles at 95∘C for 30 s 60∘Cfor 30 s and 72∘C for 45 s and a final extension at 72∘Cfor 10min PCR products were purified using the PCRDNA Purification Kit (Applied Biosystems California USA)and sequenced using 20 ng purified DNA and 13 120583L ofprimer sets in a final volume of 20120583L After amplifica-tion products were purified according to the protocol ofthe BigDye Terminator Purification X Kit (Applied Biosys-tems California USA) and sequenced on a 3130 sequencer

GeneticAnalyzer (Applied Biosystems California USA)Thesequences were compared to the 16S rDNA gene sequencesof Staphylococcus species available at the GenBank database(httpwwwncbinlmnihgovGenbankindexhtml) Multi-ple sequence alignments were performed using ClustalW (Kyoto University Bioinformatics Center httpwwwgenomejptoolsclustalw)

27 Sequencing of Enterotoxin PCR Products PCR productswere purified using the PCR DNA Purification Kit (AppliedBiosystems California USA) and sequenced using 10 ngof purified DNA and 32 pmoles of each primer set in afinal volume of 20 120583L After amplification in the sameconditions as the PCR step (Section 233) products werepurified according to the BigDye Terminator PurificationX Kit protocol (Applied Biosystems California USA)and sequenced on a 3130 sequencer Genetic Analyzer(Applied Biosystems California USA) The sequences werecompared to Staphylococcus aureus and Staphylococcuspasteuri gene sequences available at the GenBank database(httpwwwncbinlmnihgovGenbankindexhtml) Multi-ple sequence alignments were performed using ClustalW (Kyoto University Bioinformatics Center httpwwwgenomejptoolsclustalw)

28 Phylogenetic Analyses Phylogenetic relationshipsbetween the CNS strains were performed by sequencealignments using the Clustal X 20 software package [24]The phylogenetic trees were constructed using the softwareMega 60 and UPGMAmethods [25]

29 Antibiotic Susceptibility Tests An inoculumof each strainequivalent to a 05 McFarland scale was swabbed onto aMueller-Hinton agar plate (BD BBL Franklin Lakes NewJersey USA) and the antibiotic disc was then placed on theplate followed by overnight incubation at 37∘CThe inhibitionzone was interpreted according to the Clinical LaboratoryStandard (CLSI) Guidelines formerly known as the NationalCommittee for Clinical Laboratory Standards The testedantibioticswere penicillinG (10U) oxacillin (1120583g) neomycin(30 120583g) sulfamethoprim (5 120583g) clindamycin (2120583g) gen-tamicin (10 120583g) cefoxitin (30 120583g) rifampicin (5 120583g) ery-thromycin (15 120583g) tetracycline (30 120583g) vancomycin (30120583g)ciprofloxacin (5120583g) sulfazothrim (23120583g) cefepime (30 120583g)linezolid (30 120583g) and chloramphenicol (30 120583g)

210 Minimal Inhibitory Concentration (MIC) Determina-tions The MICs of vancomycin linezolid methicillin andampicillin were determined by the macrodilution brothmethod based on CLSI recommendations using in-house-prepared panels [26] Antibiotic concentrations of 003 0060125 025 05 10 and 20mgmLminus1 were tested One mLof broth was transferred to the tubes and 100 120583L of thebacteria suspension was adjusted to 106 CFUmL in saline085 according to a 05 McFarland scale and transferredto tubes containing 1mL of each antimicrobial Strains weregrown in Mueller-Hinton broth (BD BBL Franklin LakesNew Jersey USA) and the MIC was estimated as the lowest

BioMed Research International 5

antibiotic concentration that inhibits visible growth after 24 h[26]

3 Results and Discussion

31 Isolation and Identification of Coagulase-Negative Strainsfrom Salami Sixty-five presumable coagulase-negativestaphylococci microorganisms from salami were isolated bycolony morphology coagulase slide test subsequent tubetest and biochemical tests The sequencing of the V5 regionof the 16S rDNA fragment of the strains was discriminativeenough to differentiate the Staphylococcus isolated fromsalami at the subspecies level with the exception of 1 strainidentified up to the Staphylococcus spp genus

Nineteen distinct strains were identified as CNS in salamiwith 08 of 19 (42) identified as S saprophyticus thepredominant species followed by 05 strains of S xylosus(26) 02 strains of S carnosus (11) and 01 strain of eachof the following species S succinus S sciuri S epidermidisand S hominis (5 each) (Table 2)

The 08 S saprophyticus strains were identified asKJ6991511 AB6977171 JX4901221 KJ0046231 andHQ6995101 EU4309921 HF9372521 and KJ9496061 withthe latter two and S sciuri JX9664361 being homologous(96ndash98) to strains from the environment Five S xylosusstrains CP0072081 KF1980801 CP0087241 AM8827001and KC4565901 and a single S succinus strain KC3298241were identified The last three S xylosus strains and theS succinus strain are homologous (97-98) to strainsfound in fermented meat or meat starter cultures Sxylosus CP0072081 showed homology (98) to potentialopportunistic pathogenic strains from mammal species

The S carnosus strains identified were KJ8620021 andNR1164341 and the latter as well as the single S hominisJX5199881 (96-97) identified were homologous (97) tospecies from human microbiota

S carnosus and S xylosus are commonly used as com-mercial starter cultures for sausage manufacturing [26] butS succinus has also been observed in dry fermented sausagesand its use as a starter culture has been already proposed [27]

The diversity of CNS microbiota found in the samplesanalyzed in the present study could be related to the originof the salami The microbiota from the artisanal salamishowed greater biodiversity when compared to the commer-cial salami In commercial salami S saprophyticus was thepredominant species but S xylosus and S carnosus alsoobserved in commercial salami are commonly isolated fromstarter cultures [26] The predominance of S saprophyticusfollowed by S xylosus has also been reported in salamifrom South Italy [28] similar to Belgian sausages where Ssaprophyticus was the most frequently detected species [29]

S carnosus was not observed in artisanal salami but Ssuccinus S epidermidis and S hominis were detected

The biodiversity of the CNS staphylococci species foundin microbiota depends on the kind of meat-fermented prod-uct but CNS strains such as S saprophyticus S auricularisS xylosus S capitis S hominis S carnosus S haemolyticusS warneri S equorum S cohnii S capitis and S intermediushave been described inNapoli-type salami Sremska sausages

dry sausages raw meat and naturally fermented meat [30ndash33]

During the last decades S epidermidis and S sapro-phyticus have been described as emerging pathogens [34]S saprophyticus is considered a frequent contaminant offermented sausages and raw meats and has been isolatedfrom rectal swabs of cattle carcasses and pigs In humans themain reservoir of S saprophyticus is the gastrointestinal tract[35] S saprophyticus and S epidermidis can be opportunisticpathogens isolated from the human urinary tract and thepresence of these species in food should be taken into accountconcerning possible contamination of the starter inoculumandor improvements in the salami manufacturing process[13]

Other CNS species observed in the present study aremainly associated with ordinary food contaminants withS epidermidis and S hominis being the dominant speciesin human skin and occasionally isolated from the skin ofdomestic animals [36]

32 Phylogenetic Relationships of the CNS Identified in SalamiCNS strains can be grouped into four species groupssaprophyticus simulans epidermidis and haemolyticus Fre-quently the saprophyticus species group includes S xylosusand S saprophyticus while the simulans species group iscomprised of S carnosus and S piscifermentans the epi-dermidis species group is composed of S epidermidis Scapitis S caprae and S saccharolyticus and the haemolyticusspecies group encompasses S haemolyticus S hominis andS devriesei [37 38] In the present study the main clustergrouped several S saprophyticus strains namely KJ6991511AB6975171 KJ9496061 JX4901221 and KJ0046231 four Sxylosus strains CP0087241 CP0072081 AM8827001 andKC4565901 and the S succinusKC3298241 strain (Figure 1)These strains are homologous to those from fermented meatmicrobiota

The subclusters of those clusters showed mismatches ofspecies belonging to the four species groups The first sub-cluster grouped S epidermidis HF0882111 S saprophyticusHQ6995101 and S carnosusNR1164341 and the second sub-cluster grouped S hominis JX5199881 and the S saprophyticusEU4309921 all of them originally isolated from animalsand human beings The third subcluster includes speciespreviously isolated from the marine environment such as SxylosusKF1980801 while S carnosusKJ8620021 is utilized asa probiotic organism in foodsThe fourth cluster grouped theS sciuri JX9664361 and S saprophyticus HF9372521 strainswhich are homologous to species found in soil

The CNS strains clustered into groups near the bottomof the phylogenetic tree are mostly strains found in artisanalsalami whereas the species at the top of the tree are mainlyCNS strains found in commercial salami (Figure 1)

The close similarities between the S saprophyticusAB6975171 and KJ9496061 strains the S succinusKC3298241 S xylosus AM8827001 KC4565901 andCP0087241 strains the S saprophyticus KJ0046231 andKJ6991511 strains and S xylosus CP0072081 are supportedby a bootstrap value of 100 The interspecies similaritieswere over 90 which demonstrates close phylogenies

6 BioMed Research International

Table2Genotypicandph

enotypiccharacteriz

ationof

CNSstrainsfrom

salami

Strainsidentificatio

nandcharacteriz

ation

Salamiorig

inStaphylococcus

species

Genotypic

Phenotypic

GenBa

nkaccessionnu

mbera

ndsim

ilarity(

)Presence

ofenterotoxingenes

mRN

Adetection

Enterotoxinprod

uctio

n(ngm

Lminus1)

Com

mercial

Staphylococcus

sppKF

1354451(96)

seaseb

mdashmdash

Scarnosus

KJ8620021(96)

seasebsedsehseiand

selj

mdash12plusmn01

Scarnosus

NR1164341(98)

sebsecsedandsee

see

03

Ssaprophyticus

AB6

977171

(98)

sehseiandselm

seiselm

03

Ssaprophyticus

EU4309921(99)

seasebselm

seln

selo

and

tstH1

sebselm

and

selo

13plusmn01

Ssaprophyticus

HQ6995101(97)

mdashmdash

mdashSsaprophyticus

JX4901221(99)

seaseh

seh

05plusmn01

Ssaprophyticus

KJ00

46231(96)

sec

mdash04

Ssaprophyticus

KJ9496061(98)

seasebseca

ndseh

seasebandseh

mdashSsciuriJX9664361(98)

sedsei

sei

10plusmn01

SxylosusA

M8827001(97)

mdashmdash

mdashSxylosusCP

0072081(99)

mdashmdash

mdashSxylosusC

P0087241(98)

seatstH1

sea

14SxylosusK

C4565901(98)

mdashmdash

mdash

Artisa

nal

SepidermidisKF

6005891(97)

secsedseesegsehandsei

seesei

07plusmn01

ShominisJX5199881(97)

seasebsecsedseljselm

seln

andselo

selnselo

09plusmn01

Ssaprophyticus

HF9

372521(97)

seb

seb

05

Ssaprophyticus

subspbovisK

J6991511

(98)

secselm

seln

andtstH1

seln

09plusmn01

Ssuccinus

KC3298241(99)

seasebsecseljandseln

sebsec

13SxylosusK

F1980801(97)

secselm

andtstH1

mdash05plusmn01

Thep

resenceo

fenterotoxin

genesseasebsecsedseesegsehsei

seljselkselm

seln

selo

selq

selrselu

and

tstH1w

asteste

dby

PCRusingthes

pecific

seto

fprim

ers

sea-seee

nterotoxin

prod

uctio

nwas

evaluatedby

immun

e-sorbentassays(EL

ISA)u

singthed

etectio

nkitR

IDASC

REEN

SETAB

CD

EV

aluesa

redisplay

edas

them

eansplusmnSD

ofassays

perfo

rmed

indu

plicate

mRN

Atranscrip

tsfora

llenterotoxingenesw

eree

valuated

byrealtim

eRT-PC

Rtests

CT

values

ared

isplayedas

meansplusmnSD

ofRT

-PCR

tests

perfo

rmed

indu

plicateseamdash

SxylosusC

P00872413

0plusmn04sebmdash

SxylosusC

P008724133plusmn20Ssaprophyticu

sJX4901221302plusmn04Ssaprophyticus

subspbovisK

J6991511

32plusmn01SxylosusK

F19808013

6plusmn10

and

ShominisJX519988134plusmn10secmdashSsaprophyticus

subspbovisK

J6991511

31plusmn11seemdashSsaprophyticus

subspbovisK

J6991511306plusmn05

Ssuccinus

KC3298241285plusmn01andSsaprophyticus

HF9

3725213

2plusmn04sehmdash

SxylosusC

P0087241 31plusmn10Ssaprophyticu

sJX9

6643613

32plusmn06seimdash

Ssaprophyticus

AB6

977171

30plusmn10

Ssaprophyticu

sJX9664361306plusmn10

seln

mdashShominisJX5199881303plusmn10

Ssaprophyticu

sEU430992131plusmn03andselomdashShominisJX519988132plusmn10

BioMed Research International 7

O S succinus KC3298241Δ S xylosus AM8827001

O S saprophyticus KJ6991511Δ S saprophyticus AB6977171

Δ S xylosus CP0087241Δ S xylosus KC4565901Δ S saprophyticus KJ9496061Δ S saprophyticus JX4901221Δ S saprophyticus KJ0046231

Δ S xylosus CP0072081Δ S carnosus NR1164341Δ S saprophyticus HQ6995101

O S epidermidis KF0882111O S hominis JX5199881Δ S saprophyticus EU4309921

O S xylosus KF1980801Δ S carnosus KJ8620021Δ S sciuri JX9664361O S saprophyticus HF9372521

04 03 02 01 00

Figure 1 Phylogenetic tree generated from the multiple alignments of the 16S rDNA sequences of CNS strains found in salami usingthe ClustalX 20 software The phylogenetic tree was constructed by using the Mega 60 software and the unweighted pair group method(UPGMA) Bootstrap values ranged from 00 to 04 Strains found in commercial (Δ) or artisanal salami (O)

between these CNS strains Suzuki et al 2012 demonstrateda bootstrap value higher than 90 for interspecies similaritiesbetween S saprophyticus S epidermidis S hominis and Scarnosus

33 Genotypic and Phenotypic Characterization of CNSStrains Twelve distinct combinations of staphylococcalenterotoxins geneswere found in the 15CNS strains compris-ing SEs AndashE GndashJ and also the enterotoxin-like toxins (SElL)KndashR and U (Table 2)

Fifteen strains (79) carried at least one gene encodingenterotoxins in their genomes (Figure 2) The seb and secgenes were the most predominant harbored by 57 ofthe strains followed by sea carried by 50 whereas thesedsehselm genes showed intermediate incidence harboredby 33 of the strains while seiseln and tsH1 were found in27 of strains Finally see selj and selo showed low incidence(13) and seg was carried by only 1 of strains (Table 2)

The relationships between superantigenic toxin geno-types and toxin gene-encoding mobile genetic elementsin CNS strains were evaluated Distinct combinations ofSaPI and plasmids or plasmids and genes of egc operonenterotoxins were found in the CNS strains obtained fromsalami

Four strains presented only the classical enterotoxingenes namely S saprophyticus KJ0046231 and HF9372521S xylosus CP0087241 and S carnosus NR1164341 Anotherstrain S saprophyticus AB6977171 presented only the newlydescribed enterotoxins seh sei and selm and finally 10 strainspresented a combination of classical and newly described

enterotoxin genes in their genomes with at least one of eachenterotoxin type (Table 2)

Previous studies have shown that sea is the most com-mon toxin associated with Staphylococcus food poisoningfollowed by sed and see with SEHand SEI being considered asplaying only a minor role [39] Among the 15 enterotoxigenicstrains found in salami 73 of them harbored at least the seagene or combinations of these 05 genes Strains S carnosusKJ8620021 found in commercial salami and S epidermidisKF6005891 found in artisanal salami carry 04 of these genesThe sea and seb enterotoxin genes are known to occupy thesame locus on the chromosome which may explain whythese enterotoxins are commonly found together in foodpoisoning outbreaks [36] The combination of sea and sebgenes was found in 05 strains S saprophyticus EU4309921 Scarnosus KJ8620021 S hominis JX5199881 S saprophyticusKJ9496061 and S succinus KC3298241 A single strain Scarnosus KJ8620021 showed the combination of sea and seigenes

Additionally some CNS are able to produce TSST-1 aloneor in combination with other enterotoxins Herein 04 of the19 strains (21) S saprophyticus EU4309921 S saprophyti-cus subsp bovis KJ6991511 and S xylosus CP0087241 andKF1980801 were shown to harbor the tstH1 combined withse andor sel enterotoxin genes (Table 2)

The staphylococci enterotoxins genes seg and sei [40] arepart of a chromosomal operon gene cluster (egc) comprisingfive genes designated as selo selm sei seln and seg Twoof the CNS strains determined in the present study Ssaprophyticus EU4309921 and S hominis JX5199881 were

8 BioMed Research International

sed

seb

sec

see

sea

M 2 3 4 51

500bp

(a)

sea

sebsebseb

M 1 2 3 4 5

(b)

sei

sehseg

1M

(c)

seh sehseh

M 1 2 3

(d)

1 2 4M 3 5

(e)

selm

seljseln

selo

21M

(f)

seln

selm

M 1 2

(g)

Figure 2 Uniplex duplex and multiplex PCR screening for the detection of enterotoxin genes in CNS strains from salami (a) Lane M100 bp DNA ladder plus (Fermentas Foster City CA USA) lane 1 S aureus ATCC 29231 harboring sea gene lane 2 S aureus NCTC 10654harboring seb gene lane 3 S aureus ATCC19095 harboring the sec gene lane 4 S aureus ATCC 13563 harboring the sed gene and lane 5 Saureus ATCC 27664 harboring the see gene (b) Lane M 100 bp DNA ladder plus lane 1 Staphylococcus spp lane 2 S carnosus NR116434lane 3 S carnosus KJ862002 lane 4 S carnosus KJ862002 and lane 5 S carnosus KJ8620021 (c) Lane M 100 bp DNA ladder plus lane1 S aureus ATCC 19095 harboring seg seh and sei genes (d) Lane M 100 bp DNA ladder plus lane 1 S saprophyticus AB6977171 lane 2S epidermidis KF 6005891 and lane 3 S sciuri JX9664361 (e) Lane M 100 bp DNA ladder plus lane 1 S xylosus KF1980801 lane 2 SsaprophyticusKJ6991511 (f) LaneM 100 bp DNA ladder plus lane 1 S aureusATCC 27154 harboring selj slem seln and selo genes (g) LaneM 100 bp DNA ladder plus lane 1 S xylosus KF1980801 and lane 2 S saprophyticus subsp bovis KJ6991511

BioMed Research International 9

shown to carry the selm seln and selo genes and the selmand seln sequences were detected in the genome of a singlestrain S saprophyticus subsp bovisKJ6991511 S epidermidisKF6005891 showed a combination of seg and sei genesand 03 strains harbored a single gene sei or selm or selnperhaps due to the high degree of genetic polymorphism inthe chromosomal assembly [41] Indrawattana et al 2013detected the seg-sei-selm-seln-selo of the highly prevalent egclocus was 261 in contrast to this study where 13 CNSpresents the combination of selm seln and selo genes [42]

The CNS strains clustered into groups near the bottom ofthe phylogenetic tree carried the classical enterotoxin geneswhile the species at the top of the tree showed high diversityamong the enterotoxin genes combining the classical and thenewly described genes in their genomes (Figure 1)

To assess the risk of staphylococcal food poisoning theability of the identified strains in harboring the se andsel genes and in expressing and producing enterotoxins invitro was evaluated The mRNA for each enterotoxin genewas evaluated by real time RT-PCR assays and enterotoxincontent was estimated by a sandwich enzyme immunoassayfor the combined detection of Staphylococcus enterotoxins(SET) A B C D and E

The fifteen enterotoxigenic CNS strains were able toexpress the classical enterotoxins SEA seb sec sed and seein concentrations ranging from 03 ngmLminus1 to 14 ngmLminus1as assessed by the in vitro assays (Table 2) The S sapro-phyticus AB6977171 KJ6991511 JX4901221 KJ0046231 andHF9372521 strains S xylosus KF1980801 and S carnosusNR1164341 produced low amount of enterotoxins lower than05 ngmLminus1 S epidermidisKF6005891 S saprophyticus bovisKJ6991511 and S hominis JX5199881 produced intermediateamount of enterotoxins ranging from 07 to 090 ngmLminus1while S sciuri JX9664361 S saprophyticus EU4309921 Scarnosus KJ8620021 S xylosus CP0087241 and S succi-nus KC3298241 produced enterotoxins in concentrationsge10 ngmLminus1

Although the sandwich enzyme immunoassay is consid-ered themost sensitivemethod to detect seandashsee enterotoxinsable to detect 0125 ngmLminus1 differences in the specificity andsensitivity of the assays for the detection of staphylococcalenterotoxins from foods are expected [43] A single strainStaphylococcus spp KF1354451 which harbors both the seaand seb genes was unable to produce seandashsee enterotoxins

The mRNA levels evaluated by the real time RT-PCRassays for enterotoxins were detected in 12 of 15 strains(86) (Table 2) Transcripts for the classical enterotoxinsgenes were detected when S saprophyticus HF9372521 Scarnosus NR1164341 S xylosus CP0087241 and S succinusKC3298241 were assayed Transcripts for newly describedenterotoxins were observed in S saprophyticus JX4901221and AB6977171 S sciuri JX9664361 S saprophyticus subspbovis KJ6991511 and S hominis JX5199881 Transcripts fromboth the classical and newly described enterotoxins weredetected in S saprophyticus EU4309921 and KJ9496061 andS epidermidis KF6005891

No mRNA transcripts were obtained for S sapro-phyticus KJ00462321 and S carnosus KJ8620021 although

enterotoxin production was detected by the enzyme-linkedimmunosorbent tests (Table 2)

The 04 strains that carry tstH1 in their genome Ssaprophyticus EU4309921 and KJ6991511 and S xylosusCP0087241 and KF980801 were not able to produce mRNAfor TSST-1 in the assay conditions

The production of classical enterotoxins in vitro(immunologic test) matched the results shown by real timeRT-PCR assays for the following strains S saprophyticusJX4901221 S sciuri JX9664361 S saprophyticusEU4309921S saprophyticus HF9372521 S saprophyticus subsp bovisKJ6991511 S hominis JX5199881 S epidermidis KF6005891and S succinus KC3298241

Ten strains namely S saprophyticusAB6977171 S sapro-phyticus EU4309921 S saprophyticus HF9372521 S sapro-phyticus KJ6991511 S saprophyticus KJ9496061 S succinusKC3298241 S hominis JX5199881 S xylosus KF1980801and S xylosus CP0087241 and S epidermidis KF6005891expressed mRNA for multiple se andor sel genes There isa differential transcription among these genes where themost frequent among the classical ones were seb and seeseatranscribed by 04 and 02 strains respectively and the mostfrequent among the newly described genes were sei andsehselnselo transcribed by 03 and 02 strains respectively

No mRNA for the sec gene was detected although SsaprophyticusAB6977171 was able to produce the enterotoxinin vitro as detected by the ELISA assays

34 Enterotoxin Gene Homologies between Salami CNSand CPS Strains The nucleotide sequencing of six entero-toxin genesmdashtwo of them sec and see encoding classi-cal enterotoxinsmdashand four of them encoding the newlydescribed genes seg seh selm and seln fromCNS strains wascompared to the enterotoxin genes from S aureus or S pas-teuri two coagulase-positive staphylococci strains (Figure 3)The homology between the CNS and CPS enterotoxin genesvaried from 65 to 98 The homology of sec seg sehselm and seln between CNS from salami and S aureus was98 60 98 65 and 70 respectively The homologybetween see from CNS found in salami and in S pasteuri was98

Although further studies should be performed it seemsthat the sequences encoding enterotoxins can be conservedamong coagulase-negative and coagulase-positive staphy-lococci as shown in the phylogenetic analysis of the 06enterotoxin genes (Figure 4)

35 Antimicrobial Multiresistance of CNS Strains (MRCNS)Another safety hazard associated with CNS strains besidesthe ability of producing enterotoxins in food matrices is theantimicrobial resistance to antimicrobial agents commonlyused to treat staphylococci infections The antimicrobialresistance carried by CNS strains from food matrices can bespread to the population by the consumption of an apparentlysafe food

Among the 19 CNS strains identified in salami 14 showedmultiresistance to antimicrobial agentsThree strains showedthe highest MAR indices 093 and 080 07 strains presented

10 BioMed Research International

lowast lowastlowastlowastlowastlowast lowast lowast lowastlowast lowast lowast lowastlowast lowastlowastlowastlowast lowastlowastlowast lowast

lowastlowast lowast lowast lowastlowastlowastlowast lowast lowast lowastlowast lowast

lowastlowastlowastlowast lowast lowast lowast

lowastlowast lowast lowast lowastlowast lowastlowast lowast lowastlowast lowastlowastlowastlowastlowast lowastlowast lowast lowastlowast lowastlowast

lowastlowast lowastlowastlowast lowast lowast lowast lowast lowast lowastlowastlowastlowastlowast

lowastlowast lowastlowast lowastlowast lowastlowast lowast lowast lowastlowast lowast lowast lowastlowastlowast lowast lowastlowast lowastlowastlowast

lowast lowast lowast lowast lowast lowast lowastlowast lowast lowastlowastlowast lowast lowastlowastlowast lowast lowast lowastlowastlowast lowast lowastlowastlowast lowast

lowast lowast lowast lowastlowastlowastlowast lowast lowastlowastlowast lowast lowast lowastlowastlowast lowast lowast lowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast lowast lowast lowast lowast lowastlowast lowast lowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

Enterotoxin C (sec) GATAAAAATGAATAAGAGTCGATTTATTTCATGCGTAATTTTGATATTCGCACTTATACT------------------------------------------------------------

AGTTCTTTTTACACCCAACGTATTAGCAGAGAGCCAACCAGACCCTACGCCAGATGAGTT------------------------------------------------------------

GCACAAAGCGAGTAAATTCACTGGTTTGATGGAAAATATGAAAGTTTTATATGATGATCA------------------------------------------------------------

TTATGTATCAGCAACTAAAGTTAAGTCTGTAGATAAATTTTTGGCACATGATTTAATTTA------------------------------------------------------------

TAACATTAGTGATAAAAAACTGAAAAATTATGACAAAGTGAAAACAGAGTTATTAAATGA------------------------------------------------------------

AGGTTTAGCAAAGAAGTACAAAGATGAAGTAGTTGATGTGTATGGATCAAATTACTATGT------------------------------------------------------------

AAACTGCTATTTTTCATCCAAAGATAATGTAGGTAAAGTTACAGGTGGCAAAACTTGTAT------------------------------------------------------------

GTATGGAGGAATAACAAAACATGAAGGAAACCACTTTGATAATGGGAACTTACAAAATGT------------------------------------------------------------

ACTTATAAGAGTTTATGAAAATAAAAGAAACACAATTTCTTTTGAAGTGCAAACTGATAA------------------------------------------------------------

GAAAAGTGTAACAGCTCAAGAACTAGACATAAAAGCTAGGAATTTTTTAATTAATAAAAA------------------------------------------------------------

AAATTTGTATGAGTTTAACAGTTCACCATATGAAACAGGATATATAAAATTTATTGAAAA----TTGTATGAGTTTAACAGTTCACCATATGAAACAGGATATATAAAATTTATTGAAAA

TAACGGCAATACTTTTTGGTATGATATGATGCCTGCACCAGGCGATAAGTTTGACCAATCTAACGGCAATACTTTTTGGTATGATATGATGCCTGCACCAGGCGATAAGTTTGACCAATC

TAAATATTTAATGATGTACAACGACAATAAAACGGTTGATTCTAAAAGTGTGAAGATAGATAAATATTTAATGATGTACAACGACAATAAAACAGTTGATTCTAAAAGTGTGAAGATAGA

AGTCCACCTTACAACAAAGAATGGATAATGTTAATCCGATTTTGATATAAAAAGTGAAAGAGTCCACCTTACAACAAAGAATGGATAATGTAATCCGATTATGGACAAGTGTGCTTTTTT

Enterotoxin C TATTAGATATATTTGAAAGGTAAGTACTTCGGTGCTTGCCTTTTTAGGATGCATATATATEnterotoxin C CNS

Enterotoxin CEnterotoxin C CNS

Enterotoxin CEnterotoxin C CNSEnterotoxin CEnterotoxin C CNSEnterotoxin CEnterotoxin C CNSEnterotoxin CEnterotoxin C CNSEnterotoxin CEnterotoxin C CNSEnterotoxin CEnterotoxin C CNSEnterotoxin CEnterotoxin C CNSEnterotoxin CEnterotoxin C CNS

Enterotoxin CEnterotoxin C CNSEnterotoxin CEnterotoxin C CNS

Enterotoxin CEnterotoxin C CNS

Enterotoxin CEnterotoxin C CNS

Enterotoxin CEnterotoxin C CNS

TTTGTGATCGTCGGCCA-----ACCGATACCGTCGTATTCTTGTACCGCGCAAGGCGAAGlowast lowast lowastlowastlowast lowast lowast lowast lowast lowastlowast lowast lowastlowastlowast lowast lowast lowast lowast

Enterotoxin E (see) Enterotoxin E TGCTTTAGGCGGGGTCTTACAGCATATTATCGTAGAAGATGCACAAGCTGCACGTAAAGCS pasteuri TGCTTTAGGCGCATCGTTACAACATGTCATCGTAGAAGATGAAAAAGATGGTCGTCAGGC

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast lowastlowastlowastlowast lowastlowastlowast lowast lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast lowast lowastlowastlowast lowastlowast lowastlowastlowast lowast lowastlowastlowast

Enterotoxin E ATTAGAATTCTTGAAATCAAAACA-AGCAGGACGTGCTACATTTCTACCATTAAATGTCAS pasteuri

Enterotoxin ES pasteuri

GATACAATT-TTTAAAACAACGTAGTTTAGGACGTGCCACATTCCTACCTTTAAATGTTA lowastlowast lowastlowastlowastlowast lowastlowast lowastlowastlowast lowastlowastlowast lowast lowastlowastlowastlowastlowastlowastlowastlowastlowast lowastlowastlowastlowastlowast lowastlowastlowastlowastlowast lowastlowastlowastlowastlowastlowastlowastlowast lowast

TCAAACCTAGAAATATTTCAACCAAGATATATlowast lowastlowastlowastlowast lowastlowastlowast lowastlowastlowastlowastlowast

Enterotoxin G (seg) Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

---------------------------attaagaaatactt--ccattttaatactcttaGGGGATATTTCCGGGAAACCGGGGCTAATACTGGATAACTTTTCTCTCCGAACGAAGAGA

gaatcaacaactttattatctccg--taaat-----atttaaaaacttatatggaacaaaGATTCAACGATGGTCTTGGTTTTCAATTAAGATGAACCCGCGGCGCTTAGCTGGTGGGAC

aggtactagtt-----------------------------------------cttttttaAGGTCACGGCTCACCCAAGCCCCCATGCGTATACGACCTGGAAGGGTGATCGGCCACATT

ggaaataagtcaaaccaaaaacttgtattgttcttttcagtaaat---------------GGGACTGAGACACAGCACAAACTCCTACGGGAGGCAGCAATAGGGAATCTTCCGCAATGG

------------------------------------------------------------CCGAAAGTCTGACGGAGCAACGCCGCGAGACTGAAGAATGTCTTCGGATCGTAAAACTCT

------------------------tttatatatccagattcaaa-----tgcagaaccatGTTGTTAGGGAAGAACAAGTATCGTTCGAATAGGGCGGTACCTTGACGGTACCTAACCAG

caaactcgtatagctttttttct---------ttagtgagccagtgtcttgctttg----AAAGCTTTGCTAACTACCTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCG

-taatctagttcctgaatagtaaccatattcttatttgtagttattgta--aatccaagtGAATTATTGGGCGTAAAGCGCGCGCATGCGGTCTTTTAAATCTGATGTCAAAGCCCACGG

gattgtct----------attgtcgattgttacctgtacagt------------aattaaGCTCAACCGTGGAGGGTCATTGGAAACTGTAAGACTTGGAGTGCAGAAGAGGATAGTGGA

Figure 3 Continued

BioMed Research International 11

lowast lowast lowast lowastlowast lowastlowast lowastlowastlowast lowast lowastlowast lowast lowast lowast lowast

lowast lowast lowastlowastlowast lowastlowast lowast lowastlowastlowast lowast lowastlowast lowastlowastlowast lowast lowastlowast lowastlowast lowast lowast lowastlowast lowast

lowastlowast lowast lowast lowastlowastlowastlowastlowast lowast lowast lowastlowast lowast lowast lowast lowast lowast lowastlowastlowast

lowastlowast lowast lowastlowast lowastlowastlowast lowastlowast lowastlowast lowast lowast lowast lowast lowastlowast lowastlowast lowast lowast lowast lowastlowast

lowastlowast lowastlowastlowastlowast

lowast lowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowast lowast lowastlowastlowastlowast lowast lowastlowast lowastlowast lowast lowastlowastlowastlowast lowast lowastlowast lowast lowast lowastlowastlowastlowastlowastlowastlowast

lowastlowast lowast lowast lowast lowast lowastlowastlowast lowastlowast lowastlowast lowast lowastlowastlowastlowastlowastlowastlowastlowast lowastlowastlowast

lowast lowast lowast lowast lowast lowast lowastlowast lowastlowast lowast lowast lowastlowastlowast lowast lowast lowastlowastlowast lowast lowast

lowastlowast lowastlowastlowastlowastlowastlowast lowastlowast lowastlowastlowast lowastlowast lowast lowastlowast lowastlowast lowastlowastlowast lowast lowast

lowastlowast lowast lowastlowastlowast lowast lowastlowastlowastlowastlowast lowast lowast lowast lowastlowastlowast lowast lowastlowast lowast

lowastlowast lowast lowast lowast lowast lowastlowast lowast lowastlowast lowast lowastlowast lowast lowastlowast lowastlowastlowastlowast lowast lowast lowastlowast

lowast lowastlowastlowast lowast lowastlowast lowast lowast lowastlowast lowast lowastlowast lowast lowastlowastlowastlowastlowastlowast lowast

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

tttatctctttcattttctgaactattaaa----tgtaagaccacc---atacatacaacATTCCACGTGTAACGGTGAAAATGAGTAGAGATGTGGAGGAACACCAGTGGAGAAACGCG

aacctccaaaattttggtttatatccggttc-----------agatttaggtattatacaACTCTCTGGTCTGTTACTGACGCTGAGGCGCGAAAGCGTGGGAGCAAACAGGATTATATA

tgtataaaaatatggaacgccaaaaatgtctacttttttatctttataattgttagctaaCCCCTGGTAGTCCAAGCCGTAAACGATGAGTGCTAGTGTTACAGGGT------------T

ttctgtattttctaattcagttttaacctcattataactcttatactcaattggaaaaatTCCGCCCTTTACTGCTGCAGCTAACG---CATTAG-GCACTCCGCCTGGGGAGTACAACT

taaatcatgagataaaaactgtctagaattaataactcctcttccttcaacaggtggagaGCAA------GGTTGAAACTCAAAGCA----TTGACCGGGGGCC---CGATAAGCTGAGG

cgtataaagattcattacat----tacccatagttcccttattgtttttataatcacttaTACATGGTGTTTAATTCCAAACCACGCCAAGAACCCTCACGAGGTCTTGACATCCTCTGG

ctttattt----------------------------------------------------ACCTCCCTAGAGAATAAGGGCTTCCCCGTCGGGGATGAAATGAACAGGTGGTGCATGGAT

-------agttcgtc---AGTCTTCAGCTCGTGCAT

Enterotoxin H (seh)

------------------------------------------------------------ATCTATGAATATAAATCTTTTAATATTGGTTTAATATAAATATTCCATTTCTAGAAACCT

------------------------------------------------------------ATATTTTATGTTTGTATAAATTCATAAATTGTTCGAAATTTATATATTAAAAGTTGTTTT

------------------------------------------------------------TGTTGTTTTATAATTAAGTTAATGAAATATATTGAGGAGTTTTAAAATGATTAATAAAAT

------------------------------------------------------------TAAAATATTATTTTCGTTTTTAGCATTATTACTTTCATTCACATCATATGCGAAAGCAGA

------------ -----------TTACGATTTAGCTTTAGCTAATGCATATGGTCAATAAGATTTACACGATAAAAGTGAGTTAACAGATTTAGCTTTAGCTAATGCATATGGTCAATA

TAATCACCCATTCATTAAAGAAAATATTAAGAGTGATGAAATAAGTGGAGAAAAAGATTTTAATCACCCATTCATTAAAGAAAATATTAAGAGTGATGAAATAAGTGGAGAAAAAGATTT

AATATTTAGAAATCAAGGTGATAGTGGCAATGATTTGAGAGTAAAGTTTGCAACTGCTGAAATATTTAGAAATCAAGGTGATAGTGGCAATGATTTGAGAGTAAAGTTTGCAACTGCTGA

TTTAGCTCAGAAGTTTAAAAATAAAAATGTAGATATATATGGGGCATCTTTTTATTATAATTTAGCTCAGAAGTTTAAAAATAAAAATGTAGATATATATGGGGCATCTTTTTATTATAA

GTGTGAAAAAATAAATGAAAATATTTCTGAATGTCTATATGGAGGTACAACACTAAATAAGTGTGAAAAAATAAGTGAAAATATTTCTGAATGTCTATATGGAGGTACAACACTAAATAG

TGAAAAATTGGCACAGGAAAGGGTGATTGGTGCTAATGTTTGGGTAGATGGTATTCAAAATGAAAAATTGGCACAGGAAAGGGTGATTGGTGCTAATGTTTGGGTAGATGGTATTCAAAA

AGAAACAGAATTAATACGAACAAATAAGAAAAATGTGACATTGCAAGAATTAGATATAAAAGAAACAGAATTAATACGAACAAATAAGAAAAATGTGACATTGCAAGAATTAGATATAAA

Enterotoxin H CNS GATCAGAAAAATATTGTCCGATAAATATAAAATTTATTATAAAGACAGCGAAATAAGGG-Enterotoxin H

Enterotoxin H CNSEnterotoxin H

Enterotoxin H CNSEnterotoxin HEnterotoxin H CNSEnterotoxin HEnterotoxin H CNSEnterotoxin HEnterotoxin H CNSEnterotoxin HEnterotoxin H CNSEnterotoxin H

Enterotoxin H CNSEnterotoxin H

Enterotoxin H CNSEnterotoxin H

Enterotoxin H CNSEnterotoxin H

Enterotoxin H CNSEnterotoxin H

Enterotoxin H CNSEnterotoxin H

GATCAGAAAAATATTGTCCGATAAATATAAAATTTATTATAAAGACAGCGAAATAAGTAA

Enterotoxin N (seln) TGATTTAGCTTTAGCTAATGCATATGGTCAATAT-AATCACCCATTCATTAAAGAAAA-----TAGATATTTATGGACTGTATTATGGAAATAAATGTGTAGGCTTAACTGAAGAAAAAA

TATTAAGAGTGATGAAATAAGTGGAGAAAAAGATTTAATATTTAGAAATCAAGGTGATAGCATCATGCTTATACGGAGGAGTTAC------GA--TATATGATGGAAATCAATTAGATGA

TGGCAATGATTTGAGAGTAAAG--------------TTTGCAA-CTGCTGATTTAGCTCAAGAGAGAGTTATAGGCGTTAATGTATTTAAAGATGGTATCCAACAAGAAGGTTTTGTTAT

GAAGTTTAAAAATAAAAATGTAGATATATATGGGGCATCTTTTTATTATAAGTGTGAAAAAAAAACTAAAAAGGCTAAAGTAACAGTACAA------GAATTAGATACTAAAGTTCGATT

AATAAGTGAAAATATTTCTGAATGTCTATATGGAGGTACAACACTAAATAATGAAAAATT------------TAAATTAGAAAATTTATATAAAATATACAATAAAGATACCGGTAACAT

Enterotoxin N CNSEnterotoxin N

Enterotoxin N CNSEnterotoxin N

Enterotoxin N CNSEnterotoxin N

Enterotoxin N CNSEnterotoxin N

Enterotoxin N CNSEnterotoxin N

Figure 3 Continued

12 BioMed Research International

lowastlowastlowast lowastlowast lowastlowast lowastlowast lowast lowastlowast lowastlowastlowast lowastlowast lowast lowastlowastlowastlowast

lowastlowast lowast lowastlowast lowast lowast lowastlowast lowastlowastlowastlowastlowast lowast lowast lowast lowastlowast lowast lowast lowastlowast lowast lowast

lowast lowast

lowast lowastlowast lowast lowastlowast lowast lowast lowast lowast lowast lowast lowast lowastlowast lowast lowastlowastlowastlowast lowast

lowast lowastlowast lowast lowast lowastlowast lowast lowastlowastlowast lowast lowastlowast lowast lowastlowast lowastlowast lowast lowast

lowastlowastlowast lowast lowast lowastlowast lowastlowast lowast lowast lowast

lowast lowast lowast lowastlowastlowast lowastlowastlowast lowastlowast lowast lowastlowastlowastlowastlowast lowastlowastlowast lowast

lowastlowast lowast lowast lowastlowast lowastlowastlowast lowastlowast lowast lowast lowastlowastlowastlowast lowastlowast

lowast lowast lowast lowastlowast lowast lowastlowast lowastlowastlowastlowastlowastlowast lowastlowast lowast lowastlowast lowastlowastlowast

lowast lowast lowast lowast lowastlowast lowastlowastlowastlowast lowast lowast lowastlowastlowast lowast lowast lowast lowast lowast

lowast lowast lowast lowast lowastlowastlowast lowast lowast lowastlowast lowastlowast lowast lowastlowastlowast lowast lowastlowastlowast lowast

lowast lowast lowastlowastlowast lowast lowastlowast lowastlowastlowast lowastlowastlowastlowastlowast lowastlowast lowast lowastlowastlowast lowast lowast lowastlowastlowast

lowastlowastlowast lowast lowastlowast lowastlowast lowastlowast lowast lowast lowast lowast lowastlowast lowastlowast lowastlowast lowast lowast

lowast lowast lowast lowast lowastlowast lowast lowast lowastlowast lowastlowast lowast lowastlowastlowast lowastlowastlowastlowast lowast lowastlowastlowastlowastlowast lowast

lowastlowast lowast lowast lowastlowastlowast lowastlowast lowast lowastlowast lowast lowastlowast lowastlowast lowastlowast lowast lowast lowast

lowastlowastlowastlowastlowast lowastlowast lowastlowast lowast lowastlowast lowast lowast lowast lowast lowastlowast lowastlowast

lowast lowast lowastlowast lowastlowast lowastlowastlowastlowastlowastlowast lowastlowast lowast lowast lowast lowast lowast lowast lowast lowast lowast lowast lowast

lowastlowast lowastlowast lowast lowastlowastlowastlowast lowast lowast lowastlowastlowast lowastlowast lowast lowastlowastlowast lowast

lowastlowastlowastlowast lowastlowast lowast lowast lowast lowast lowastlowast lowast lowast lowast lowastlowastlowastlowastlowast lowastlowast lowastlowastlowast lowast

lowast lowastlowastlowast lowastlowastlowastlowastlowast lowast lowast lowast lowastlowast lowast lowast lowastlowast lowast lowastlowastlowast lowastlowast lowast

lowastlowast lowast lowast lowast lowastlowast lowast lowastlowast lowastlowast lowastlowast lowast lowastlowastlowastlowast

ATTAATACGAACAAATAAGAAAAATGTGACATTGCAAGAATTAGATATAAAGATCAGAAA-TTAT----------TATGATTTATATAAC--------------ATAAAAGGTTCAG---

AATATTGTCCGATAAATATAAAATTTATTATAAAGACAGCGAAATAAGGTAAAAGG------TAGGAGCAGAGTTTTTTCAATTTTATAGTGATAATAGAACAGTTAGCTCATCTAATTA

Enterotoxin N CNS -TAAA---Enterotoxin N

lowast lowastlowastlowastlowastlowast lowastlowast lowast lowast lowast lowastlowast lowast lowastlowastlowastlowastlowastlowast lowastlowast lowastlowast

GGCACAGGAAAGGGTGATTGGTGCTAATGTTTGGGTAGATGGTATTCAAAAAGAAACAGAACAA----AAAGGATGCATTTTCTTT-----CATTCTAATAATCATCAAAATCAATCATT

Enterotoxin N CNSEnterotoxin N

Enterotoxin N CNSEnterotoxin N

Enterotoxin N CNSEnterotoxin N

TCATATCG

Enterotoxin M (selm)

ttgctattcgcaaaatcatatcgcaaccgctgatgtcggagtt--ttgaatcttaggaacEnterotoxin M CNSEnterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

--------------AGTATGTTTAAATCCCCGCTCACAGTTATGTTTAACTCTTCCTTTC

tattatggta-gctatccaattgaagaccaccaaagtattaatcctgaaaataatcatctTTTTCACACCGCCCCGTCGGGTGTTGCGCACGGGGTTGTTTAACCCCCCCGGAAAAAATT

ttcgcatcaattagttttttctatggataattcgacagtaacagctgaatttaagaacgtEnterotoxin M CNS

Enterotoxin M CNS

TTCCCTCCCTCCTCCTTACTC---------------CCT----------------CCCCT

tgatgatgtaaaggaattcaaaaatcatgctgtagatgtatatggtctaagttatagtggEnterotoxin M CNS TTTTCCTACAAATCCTTTCTCCCCTCCCGGAGTAGAAGTAAA------------------

atattgtttgaaaaacaaatatatatacggtggagttacattagcaggtgat-tatttagEnterotoxin M CNS -------------------TAAAACAATACCCCTACCACCTTAACAATTTTTTTATTGAG

agaaatctagacgtattcct-attaatctttgggttaatggagaacatcaaactatatctEnterotoxin M CNS CCCCTCCCCCCCCTTCTCATTTTTTCTCTTTGAC-----------GTTCCCCCCATTTCT

actgacaaagtatcaactaataaaa-agttagtaacagctcaagaaattgatactaaattEnterotoxin M CNS GCCAATGCTTAACCCACCCTTAAAGGCCATCGGCACATATTAGACACTCC-----CCACC

aagaagatatctacaagaagaatataatatttatggctttaatgatac---aaataaaggEnterotoxin M CNS CGCCCCCTCTTTTTATTAAGGACACAACTCTTTTTATTTTCCCCCAAGAACTAATTCTGA

a-agaaattatggtaataagtcaaaatttagttctgga---------tttaatgcaggaaEnterotoxin M CNS AAAAAAACGTTCCGCTTAGTTCAGAATTTCCTTTGAGCCTTTTATCCTTTTGTACGCGAA

aaatattatttca--------tttgaatgatggttcatcattttcttatgacttatttga

Enterotoxin M CNS AAACCATCAGCCATTGAATCAGTTATCCCATTGCCCTAACTAACCCAATATCTAATCTAA

tactggaacaggacaagctgaaagtttctt-aaaaatatataatgacaacaaaactgtcgEnterotoxin M CNS TTTTCGCCTACGAACGAATAATAGCTTGTGGCGAAAAATATCCAGGATTATCTACTGTAG

aaact--gaaaaattccatttagatgtagaaatatcttataaggacgaaagttgaagtatEnterotoxin M CNS CGCCTCGCCAGACGTCCTTTGAATTGA--ATATGACTCCTACTCCCCCCCGCACCGTCAA

ttcaatcataacttagtaaaggaaatgccatgaaaaaatttaaatatagtttt---atatEnterotoxin M CNS TTCAAACA---------------GGTGGAAGTTAATACTGAATAGTAGGTCTTCTTCCCG

tagtttttatattactttttaacattaaagatcttacgtatgct---caaggtgatattgEnterotoxin M CNS TGCTATTCTTAGAACTTTTAAATTATGTAAACCCAATTTTTAGTGCACTCTTTCTCCCTC

gtg-taggtaacttaagaaatttctatacaaaacatgattatatagatttaaaaggcgtcEnterotoxin M CNS GTTACTCTTAGATGGAGAAGCGTTAAGACAGAAGCTTTGTAT-------------TCCCA

acagataaaaa-cctacctattgcaaatcaacttgaattttcaac-----aggt----acEnterotoxin M CNS GCAGAGTGCAACCTTTCACAATGAAGTTTTTATCTAATTTACACGCCTTTAGGCTCCAAA

caatgatttgatctcagaatctaataattgggacgaaataagtaaatttaaaggaaagaaEnterotoxin M CNS TACGTATTAAATCTCCTCCTGT--TTATCTGACCCTAAG-ATAAAACACGAAAAATTATG

actggatatttttggcattgattataatggtccttgtaaatctaaatacatgtatggaggEnterotoxin M CNS CGTGCCCACTCATCCTAT-------TACTTTCATTATATCTATAAAA-------------

Figure 3 Alignment of the enterotoxin gene sequences found in coagulase-negative staphylococci (CNS) and in coagulase-positivestaphylococci (CPS)

BioMed Research International 13

Enterotoxin (seg) S aureus AY1587031

Enterotoxin (sec) CNS

Enterotoxin (see) S pasteuri NC0227371

Enterotoxin (see) CNS

Enterotoxin (seh) CNS

Enterotoxin (selm) S aureus AF2857601

Enterotoxin (seln) CNS

Enterotoxin (seh) S aureus AJ9375481

Enterotoxin (seln) S aureus AY1587031

Enterotoxin (sec) S aureus X516611

Enterotoxin (selm) CNS

Enterotoxin (seg) CNS

150 100

100

100

100

100

99

98

96

97

72

50 0

Figure 4 Phylogenetic tree generated from the multiple alignments of the enterotoxin sequences of CNS and CPS strains using the ClustalX20 software package The phylogenetic tree was constructed using the Mega 60 software and the unweighted pair group method (UPGMA)

MAR indices varying from 066 to 046 and the remaining04 strains showed MAR indices le026 (Table 3)

The 14 MRCNS strains were resistant to 120573-lactams(oxacillin penicillin andor cefoxitin) and to vancomycincorresponding to 73 of the total CNS strains identifiedwhile 09 strains (64) showed resistance to tetracycline andgentamicin 08 strains (57) were resistant to neomycinerythromycin and chloramphenicol 07 strains (50) wereresistant to sulfamethoprim 05 strains (36) were resistantto linezolid 03 strains (21) were resistant to rifampicin 02strains (14) were resistant to ciprofloxacin and cefepimeand 01 strain (7) was resistant to clindamycin (Table 3)

The multiresistance of CNS strains demonstrated hereinis consistent with previous studies on coagulase-negativeand coagulase-positive staphylococci that have found severalresistant and multiresistant Staphylococcus aureus strains inraw milk meat and fermented meat products [43]

Surprisingly the resistance to chloramphenicol is verysimilar to that estimated for MRCNS strains isolated fromhuman clinical samples Indeed there is no direct correla-tion between the researched features and the origin of thestaphylococci strains since different virulence factors arewidespread such as antibiotic resistance [44] reinforcing thefact that the ability of foodmatrices strains to produce SE andSEl and their multiresistance character must be consideredwhen evaluating the safety hazards of food poisoning

S epidermidis KF6005891 and S hominis JX5199881showed the highestMAR indexes (093 and 080 resp)Thesestrains found in artisanal salami are homologous to strainsisolated from human skin microbiota and should be carefullyconsidered among the potential pathogenic staphylococcifound in food from animal origins which can be caused bycontamination by poor hygienic conditions during salamimanufacturing

Depending on the conditions some species of coagulase-negative staphylococci can present health risks since theyhave shown resistance to antibiotics of therapeutic impor-tance such as beta lactams [15] However multiresistantstrains like S carnosusKJ18620021 and S xylosusCP0072081and CP0087241 were intentionally introduced in the foodmatrix since they are part of the culture starter used inItalian-salami manufacturing As previously discussed foodcan be a reservoir of multiresistant microorganisms that canspread by the consumption of an apparently safe food Dueto the intensive and indiscriminate use of antibiotics forhuman and veterinarian therapeutic purposes multiresistantstaphylococci strains are being selected and reproduced infood matrices [45]

The cefoxitin and oxacillin disk diffusion tests are rec-ommended for determining the resistance and susceptibilitybreakpoint for MRSA surveillance cultures [46] In thisstudy there was a good correlation between the disc testzone diameters for oxacillin and cefoxitin (Table 3) TheMICs for ampicillin andmethicillinoxacillincefoxitin for allsusceptible CNS strainswere determined by the diffusion disctest using 003 to 2mgmLminus1 of each antimicrobial agent

S saprophyticus HF9372521 showed an MIC of003mgmLminus1 formethicillin strain S carnosusKJ8620021 Sxylosus strains CP0087241 and CP0072081 S saprophyticusJX4901221 S hominis JX5199881 and S succinusKC3298241 were resistant to 006mgmLminus1 and two strainsS saprophyticus KJ6991511 and S saprophyticus KJ0046231showed an MIC of 05mgmLminus1 (Table 4) Two of themethicillin-resistant strains S saprophyticus KJ0046231 andS xylosus CP0072081 harbor no enterotoxin gene

Five strains S saprophyticus JX4901221 S carnosusKJ8620021 S xylosusCP0087241 S xylosusKF1980801 andS saprophyticus KJ0046231 showed an MIC for ampicillin

14 BioMed Research International

Table 3 Multiple resistance to antimicrobial as found in CNS strains from salami

Salami origin CNS strains Antimicrobial agent resistance Multiple antimicrobialresistance (MAR) indexlowast

Commercial Staphylococcus spp KF1354451 CIP CLO CPM GEN NEO OXA PENSXT TET and VAN 066

Commercial S carnosus KJ8620021 CIP GEN LZD NEO OXA SXT andTET 046

Commercial S saprophyticus AB6977171 CFO CLO ERI PEN OXA TET andVAN 046

Commercial S saprophyticus EU4309921 CFO CLO ERI GEN LZD NEO OXAPEN SXT TET and VAN 080

Commercial S saprophyticus JX4901221 CFO CLO ERI GEN NEO PEN OXASXT TET and VAN 066

Commercial S saprophyticus KJ0046231 CFO OXA and PEN 020

Commercial S sciuri JX9664361 CFO CLO GEN NEO OXA PEN andTET 046

Commercial S xylosus CP0072081 OXA LZD PEN and VAN 026

Commercial S xylosus CP0087241 CFO CLO ERI GEN NEO OXA PENTET and VAN 060

Artisanal S epidermidis KF6005891 CFO CLI CLO CPM ERI GEN LZDNEO OXA PEN RIF SXT and TET 093

Artisanal S hominis JX5199881 CIP CPM CPO ERI GEN LZD NEOOXA PEN RIF SXT and VAN 080

Artisanal S saprophyticusHF9372521 CFO OXA PEN and SXT 026Artisanal S saprophyticus KJ6991511 OXA PEN and VAN 020

Artisanal S succinus KC3298241 CFO CLO ERI GEN OXA PEN RIFSXT TET and VAN 066

Artisanal S xylosus KF1980801 CFO CLO ERI GEN NEO OXA PENTET and VAN 060

lowastTheMAR index of an isolate is defined as 119886119887 where 119886 represents the number of antimicrobials to which the isolate was resistant and 119887 represents the numberof antimicrobials to which the isolate was subjectedS aureus strains ATCC WB81 (sea) ATCC 13563 (sed) and ATCC 27664 (see) showing a MAR index of 05 and S aureus strains ATCC14458 (seb) andATCCWB72 (sec) and S xylosus ATCC 29971 showing a MAR index of 03 were used as reference strainsCPM cefepime CFO cefoxitin CLO chloramphenicol CIP ciprofloxacin CLI clindamycin ERI erythromycin GEN gentamycin NEO neomycin LZDlinezolid RIF rifampicin TET tetracycline OXA oxacillin PEN penicillin SXT sulfamethoprim and VAN vancomycin

Table 4 Minimal inhibitory concentration (MIC) of compounds used in antimicrobial therapy against staphylococci infections

Salami origin GenBank accession number and similarity () MIC mgmLminus1

Methicillin Ampicillin Vancomycin Linezolid

Commercial

S carnosus KJ8620021 006 003 mdash mdashS xylosus CP0087241 006 003 006 mdash

S saprophyticus JX4901221 006 003 05 mdashS saprophyticus AB6977171 mdash mdash 05 mdashS saprophyticus EU4309921 mdash mdash 025 0125

S succinus KC3298241 006 025 003 mdash

Artisanal

S hominis JX5199881 006 05 mdash mdashS saprophyticus HF9372521 003 mdash mdash mdash

S xylosus KF1980801 003 05 mdashS saprophyticus KJ6991511 05 025 003 mdashS saprophyticus KJ0046231 05 003 003 mdash

S xylosus CP0072081 006 mdash mdash 025Strains S epidermidis KF6005891 and Staphylococcus spp KF1354451 were not susceptible to the antimicrobial concentrations tested in the present study

BioMed Research International 15

of 003mgmLminus1 two strains S succinus KC3298241 and Ssaprophyticus KJ6991511 showed an MIC of 025mgmLminus1and S hominis JX5199881 showed an MIC of 05mgmLminus1(Table 4) S saprophyticus KJ0046231 also showed an MICof 003mgmLminus1 but does not harbor enterotoxin genes It isimportant to note that most of the CNS strains that showedresistance to ampicillin were also resistant to methicillin ashighlighted by the MIC tests

Five strains S saprophyticus HF9372521 S saprophyti-cus KJ6991511 S saprophyticus JX4901221 S saprophyticusKJ0046231 and S succinus KC3298241 showed an MICfor vancomycin of 003mgmLminus1 one strain S xylosusCP0087241 showed an MIC of 006mgmLminus1 one strain Ssaprophyticus EU4309921 showed an MIC of 025mgmLminus1and two strains S xylosus KF1980801 and S saprophyticusAB6977171 showed an MIC of 05mgmLminus1 (Table 4)

Additionally five CNS strains S xylosus CP0087241S saprophyticus JX 4901221 S succinus KC3298241 Ssaprophyticus KJ6991511 and S saprophyticus KJ0046231showed resistance tomethicillin as demonstrated by theMICdeterminations (Table 4)

Previous studies have demonstrated that linezolid isactive against Gram-positive bacteria including methicillin-resistant staphylococci [47] In the present study 05 strainsshowed linezolid resistance in the disc diffusion test but itwas impossible to establish MIC values for S epidermidisKF6005891 Staphylococcus hominis JX5199881 and Staphy-lococcus carnosus KJ8620021The remaining strains S sapro-phyticus EU4309921 and S xylosus CP0072081 presentedan MIC for linezolid of 0125mgmLminus1 and 025mgmLminus1respectively

The MIC of 003mgmLminus1 for penicillin is in accordancewith the previous values estimated for resistant S aureusstrains found in several food matrices such as meat dairyproducts and ready-to-eat food [48]

There is still a lack of information on the antimicro-bial resistance of staphylococci strains from food matricesalthough they are the most worrisome vehicles of dissemi-nation of antibiotic-resistant pathogens [49]

The high resistance found for salami staphylococci strainscan be ascribed to their inappropriate use as growth promot-ers of antimicrobial agents like oxacillin vancomycin chlo-ramphenicol neomycin and erythromycin which are com-monly used in veterinary medicine to treat infections [50]Antimicrobial therapy of infections staphylococci is basedon results of susceptibility tests in vitro [51] Methicillin-resistant coagulase-negative Staphylococcus spp found inready-to-eat products such as meats fish and dairy productsalso offer risks and although they are not classical foodpoisoning bacteria their presence in food offers significantrisks to public health due to the possible spread of antibioticresistance [52]

The standardization of salami quality should includediagnostic methods to screen and quantify the presence ofclassical and newly described enterotoxins directly in thefood matrices as a routine procedure to be conducted by themeat product industry in Brazil

The safety of salami consumption could also be enhancedby the inclusion of amicrobial barrier such as the inclusion ofprobiotic strains producing natural antibiotics or competitiveflora or even the addition of natural bioagents against spoilageor pathogenic microorganisms

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors acknowledge the financial support fromFundacao Carlos Chagas Filho de Amparo a Pesquisa doEstado do Rio de Janeiro (FAPERJ Rio de Janeiro Brazil)Conselho Nacional de Desenvolvimento Cientıfico eTecnologico (CNPq Brasılia Brazil) and Coordenacao deAperfeicoamento de Pessoal de Nıvel Superior (CAPESBrasılia Brazil)

References

[1] ICMSF International Comission on Microbiological Specifica-tions for FoodsMicroorganisms in FoodsTheir Significance andMethods of Enumerations University of Toronto Press TorontoCanada 3rd edition 1983

[2] J F Veras L S Carmo L C Tong et al ldquoA study of theenterotoxigenicity of coagulase-negative and coagulase-positivestaphylococcal isolates from food poisoning outbreaks inMinasGerais Brazilrdquo International Journal of Infectious Diseases vol12 no 4 pp 410ndash415 2008

[3] K Omoe D-L Hu H Takahashi-Omoe A Nakane and KShinagawa ldquoComprehensive analysis of classical and newlydescribed staphylococcal superantigenic toxin genes in Staphy-lococcus aureus isolatesrdquo FEMS Microbiology Letters vol 246no 2 pp 191ndash198 2005

[4] J Kadariya T C Smith and D Thapaliya ldquoStaphylococcusaureus and staphylococcal food-borne disease an ongoing chal-lenge in public healthrdquoBioMedResearch International vol 2014Article ID 827965 9 pages 2014

[5] KOmoeD-LHuH KOno et al ldquoEmetic potentials of newlyidentified staphylococcal enterotoxin-like toxinsrdquo Infection andImmunity vol 81 no 10 pp 3627ndash3631 2013

[6] J K McCormick J M Yarwood and P M SchlievertldquoToxic shock syndrome and bacterial superantigens an updaterdquoAnnual Review of Microbiology vol 55 pp 77ndash104 2001

[7] T Baba F Takeuchi M Kuroda et al ldquoGenome and virulencedeterminants of high virulence community-acquired MRSArdquoThe Lancet vol 359 no 9320 pp 1819ndash1827 2002

[8] Associacao Brasileira de Industria Produtora e Exportadorada Carne Suınas (ABIPECS) ldquoStatistics slaughter ranking2000ndash2014rdquo 2014 httpabpa-brcombrsetoressuinoculturapublicacoesrelatorios-anuais

[9] N Zdolec M Hadziosmanovic L Kozacinski et al ldquoMicrobialand physicochemical succession in fermented sausages pro-duced with bacteriocinogenic culture of Lactobacillus sakei andsemi-purified bacteriocin mesenterocin Yrdquo Meat Science vol80 no 2 pp 480ndash487 2008

16 BioMed Research International

[10] F Ravyts L D Vuyst and F Leroy ldquoBacterial diversity andfunctionalities in food fermentationsrdquo Engineering in Life Sci-ences vol 12 no 4 pp 356ndash367 2012

[11] TOhlsson andN BengtssonTheHurdle ConceptMinimal Pro-cessing Technologies in the Food IndustryWoodhead Publishing2002

[12] S Bernardi B B Golineli and C J Contreras-CastilloldquoRevisao aspectos da aplicacao de culturas starter na producaode embutidos carneos fermentadosrdquo Brazilian Journal of FoodTechnology vol 13 no 2 pp 133ndash140 2010

[13] F Irlinger ldquoSafety assessment of dairy microorganismscoagulase-negative staphylococcirdquo International Journal of FoodMicrobiology vol 126 no 3 pp 302ndash310 2008

[14] B Ghebremedhin F Layer W Konig and B Konig ldquoGeneticclassification and distinguishing of Staphylococcus species basedon different partial gap 16S rRNA hsp60 rpoB sodA and tufgene sequencesrdquo Journal of Clinical Microbiology vol 46 no 3pp 1019ndash1025 2008

[15] I Babic K Markov D Kovacevic et al ldquoIdentification andcharacterization of potential autochthonous starter culturesfrom aCroatian lsquobrandrsquo product lsquoslavonski kulenrsquordquoMeat Sciencevol 88 no 3 pp 517ndash524 2011

[16] MMehrotra GWang andWM Johnson ldquoMultiplex PCR forthe detection of genes for Staphylococcus aureus enterotoxinsexfoliative toxins toxic shock syndrome toxin and methicillinresistancerdquo Journal of Clinical Microbiology vol 38 no 3 pp1032ndash1041 2000

[17] WM Johnson SD Tyler E P Ewan F EAshtonDR Pollardand K R Rozee ldquoDetection of genes for enterotoxins exfolia-tive toxins and toxic shock syndrome toxin 1 in Staphylococcusaureus by the polymerase chain reactionrdquo Journal of ClinicalMicrobiology vol 29 no 3 pp 426ndash430 1991

[18] J Mclauchlin G L Narayanan VMithani andG OrsquoNeill ldquoThedetection of enterotoxins and toxic shock syndrome toxin genesin Staphylococcus aureus by polymerase chain reactionrdquo Journalof Food Protection vol 63 no 4 pp 479ndash488 2000

[19] J P Rosec and O Gigaud ldquoStaphylococcal enterotoxin genes ofclassical and new types detected by PCR in Francerdquo Interna-tional Journal of Food Microbiology vol 77 no 1-2 pp 61ndash702002

[20] K Omoe D-L Hu H Takahashi-Omoe A Nakane andK Shinagawa ldquoIdentification and characterization of a newstaphylococcal enterotoxin-related putative toxin encoded bytwo kinds of plasmidsrdquo Infection and Immunity vol 71 no 10pp 6088ndash6094 2003

[21] N Sergeev D Volokhov V Chizhikov and A Rasooly ldquoSimul-taneous analysis of multiple staphylococcal enterotoxin genesby an oligonucleotide microarray assayrdquo Journal of ClinicalMicrobiology vol 42 no 5 pp 2134ndash2143 2004

[22] S Holtfreter D Grumann M Schmudde et al ldquoClonal distri-bution of superantigen genes in clinical Staphylococcus aureusisolatesrdquo Journal of Clinical Microbiology vol 45 no 8 pp2669ndash2680 2007

[23] W J Mason J S Blevins K Beenken NWibowo N Ojha andM S Smeltzer ldquoMultiplex PCR protocol for the diagnosis ofstaphylococcal infectionrdquo Journal of Clinical Microbiology vol39 no 9 pp 3332ndash3338 2001

[24] E Rahimi and H G Safai ldquoDetection of classical enterotoxinsof Staphylococcus aureus strains isolated frombovine subclinicalmastitis in Isfahan Iranrdquo Veterinary Microbiology vol 141 no3-4 pp 393ndash394 2010

[25] M A Larkin G Blackshields N P Brown et al ldquoClustalW andClustal X version 20rdquo Bioinformatics vol 23 no 21 pp 2947ndash2948 2007

[26] P H A Sneath and R R Sokal Unweighted Pair Group Methodwith Arithmetic Mean Numerical Taxonomy W H FreemanSan Francisco Calif USA 1973

[27] NCCLS ldquoPerformance standards for antimicrobial susceptibil-ity testing 14th informational supplementrdquo Tech Rep M 100-S14 NCCLS 2004

[28] S Corbiere Morot-Bizot S Leroy and R Talon ldquoMonitoringof staphylococcal starters in two French processing plantsmanufacturing dry fermented sausagesrdquo Journal of AppliedMicrobiology vol 102 no 1 pp 238ndash244 2007

[29] R Talon S Leroy I Lebert et al ldquoSafety improvement andpreservation of typical sensory qualities of traditional dryfermented sausages using autochthonous starter culturesrdquo Inter-national Journal of Food Microbiology vol 126 no 1-2 pp 227ndash234 2008

[30] G Mauriello A Casaburi G Blaiotta and F Villani ldquoIsolationand technological properties of coagulase negative staphylo-cocci from fermented sausages of Southern ItalyrdquoMeat Sciencevol 67 no 1 pp 149ndash158 2004

[31] M Janssens N Myter L De Vuyst and F Leroy ldquoSpeciesdiversity and metabolic impact of the microbiota are low inspontaneously acidified Belgian sausages with an added starterculture of Staphylococcus carnosusrdquo Food Microbiology vol 29no 2 pp 167ndash177 2012

[32] G Blaiotta D Ercolini G Mauriello G Salzano and F VillanildquoRapid and reliable identification of Staphylococcus equorum bya species-specific PCR assay targeting the sodA generdquo Systematicand Applied Microbiology vol 27 no 6 pp 696ndash702 2004

[33] L Kozacinski E Drosinos F Caklovica L Cocolin J Gasparik-Reichardt and S Veskovic ldquoInvestigation of microbial associa-tion of traditionally fermented sausagesrdquo Food Technology andBiotechnology vol 46 no 1 pp 93ndash106 2008

[34] S Leroy P Giammarinaro J-P Chacornac I Lebert and RTalon ldquoBiodiversity of indigenous staphylococci of naturallyfermented dry sausages and manufacturing environments ofsmall-scale processing unitsrdquo Food Microbiology vol 27 no 2pp 294ndash301 2010

[35] E Coton M-H Desmonts S Leroy et al ldquoBiodiversity ofCoagulase-Negative Staphylococci in French cheeses dry fer-mented sausages processing environments and clinical sam-plesrdquo International Journal of Food Microbiology vol 137 no 2-3 pp 221ndash229 2010

[36] S Hosseinzadeh and H D Saei ldquoStaphylococcal species associ-ated with bovinemastitis in the NorthWest of Iran emerging ofcoagulase-negative staphylococcirdquo International Journal of Vet-erinary Science and Medicine vol 2 no 1 pp 27ndash34 2014

[37] A Piette and G Verschraegen ldquoRole of coagulase-negativestaphylococci in human diseaserdquo Veterinary Microbiology vol134 no 1-2 pp 45ndash54 2009

[38] T M Moura F S Campos P A DrsquoAzevedo et al ldquoPrevalenceof enterotoxin-encoding genes and antimicrobial resistancein coagulase-negative and coagulase-positive Staphylococcusisolates from black pudding in Southern Brazilrdquo Revista daSociedade Brasileira deMedicina Tropical vol 45 no 5 pp 579ndash585 2012

[39] H I Suzuki T Lefebure P P Bitar and M J StanhopeldquoComparative genomic analysis of the genus Staphylococcusincluding Staphylococcus aureus and its newly described sister

BioMed Research International 17

species Staphylococcus simiaerdquo BMC Genomics vol 13 article38 2012

[40] R P Lamers G Muthukrishnan T A Castoe S Tafur A MCole and C L Parkinson ldquoPhylogenetic relationships amongStaphylococcus species and refinement of cluster groups basedon multilocus datardquo BMC Evolutionary Biology vol 12 article171 2012

[41] I V Pinchuk E J Beswick and V E Reyes ldquoStaphylococcalenterotoxinsrdquo Toxins vol 2 no 8 pp 2177ndash2197 2010

[42] S R Monday and G A Bohach ldquoGenes encoding staphylo-coccal enterotoxins G and I are linked and separated by DNArelated to other staphylococcal enterotoxinsrdquo Journal of NaturalToxins vol 10 no 1 pp 1ndash8 2001

[43] D Y Thomas S Jarraud B Lemercier et al ldquoStaphylococcalenterotoxin-like toxins U2 and V two new staphylococcalsuperantigens arising from recombination within the entero-toxin gene clusterrdquo Infection and Immunity vol 74 pp 4724ndash4734 2006

[44] N Indrawattana O Sungkhachat N Sookrung et al ldquoStaphylo-coccus aureus clinical isolates antibiotic susceptibility molecu-lar characteristics and ability to form biofilmrdquo BioMed ResearchInternational vol 2013 Article ID 314654 11 pages 2013

[45] V Pereira C Lopes A Castro J Silva P Gibbs and P TeixeiraldquoCharacterization for enterotoxin production virulence factorsand antibiotic susceptibility of Staphylococcus aureus isolatesfrom various foods in Portugalrdquo Food Microbiology vol 26 no3 pp 278ndash282 2009

[46] I Anacarso C Condo C Sabia and et al ldquoAntimicrobialresistance and other related virulence factors in staphylococcusspp isolated from food environmental and humans in ItalyrdquoUniversal Journal Microbiology Research vol 1 no 1 pp 1ndash92013

[47] B Martın M Garriga M Hugas S Bover-Cid M T Veciana-Nogues and T Aymerich ldquoMolecular technological and safetycharacterization of Gram-positive catalase-positive cocci fromslightly fermented sausagesrdquo International Journal of FoodMicrobiology vol 107 no 2 pp 148ndash158 2006

[48] NM Broekema T Van Tam T AMonson S AMarshall andD M Warshauer ldquoComparison of cefoxitin and oxacillin diskdiffusionmethods for detection ofmecA-mediated resistance inStaphylococcus aureus in a large-scale studyrdquo Journal of ClinicalMicrobiology vol 47 no 1 pp 217ndash219 2009

[49] E Cercenado F Garcıa-Garrote and E Bouza ldquoIn vitro activityof linezolid against multiply resistant Gram-positive clinicalisolatesrdquo Journal of Antimicrobial Chemotherapy vol 47 no 1pp 77ndash81 2001

[50] A Aydin K Muratoglu M Sudagidan K Bostan B Okukluand SHarsa ldquoPrevalence and antibiotic resistance of foodborneStaphylococcus aureus isolates in Turkeyrdquo Foodborne Pathogensand Disease vol 8 no 1 pp 63ndash69 2011

[51] D Sergelidis A Abrahim T Papadopoulos et al ldquoIsolation ofmethicillin-resistant Staphylococcus spp from ready-to-eat fishproductsrdquo Letters in Applied Microbiology vol 59 no 5 pp500ndash506 2014

[52] I PhillipsM Casewell T Cox et al ldquoDoes the use of antibioticsin food animals pose a risk to human health A critical reviewof published datardquo Journal of Antimicrobial Chemotherapy vol53 no 1 pp 28ndash52 2004

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Page 2: Research Article Safety Evaluation of the Coagulase ...downloads.hindawi.com/journals/bmri/2015/483548.pdf · Research Article Safety Evaluation of the Coagulase-Negative Staphylococci

2 BioMed Research International

The toxic shock syndrome toxin-1 (TSST-1) is also amember of the SE-related toxin family and has the ability tostimulate large populations of T cells containing a particularV120573 element in their T-cell receptors (TCR) Like othersuperantigenic toxins it bypasses normal antigen presenta-tion by binding to class II major histocompatibility complexmolecules on antigen-presenting cells and to specific variableregions on the beta-chain of the T-cell antigen receptorThrough this interaction a massive proliferation of T cells atorders ofmagnitude above antigen-specific activation occursresulting in a massive cytokine release that is believed to beresponsible for the most severe features of TSST [6]

Enterotoxin (SE) genes are encoded in mobile geneticelements such as plasmids prophages and Staphylococcuspathogenic islands (SaPIs) [7]

Salami is a kind of dry sausage obtained by the microbialfermentation of raw pork meat using Staphylococcus startercultures as technological accessories to ferment the productand give it its organoleptic characteristics In Brazil Italiantype salami similar to salami produced in Southern Europeis avidly consumed with a production trade of 13093 tonsbetween 2000 and 2014 [8]

S xylosus S equorum and S carnosus are part of thestarter culture microbiota that participate in the reactionsrequired for creating the flavor and aroma during the matur-ing period of fermented meat production [9] In additionother species such as S epidermidis S pasteuri S sciuri andS succinus may also occasionally be present in meaningfulamounts [10]

However even the combination of physical and chemicalbarriers cannot always guarantee the stability and microbialsafety of starter cultures Contamination of salami fermen-tation starter culture microbiota by pathogenic coagulase-negative staphylococci (CNS) strains is perhaps the mostharmful factor in the production of cured meat productssince these pathogens are able to produce heat-stable entero-toxins with superantigenic activities in food matrices [11 12]

Staphylococci species are not usually identified at thespecies level by routine laboratory testing and commercialkits since phenotypic discrimination cannot reliably identifythese species due to the variable expression of some phe-notypic traits [13] For this purpose molecular techniquesincluding nucleotide sequencing within the 16S rDNA hsp60tuf sodA and rpoB genes have been successfully used toidentify Staphylococcus species [14]

Depending on the conditions some species of coagulase-negative staphylococci can present health risks since theyhave shown resistance to several antibiotics of therapeuticimportance such as 120573-lactams [15]

The aim of the present study was to identify the membersof the CNS microbiota from salami We sampled the salamimarketed in Brazil comparing the CNS microbiota in salamiproduced by industrial companies and in artisanal salamimanufactured by small producers The CNS strains wereidentified by sequencing of a 16S rDNA region and thephylogenic relationships between the observed species wereestablished The presence of multiple genes encoding theclassical and newly described sesel and tstH1 toxins in theCNS genomes was investigated The risk of food poisoning

was assessed by evaluating the ability of the CNS strains intranscribing and expressing the classical and newly describedenterotoxins in vitro by using real time RT-PCR and enzyme-linked immunosorbent assay (ELISA) The resistance ofthe isolated strains to antimicrobial agents of therapeuticimportance in staphylococci infections was also evaluated

2 Materials and Methods

21 Isolation of Bacterial Strains Six samples of distinctbrands of salami 03 from themeat industry and 03 from smallartisanal producers were collected in the municipality of Riode Janeiro Brazil Twenty-five grams of salami was added to225mL of 01 peptone water The suspensions were trans-ferred to homogenizer bags (Interscience SaintNom France)and coupled to a Stomacher 400 circulator (Seward Wor-thing West Sussex UK) at 260 rpm for 1min The suspen-sions were serial-diluted from 10minus6 to 100 and 100 120583L of eachdilution was transferred onto 20120583L of Baird-Parker agar con-taining egg yolk tellurite emulsion (BPAthorn RPF bioMerieuxFrance) Eighty presumptive coagulase-negative staphylo-cocci colonies were tested by Gram-staining catalase coagu-lase and thermostable DNAse activities according to BergeyrsquosManual of Systematic Bacteriology Sixty presumptive CNSstrains were stored at minus80∘C in tryptone soy agar (TSAFranklin Lakes New Jersey USA) plus 45 vv glycerol

22 DNA Preparation The strains were cultured aerobicallyovernight in 10mL Brain Heart Infusion broth (BD BBL LePont de Claix France) at 37∘C for 24 h The suggestive CNScolonies were adjusted to 106UFCmL in a spectrophotome-ter and harvested by centrifugation at 5700times g for 1minThecell pellet was used for DNA extraction using the DNeasyblood and tissue kit (Qiagen Dusseldorf Germany) fol-lowing the manufacturerrsquos instructions Genomic DNA wasquantified using the Qubit fluorometer (Invitrogen GrandIsland New York USA) and Qubit assay kits

23 PCR Tests

231 Primer Sequences and Target Genes Primer sets flank-ing the sea seb sec sed see seg seh sei selk selm seln seloselq selr selu and tstH1 sequences are listed in Table 1

232 Uniplex- Duplex- and Multiplex-PCR Tests Uniplex-PCR tests targeting the tstH1 sequence and duplex-PCRtargeting the seaseb and secsed sequences were performedPCR mixtures contained 25120583L of 20mM MgCl

2 10x PCR

buffer (Invitrogen Grand Island New York USA) 100mMdNTPmix (FermentasThermoScientific Vilnius Lithuania)02mM of each primer (Table 1) 05U Taq DNA polymerase(Invitrogen Grand Island New York USA) and 100 ngof DNA templates Uniplex- and duplex-PCR assays wereperformed under the following conditions 94∘C for 5minfollowed by 35 cycles of 94∘C for 2min 53∘C for 2min and72∘C for 1min for extension ending with a final extensionat 72∘C after 7min [16] with modifications in the annealingtemperature using a thermal cycler (MyCycler Bio-Rad

BioMed Research International 3

Table 1 Primer set for V3 16S rDNA sequencing and PCRreal time RT-PCR tests targeting the classical and newly described staphylococcalenterotoxin genes

Primers set and sequences (51015840-31015840) Gene Amplicon (bp) ReferencesSEAf TTGGAAACGGTTAAAACGAA sea 120 [17]SEA

rGAACCTTCCCATCAAAAACA

SEBf TCGCATCAAACTGACAAACG seb 478 [17]SEBr GCAGGTACTCTATAAGTGCCSECf GACATAAAAGCTAGGAATTT sec 257 [17]SECr AAATCGGATTAACATTATCCSEDf CTAGTTTGGTAATATCTCCT sed 317 [17]SEDr TAATGCTATATCTTATAGGGSEEf TAGATAAAGTTAAAACAAGC see 170 [17]SEEr TAACTTACCGTGGACCCTTCSEGf TGCTATCGACACACTACAACC seg 704 [18]SEGr CCAGATTCAAATGCAGAACCSEHf CGAAAGCAGAAGATTTACACG seh 495 [18]SEHr GACCTTTACTTATTTCGCTGTCSEIf GACAACAAAACTGTCGAAACTG sei 630 [18]SEIr CCATATTCTTTGCCTTTACCAGSElJf CAGCGATAGCAAAAATGAAACA selj 426 [19]SElJr TCTAGCGGAACAACAGTTCTGASElMf CCAATTGAAGACCACCAAAG selm 517 [20]SElMr CTTGTCCTGTTCCAGTATCASElNf ATTGTTCTACATAGCTGCAA seln 682 [20]SElNr TTGAAAAAACTCTGCTCCCASElOf AGTCAAGTGTAGACCCTATT selo 534 [20]SElOr TATGCTCCGAATGAGAATGASElKf ATGAATCTTATGATTTAATTTCAGAATCAA selk 545 [21]SElKr ATTTATATCGTTTCTTTATAAGAAATATCGSElQf GGAAAATACACTTTATATTCACAGTTTCA selq 539 [21]SElQr ATTTATTCAGTTTTCTCATATGAAATCTCSElRf AATGGCTCTAAAATTGATGG selr 363 [22]SElRr TCTTGTACCGTAACCGTTTTSElUf AATGGCTCTAAAATTGATGG selu 215 [22]SElUr ATTTGATTTCCATCATGCTCTSST-1f ATGGCAGCATCAGCTTGATA tstH1 350 [17]TSST-1r TTTCCAATAACCACCCGTTT16S rDNAf ATA AGA CTG GGA TAA CTT CGG G 16SrDNA 500 [23]16S rDNAr CTT TGA GTT TCA ACC TTG CGG TCGf forward r reverse

Hercules CAUSA)The amplified fragments were visualizedon 10 agarose gels (Sigma) stained with GelRed (dilution1 1000) (BioAmerica Tel Aviv Israel) and documented on atransilluminator (MiniLumi Imaging Bio-Systems BioAmer-ica Tel Aviv Israel)

233 Multiplex-PCR Tests Multiplex-PCR assays were per-formed by the simultaneous amplification of the see seg sehsei selj selm seln selo selk selq selr and selu sequences usingthe primer sets listed in Table 1 Each reaction contained50120583L of a mix containing 05U Taq DNA polymerase 10xPCR buffer 100mMdNTP 02120583Mof each primer and 100 ng

of DNA template DNA amplification of see seg seh andsei was carried out as follows 95∘C for 5min 35 cycles of95∘C for 30 s 53∘C for 90s and 72∘C for 90 s and a finalextension at 72∘C for 10min The DNA amplifications of theselj selm seln and selo group and the selk selq selr andselu group were carried out in the same conditions [3] PCRproducts were visualized by electrophoresis on 12 agarosegels (Uniscience do Brasil Sao Paulo Brazil) in 1x TAE (Tris-boric acid-EDTA) buffer stained by 05 120583gmLminus1 of GelRed(BioAmerica Tel Aviv Israel) and documented on a transil-luminator (MiniLumi Imaging Bio-Systems BioAmerica TelAviv Israel)

4 BioMed Research International

DNA templates from the following reference strains wereused S aureusATCC 29231 (sea) S aureusATCC 14458 (sebtstH selk selq selr and selu) S aureus ATCC 19095 (sec segseh and sei) S aureus ATCC 13563 (sed) S aureus ATCC27664 (see) and S aureus ATCC 27154 (selj selm seln andselo) and S xylosus ATCC 29971

24 Enterotoxin ExpressionAssays Theobserved strainswerecultured aerobically overnight in 10mL Brain Heart InfusionBroth (BD BBL Le Pont de Claix France) at 37∘C for 72 hBacteria supernatants were collected by centrifugation at4000timesg for 10min and used for the detection of sea seb secsed and see by an ELISA assay using a commercial detectionkit (RIDASCREEN SET A B C D E Art number R4101 R-Biopharm AG Germany) The assay was performed accord-ing to the manufacturerrsquos recommendation and as describedelsewhere [16]Themean lower limit of detection of the assaywas 025 ngmLminus1 The threshold is defined as the average ODof two negative controls plus 015 a constant established bythe kit Samples containing SEs showed absorbance valuesequal to or greater than the threshold value All experimentswere performed in duplicate

25 Real Time RT-PCR Assays Total RNA was extracted byusing the QIAGEN RiboPure Bacteria kit (Life TechnologiesCarlsbad California USA) following the manufacturerrsquosinstructions and quantified using the Qubit fluorometer(Invitrogen Grand Island New York USA) and Qubit assaykits The cDNA synthesis was performed by using the HighCapacity cDNA Reverse Transcription Kit (Applied Biosys-tems California USA) and the ABI PRISM 7500 Fast RT-PCR system (Applied Biosystems California USA) Sampleswere plated in triplicate in 96-well plates as follows 12 120583Lof the SYBR Green PCR Master Mix 1 120583L of primer mix(sea seb sec sed and see) and 45 120583L of the cDNA ultrapurewater in each well Amplification was performed under thefollowing conditions 95∘C for 15min 40 cycles at 95∘Cfor 15 s 54∘C for 30s and 72∘C for 30 s The dissociationcurve was performed at 95∘C for 15 sec 54∘C for 30 sec and95∘C for 15 sec CT means the standard deviations and thecDNA semiquantification were calculated using the Graph-Pad Prism 5 software package Calibration curves based onfive points were constructed in triplicate corresponding toserial dilutions (1 1 10 1 100 1 1000 and 1 10000) from100 ng of a DNA template stock solution

26 16S rDNA Sequencing Amplification of the V5 regionof 16S rDNA fragment was performed using 50 ng of DNAtemplates from the 65 strains found in the salami samplesPCR was performed under the following conditions 95∘Cfor 10min followed by 30 cycles at 95∘C for 30 s 60∘Cfor 30 s and 72∘C for 45 s and a final extension at 72∘Cfor 10min PCR products were purified using the PCRDNA Purification Kit (Applied Biosystems California USA)and sequenced using 20 ng purified DNA and 13 120583L ofprimer sets in a final volume of 20120583L After amplifica-tion products were purified according to the protocol ofthe BigDye Terminator Purification X Kit (Applied Biosys-tems California USA) and sequenced on a 3130 sequencer

GeneticAnalyzer (Applied Biosystems California USA)Thesequences were compared to the 16S rDNA gene sequencesof Staphylococcus species available at the GenBank database(httpwwwncbinlmnihgovGenbankindexhtml) Multi-ple sequence alignments were performed using ClustalW (Kyoto University Bioinformatics Center httpwwwgenomejptoolsclustalw)

27 Sequencing of Enterotoxin PCR Products PCR productswere purified using the PCR DNA Purification Kit (AppliedBiosystems California USA) and sequenced using 10 ngof purified DNA and 32 pmoles of each primer set in afinal volume of 20 120583L After amplification in the sameconditions as the PCR step (Section 233) products werepurified according to the BigDye Terminator PurificationX Kit protocol (Applied Biosystems California USA)and sequenced on a 3130 sequencer Genetic Analyzer(Applied Biosystems California USA) The sequences werecompared to Staphylococcus aureus and Staphylococcuspasteuri gene sequences available at the GenBank database(httpwwwncbinlmnihgovGenbankindexhtml) Multi-ple sequence alignments were performed using ClustalW (Kyoto University Bioinformatics Center httpwwwgenomejptoolsclustalw)

28 Phylogenetic Analyses Phylogenetic relationshipsbetween the CNS strains were performed by sequencealignments using the Clustal X 20 software package [24]The phylogenetic trees were constructed using the softwareMega 60 and UPGMAmethods [25]

29 Antibiotic Susceptibility Tests An inoculumof each strainequivalent to a 05 McFarland scale was swabbed onto aMueller-Hinton agar plate (BD BBL Franklin Lakes NewJersey USA) and the antibiotic disc was then placed on theplate followed by overnight incubation at 37∘CThe inhibitionzone was interpreted according to the Clinical LaboratoryStandard (CLSI) Guidelines formerly known as the NationalCommittee for Clinical Laboratory Standards The testedantibioticswere penicillinG (10U) oxacillin (1120583g) neomycin(30 120583g) sulfamethoprim (5 120583g) clindamycin (2120583g) gen-tamicin (10 120583g) cefoxitin (30 120583g) rifampicin (5 120583g) ery-thromycin (15 120583g) tetracycline (30 120583g) vancomycin (30120583g)ciprofloxacin (5120583g) sulfazothrim (23120583g) cefepime (30 120583g)linezolid (30 120583g) and chloramphenicol (30 120583g)

210 Minimal Inhibitory Concentration (MIC) Determina-tions The MICs of vancomycin linezolid methicillin andampicillin were determined by the macrodilution brothmethod based on CLSI recommendations using in-house-prepared panels [26] Antibiotic concentrations of 003 0060125 025 05 10 and 20mgmLminus1 were tested One mLof broth was transferred to the tubes and 100 120583L of thebacteria suspension was adjusted to 106 CFUmL in saline085 according to a 05 McFarland scale and transferredto tubes containing 1mL of each antimicrobial Strains weregrown in Mueller-Hinton broth (BD BBL Franklin LakesNew Jersey USA) and the MIC was estimated as the lowest

BioMed Research International 5

antibiotic concentration that inhibits visible growth after 24 h[26]

3 Results and Discussion

31 Isolation and Identification of Coagulase-Negative Strainsfrom Salami Sixty-five presumable coagulase-negativestaphylococci microorganisms from salami were isolated bycolony morphology coagulase slide test subsequent tubetest and biochemical tests The sequencing of the V5 regionof the 16S rDNA fragment of the strains was discriminativeenough to differentiate the Staphylococcus isolated fromsalami at the subspecies level with the exception of 1 strainidentified up to the Staphylococcus spp genus

Nineteen distinct strains were identified as CNS in salamiwith 08 of 19 (42) identified as S saprophyticus thepredominant species followed by 05 strains of S xylosus(26) 02 strains of S carnosus (11) and 01 strain of eachof the following species S succinus S sciuri S epidermidisand S hominis (5 each) (Table 2)

The 08 S saprophyticus strains were identified asKJ6991511 AB6977171 JX4901221 KJ0046231 andHQ6995101 EU4309921 HF9372521 and KJ9496061 withthe latter two and S sciuri JX9664361 being homologous(96ndash98) to strains from the environment Five S xylosusstrains CP0072081 KF1980801 CP0087241 AM8827001and KC4565901 and a single S succinus strain KC3298241were identified The last three S xylosus strains and theS succinus strain are homologous (97-98) to strainsfound in fermented meat or meat starter cultures Sxylosus CP0072081 showed homology (98) to potentialopportunistic pathogenic strains from mammal species

The S carnosus strains identified were KJ8620021 andNR1164341 and the latter as well as the single S hominisJX5199881 (96-97) identified were homologous (97) tospecies from human microbiota

S carnosus and S xylosus are commonly used as com-mercial starter cultures for sausage manufacturing [26] butS succinus has also been observed in dry fermented sausagesand its use as a starter culture has been already proposed [27]

The diversity of CNS microbiota found in the samplesanalyzed in the present study could be related to the originof the salami The microbiota from the artisanal salamishowed greater biodiversity when compared to the commer-cial salami In commercial salami S saprophyticus was thepredominant species but S xylosus and S carnosus alsoobserved in commercial salami are commonly isolated fromstarter cultures [26] The predominance of S saprophyticusfollowed by S xylosus has also been reported in salamifrom South Italy [28] similar to Belgian sausages where Ssaprophyticus was the most frequently detected species [29]

S carnosus was not observed in artisanal salami but Ssuccinus S epidermidis and S hominis were detected

The biodiversity of the CNS staphylococci species foundin microbiota depends on the kind of meat-fermented prod-uct but CNS strains such as S saprophyticus S auricularisS xylosus S capitis S hominis S carnosus S haemolyticusS warneri S equorum S cohnii S capitis and S intermediushave been described inNapoli-type salami Sremska sausages

dry sausages raw meat and naturally fermented meat [30ndash33]

During the last decades S epidermidis and S sapro-phyticus have been described as emerging pathogens [34]S saprophyticus is considered a frequent contaminant offermented sausages and raw meats and has been isolatedfrom rectal swabs of cattle carcasses and pigs In humans themain reservoir of S saprophyticus is the gastrointestinal tract[35] S saprophyticus and S epidermidis can be opportunisticpathogens isolated from the human urinary tract and thepresence of these species in food should be taken into accountconcerning possible contamination of the starter inoculumandor improvements in the salami manufacturing process[13]

Other CNS species observed in the present study aremainly associated with ordinary food contaminants withS epidermidis and S hominis being the dominant speciesin human skin and occasionally isolated from the skin ofdomestic animals [36]

32 Phylogenetic Relationships of the CNS Identified in SalamiCNS strains can be grouped into four species groupssaprophyticus simulans epidermidis and haemolyticus Fre-quently the saprophyticus species group includes S xylosusand S saprophyticus while the simulans species group iscomprised of S carnosus and S piscifermentans the epi-dermidis species group is composed of S epidermidis Scapitis S caprae and S saccharolyticus and the haemolyticusspecies group encompasses S haemolyticus S hominis andS devriesei [37 38] In the present study the main clustergrouped several S saprophyticus strains namely KJ6991511AB6975171 KJ9496061 JX4901221 and KJ0046231 four Sxylosus strains CP0087241 CP0072081 AM8827001 andKC4565901 and the S succinusKC3298241 strain (Figure 1)These strains are homologous to those from fermented meatmicrobiota

The subclusters of those clusters showed mismatches ofspecies belonging to the four species groups The first sub-cluster grouped S epidermidis HF0882111 S saprophyticusHQ6995101 and S carnosusNR1164341 and the second sub-cluster grouped S hominis JX5199881 and the S saprophyticusEU4309921 all of them originally isolated from animalsand human beings The third subcluster includes speciespreviously isolated from the marine environment such as SxylosusKF1980801 while S carnosusKJ8620021 is utilized asa probiotic organism in foodsThe fourth cluster grouped theS sciuri JX9664361 and S saprophyticus HF9372521 strainswhich are homologous to species found in soil

The CNS strains clustered into groups near the bottomof the phylogenetic tree are mostly strains found in artisanalsalami whereas the species at the top of the tree are mainlyCNS strains found in commercial salami (Figure 1)

The close similarities between the S saprophyticusAB6975171 and KJ9496061 strains the S succinusKC3298241 S xylosus AM8827001 KC4565901 andCP0087241 strains the S saprophyticus KJ0046231 andKJ6991511 strains and S xylosus CP0072081 are supportedby a bootstrap value of 100 The interspecies similaritieswere over 90 which demonstrates close phylogenies

6 BioMed Research International

Table2Genotypicandph

enotypiccharacteriz

ationof

CNSstrainsfrom

salami

Strainsidentificatio

nandcharacteriz

ation

Salamiorig

inStaphylococcus

species

Genotypic

Phenotypic

GenBa

nkaccessionnu

mbera

ndsim

ilarity(

)Presence

ofenterotoxingenes

mRN

Adetection

Enterotoxinprod

uctio

n(ngm

Lminus1)

Com

mercial

Staphylococcus

sppKF

1354451(96)

seaseb

mdashmdash

Scarnosus

KJ8620021(96)

seasebsedsehseiand

selj

mdash12plusmn01

Scarnosus

NR1164341(98)

sebsecsedandsee

see

03

Ssaprophyticus

AB6

977171

(98)

sehseiandselm

seiselm

03

Ssaprophyticus

EU4309921(99)

seasebselm

seln

selo

and

tstH1

sebselm

and

selo

13plusmn01

Ssaprophyticus

HQ6995101(97)

mdashmdash

mdashSsaprophyticus

JX4901221(99)

seaseh

seh

05plusmn01

Ssaprophyticus

KJ00

46231(96)

sec

mdash04

Ssaprophyticus

KJ9496061(98)

seasebseca

ndseh

seasebandseh

mdashSsciuriJX9664361(98)

sedsei

sei

10plusmn01

SxylosusA

M8827001(97)

mdashmdash

mdashSxylosusCP

0072081(99)

mdashmdash

mdashSxylosusC

P0087241(98)

seatstH1

sea

14SxylosusK

C4565901(98)

mdashmdash

mdash

Artisa

nal

SepidermidisKF

6005891(97)

secsedseesegsehandsei

seesei

07plusmn01

ShominisJX5199881(97)

seasebsecsedseljselm

seln

andselo

selnselo

09plusmn01

Ssaprophyticus

HF9

372521(97)

seb

seb

05

Ssaprophyticus

subspbovisK

J6991511

(98)

secselm

seln

andtstH1

seln

09plusmn01

Ssuccinus

KC3298241(99)

seasebsecseljandseln

sebsec

13SxylosusK

F1980801(97)

secselm

andtstH1

mdash05plusmn01

Thep

resenceo

fenterotoxin

genesseasebsecsedseesegsehsei

seljselkselm

seln

selo

selq

selrselu

and

tstH1w

asteste

dby

PCRusingthes

pecific

seto

fprim

ers

sea-seee

nterotoxin

prod

uctio

nwas

evaluatedby

immun

e-sorbentassays(EL

ISA)u

singthed

etectio

nkitR

IDASC

REEN

SETAB

CD

EV

aluesa

redisplay

edas

them

eansplusmnSD

ofassays

perfo

rmed

indu

plicate

mRN

Atranscrip

tsfora

llenterotoxingenesw

eree

valuated

byrealtim

eRT-PC

Rtests

CT

values

ared

isplayedas

meansplusmnSD

ofRT

-PCR

tests

perfo

rmed

indu

plicateseamdash

SxylosusC

P00872413

0plusmn04sebmdash

SxylosusC

P008724133plusmn20Ssaprophyticu

sJX4901221302plusmn04Ssaprophyticus

subspbovisK

J6991511

32plusmn01SxylosusK

F19808013

6plusmn10

and

ShominisJX519988134plusmn10secmdashSsaprophyticus

subspbovisK

J6991511

31plusmn11seemdashSsaprophyticus

subspbovisK

J6991511306plusmn05

Ssuccinus

KC3298241285plusmn01andSsaprophyticus

HF9

3725213

2plusmn04sehmdash

SxylosusC

P0087241 31plusmn10Ssaprophyticu

sJX9

6643613

32plusmn06seimdash

Ssaprophyticus

AB6

977171

30plusmn10

Ssaprophyticu

sJX9664361306plusmn10

seln

mdashShominisJX5199881303plusmn10

Ssaprophyticu

sEU430992131plusmn03andselomdashShominisJX519988132plusmn10

BioMed Research International 7

O S succinus KC3298241Δ S xylosus AM8827001

O S saprophyticus KJ6991511Δ S saprophyticus AB6977171

Δ S xylosus CP0087241Δ S xylosus KC4565901Δ S saprophyticus KJ9496061Δ S saprophyticus JX4901221Δ S saprophyticus KJ0046231

Δ S xylosus CP0072081Δ S carnosus NR1164341Δ S saprophyticus HQ6995101

O S epidermidis KF0882111O S hominis JX5199881Δ S saprophyticus EU4309921

O S xylosus KF1980801Δ S carnosus KJ8620021Δ S sciuri JX9664361O S saprophyticus HF9372521

04 03 02 01 00

Figure 1 Phylogenetic tree generated from the multiple alignments of the 16S rDNA sequences of CNS strains found in salami usingthe ClustalX 20 software The phylogenetic tree was constructed by using the Mega 60 software and the unweighted pair group method(UPGMA) Bootstrap values ranged from 00 to 04 Strains found in commercial (Δ) or artisanal salami (O)

between these CNS strains Suzuki et al 2012 demonstrateda bootstrap value higher than 90 for interspecies similaritiesbetween S saprophyticus S epidermidis S hominis and Scarnosus

33 Genotypic and Phenotypic Characterization of CNSStrains Twelve distinct combinations of staphylococcalenterotoxins geneswere found in the 15CNS strains compris-ing SEs AndashE GndashJ and also the enterotoxin-like toxins (SElL)KndashR and U (Table 2)

Fifteen strains (79) carried at least one gene encodingenterotoxins in their genomes (Figure 2) The seb and secgenes were the most predominant harbored by 57 ofthe strains followed by sea carried by 50 whereas thesedsehselm genes showed intermediate incidence harboredby 33 of the strains while seiseln and tsH1 were found in27 of strains Finally see selj and selo showed low incidence(13) and seg was carried by only 1 of strains (Table 2)

The relationships between superantigenic toxin geno-types and toxin gene-encoding mobile genetic elementsin CNS strains were evaluated Distinct combinations ofSaPI and plasmids or plasmids and genes of egc operonenterotoxins were found in the CNS strains obtained fromsalami

Four strains presented only the classical enterotoxingenes namely S saprophyticus KJ0046231 and HF9372521S xylosus CP0087241 and S carnosus NR1164341 Anotherstrain S saprophyticus AB6977171 presented only the newlydescribed enterotoxins seh sei and selm and finally 10 strainspresented a combination of classical and newly described

enterotoxin genes in their genomes with at least one of eachenterotoxin type (Table 2)

Previous studies have shown that sea is the most com-mon toxin associated with Staphylococcus food poisoningfollowed by sed and see with SEHand SEI being considered asplaying only a minor role [39] Among the 15 enterotoxigenicstrains found in salami 73 of them harbored at least the seagene or combinations of these 05 genes Strains S carnosusKJ8620021 found in commercial salami and S epidermidisKF6005891 found in artisanal salami carry 04 of these genesThe sea and seb enterotoxin genes are known to occupy thesame locus on the chromosome which may explain whythese enterotoxins are commonly found together in foodpoisoning outbreaks [36] The combination of sea and sebgenes was found in 05 strains S saprophyticus EU4309921 Scarnosus KJ8620021 S hominis JX5199881 S saprophyticusKJ9496061 and S succinus KC3298241 A single strain Scarnosus KJ8620021 showed the combination of sea and seigenes

Additionally some CNS are able to produce TSST-1 aloneor in combination with other enterotoxins Herein 04 of the19 strains (21) S saprophyticus EU4309921 S saprophyti-cus subsp bovis KJ6991511 and S xylosus CP0087241 andKF1980801 were shown to harbor the tstH1 combined withse andor sel enterotoxin genes (Table 2)

The staphylococci enterotoxins genes seg and sei [40] arepart of a chromosomal operon gene cluster (egc) comprisingfive genes designated as selo selm sei seln and seg Twoof the CNS strains determined in the present study Ssaprophyticus EU4309921 and S hominis JX5199881 were

8 BioMed Research International

sed

seb

sec

see

sea

M 2 3 4 51

500bp

(a)

sea

sebsebseb

M 1 2 3 4 5

(b)

sei

sehseg

1M

(c)

seh sehseh

M 1 2 3

(d)

1 2 4M 3 5

(e)

selm

seljseln

selo

21M

(f)

seln

selm

M 1 2

(g)

Figure 2 Uniplex duplex and multiplex PCR screening for the detection of enterotoxin genes in CNS strains from salami (a) Lane M100 bp DNA ladder plus (Fermentas Foster City CA USA) lane 1 S aureus ATCC 29231 harboring sea gene lane 2 S aureus NCTC 10654harboring seb gene lane 3 S aureus ATCC19095 harboring the sec gene lane 4 S aureus ATCC 13563 harboring the sed gene and lane 5 Saureus ATCC 27664 harboring the see gene (b) Lane M 100 bp DNA ladder plus lane 1 Staphylococcus spp lane 2 S carnosus NR116434lane 3 S carnosus KJ862002 lane 4 S carnosus KJ862002 and lane 5 S carnosus KJ8620021 (c) Lane M 100 bp DNA ladder plus lane1 S aureus ATCC 19095 harboring seg seh and sei genes (d) Lane M 100 bp DNA ladder plus lane 1 S saprophyticus AB6977171 lane 2S epidermidis KF 6005891 and lane 3 S sciuri JX9664361 (e) Lane M 100 bp DNA ladder plus lane 1 S xylosus KF1980801 lane 2 SsaprophyticusKJ6991511 (f) LaneM 100 bp DNA ladder plus lane 1 S aureusATCC 27154 harboring selj slem seln and selo genes (g) LaneM 100 bp DNA ladder plus lane 1 S xylosus KF1980801 and lane 2 S saprophyticus subsp bovis KJ6991511

BioMed Research International 9

shown to carry the selm seln and selo genes and the selmand seln sequences were detected in the genome of a singlestrain S saprophyticus subsp bovisKJ6991511 S epidermidisKF6005891 showed a combination of seg and sei genesand 03 strains harbored a single gene sei or selm or selnperhaps due to the high degree of genetic polymorphism inthe chromosomal assembly [41] Indrawattana et al 2013detected the seg-sei-selm-seln-selo of the highly prevalent egclocus was 261 in contrast to this study where 13 CNSpresents the combination of selm seln and selo genes [42]

The CNS strains clustered into groups near the bottom ofthe phylogenetic tree carried the classical enterotoxin geneswhile the species at the top of the tree showed high diversityamong the enterotoxin genes combining the classical and thenewly described genes in their genomes (Figure 1)

To assess the risk of staphylococcal food poisoning theability of the identified strains in harboring the se andsel genes and in expressing and producing enterotoxins invitro was evaluated The mRNA for each enterotoxin genewas evaluated by real time RT-PCR assays and enterotoxincontent was estimated by a sandwich enzyme immunoassayfor the combined detection of Staphylococcus enterotoxins(SET) A B C D and E

The fifteen enterotoxigenic CNS strains were able toexpress the classical enterotoxins SEA seb sec sed and seein concentrations ranging from 03 ngmLminus1 to 14 ngmLminus1as assessed by the in vitro assays (Table 2) The S sapro-phyticus AB6977171 KJ6991511 JX4901221 KJ0046231 andHF9372521 strains S xylosus KF1980801 and S carnosusNR1164341 produced low amount of enterotoxins lower than05 ngmLminus1 S epidermidisKF6005891 S saprophyticus bovisKJ6991511 and S hominis JX5199881 produced intermediateamount of enterotoxins ranging from 07 to 090 ngmLminus1while S sciuri JX9664361 S saprophyticus EU4309921 Scarnosus KJ8620021 S xylosus CP0087241 and S succi-nus KC3298241 produced enterotoxins in concentrationsge10 ngmLminus1

Although the sandwich enzyme immunoassay is consid-ered themost sensitivemethod to detect seandashsee enterotoxinsable to detect 0125 ngmLminus1 differences in the specificity andsensitivity of the assays for the detection of staphylococcalenterotoxins from foods are expected [43] A single strainStaphylococcus spp KF1354451 which harbors both the seaand seb genes was unable to produce seandashsee enterotoxins

The mRNA levels evaluated by the real time RT-PCRassays for enterotoxins were detected in 12 of 15 strains(86) (Table 2) Transcripts for the classical enterotoxinsgenes were detected when S saprophyticus HF9372521 Scarnosus NR1164341 S xylosus CP0087241 and S succinusKC3298241 were assayed Transcripts for newly describedenterotoxins were observed in S saprophyticus JX4901221and AB6977171 S sciuri JX9664361 S saprophyticus subspbovis KJ6991511 and S hominis JX5199881 Transcripts fromboth the classical and newly described enterotoxins weredetected in S saprophyticus EU4309921 and KJ9496061 andS epidermidis KF6005891

No mRNA transcripts were obtained for S sapro-phyticus KJ00462321 and S carnosus KJ8620021 although

enterotoxin production was detected by the enzyme-linkedimmunosorbent tests (Table 2)

The 04 strains that carry tstH1 in their genome Ssaprophyticus EU4309921 and KJ6991511 and S xylosusCP0087241 and KF980801 were not able to produce mRNAfor TSST-1 in the assay conditions

The production of classical enterotoxins in vitro(immunologic test) matched the results shown by real timeRT-PCR assays for the following strains S saprophyticusJX4901221 S sciuri JX9664361 S saprophyticusEU4309921S saprophyticus HF9372521 S saprophyticus subsp bovisKJ6991511 S hominis JX5199881 S epidermidis KF6005891and S succinus KC3298241

Ten strains namely S saprophyticusAB6977171 S sapro-phyticus EU4309921 S saprophyticus HF9372521 S sapro-phyticus KJ6991511 S saprophyticus KJ9496061 S succinusKC3298241 S hominis JX5199881 S xylosus KF1980801and S xylosus CP0087241 and S epidermidis KF6005891expressed mRNA for multiple se andor sel genes There isa differential transcription among these genes where themost frequent among the classical ones were seb and seeseatranscribed by 04 and 02 strains respectively and the mostfrequent among the newly described genes were sei andsehselnselo transcribed by 03 and 02 strains respectively

No mRNA for the sec gene was detected although SsaprophyticusAB6977171 was able to produce the enterotoxinin vitro as detected by the ELISA assays

34 Enterotoxin Gene Homologies between Salami CNSand CPS Strains The nucleotide sequencing of six entero-toxin genesmdashtwo of them sec and see encoding classi-cal enterotoxinsmdashand four of them encoding the newlydescribed genes seg seh selm and seln fromCNS strains wascompared to the enterotoxin genes from S aureus or S pas-teuri two coagulase-positive staphylococci strains (Figure 3)The homology between the CNS and CPS enterotoxin genesvaried from 65 to 98 The homology of sec seg sehselm and seln between CNS from salami and S aureus was98 60 98 65 and 70 respectively The homologybetween see from CNS found in salami and in S pasteuri was98

Although further studies should be performed it seemsthat the sequences encoding enterotoxins can be conservedamong coagulase-negative and coagulase-positive staphy-lococci as shown in the phylogenetic analysis of the 06enterotoxin genes (Figure 4)

35 Antimicrobial Multiresistance of CNS Strains (MRCNS)Another safety hazard associated with CNS strains besidesthe ability of producing enterotoxins in food matrices is theantimicrobial resistance to antimicrobial agents commonlyused to treat staphylococci infections The antimicrobialresistance carried by CNS strains from food matrices can bespread to the population by the consumption of an apparentlysafe food

Among the 19 CNS strains identified in salami 14 showedmultiresistance to antimicrobial agentsThree strains showedthe highest MAR indices 093 and 080 07 strains presented

10 BioMed Research International

lowast lowastlowastlowastlowastlowast lowast lowast lowastlowast lowast lowast lowastlowast lowastlowastlowastlowast lowastlowastlowast lowast

lowastlowast lowast lowast lowastlowastlowastlowast lowast lowast lowastlowast lowast

lowastlowastlowastlowast lowast lowast lowast

lowastlowast lowast lowast lowastlowast lowastlowast lowast lowastlowast lowastlowastlowastlowastlowast lowastlowast lowast lowastlowast lowastlowast

lowastlowast lowastlowastlowast lowast lowast lowast lowast lowast lowastlowastlowastlowastlowast

lowastlowast lowastlowast lowastlowast lowastlowast lowast lowast lowastlowast lowast lowast lowastlowastlowast lowast lowastlowast lowastlowastlowast

lowast lowast lowast lowast lowast lowast lowastlowast lowast lowastlowastlowast lowast lowastlowastlowast lowast lowast lowastlowastlowast lowast lowastlowastlowast lowast

lowast lowast lowast lowastlowastlowastlowast lowast lowastlowastlowast lowast lowast lowastlowastlowast lowast lowast lowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast lowast lowast lowast lowast lowastlowast lowast lowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

Enterotoxin C (sec) GATAAAAATGAATAAGAGTCGATTTATTTCATGCGTAATTTTGATATTCGCACTTATACT------------------------------------------------------------

AGTTCTTTTTACACCCAACGTATTAGCAGAGAGCCAACCAGACCCTACGCCAGATGAGTT------------------------------------------------------------

GCACAAAGCGAGTAAATTCACTGGTTTGATGGAAAATATGAAAGTTTTATATGATGATCA------------------------------------------------------------

TTATGTATCAGCAACTAAAGTTAAGTCTGTAGATAAATTTTTGGCACATGATTTAATTTA------------------------------------------------------------

TAACATTAGTGATAAAAAACTGAAAAATTATGACAAAGTGAAAACAGAGTTATTAAATGA------------------------------------------------------------

AGGTTTAGCAAAGAAGTACAAAGATGAAGTAGTTGATGTGTATGGATCAAATTACTATGT------------------------------------------------------------

AAACTGCTATTTTTCATCCAAAGATAATGTAGGTAAAGTTACAGGTGGCAAAACTTGTAT------------------------------------------------------------

GTATGGAGGAATAACAAAACATGAAGGAAACCACTTTGATAATGGGAACTTACAAAATGT------------------------------------------------------------

ACTTATAAGAGTTTATGAAAATAAAAGAAACACAATTTCTTTTGAAGTGCAAACTGATAA------------------------------------------------------------

GAAAAGTGTAACAGCTCAAGAACTAGACATAAAAGCTAGGAATTTTTTAATTAATAAAAA------------------------------------------------------------

AAATTTGTATGAGTTTAACAGTTCACCATATGAAACAGGATATATAAAATTTATTGAAAA----TTGTATGAGTTTAACAGTTCACCATATGAAACAGGATATATAAAATTTATTGAAAA

TAACGGCAATACTTTTTGGTATGATATGATGCCTGCACCAGGCGATAAGTTTGACCAATCTAACGGCAATACTTTTTGGTATGATATGATGCCTGCACCAGGCGATAAGTTTGACCAATC

TAAATATTTAATGATGTACAACGACAATAAAACGGTTGATTCTAAAAGTGTGAAGATAGATAAATATTTAATGATGTACAACGACAATAAAACAGTTGATTCTAAAAGTGTGAAGATAGA

AGTCCACCTTACAACAAAGAATGGATAATGTTAATCCGATTTTGATATAAAAAGTGAAAGAGTCCACCTTACAACAAAGAATGGATAATGTAATCCGATTATGGACAAGTGTGCTTTTTT

Enterotoxin C TATTAGATATATTTGAAAGGTAAGTACTTCGGTGCTTGCCTTTTTAGGATGCATATATATEnterotoxin C CNS

Enterotoxin CEnterotoxin C CNS

Enterotoxin CEnterotoxin C CNSEnterotoxin CEnterotoxin C CNSEnterotoxin CEnterotoxin C CNSEnterotoxin CEnterotoxin C CNSEnterotoxin CEnterotoxin C CNSEnterotoxin CEnterotoxin C CNSEnterotoxin CEnterotoxin C CNSEnterotoxin CEnterotoxin C CNS

Enterotoxin CEnterotoxin C CNSEnterotoxin CEnterotoxin C CNS

Enterotoxin CEnterotoxin C CNS

Enterotoxin CEnterotoxin C CNS

Enterotoxin CEnterotoxin C CNS

TTTGTGATCGTCGGCCA-----ACCGATACCGTCGTATTCTTGTACCGCGCAAGGCGAAGlowast lowast lowastlowastlowast lowast lowast lowast lowast lowastlowast lowast lowastlowastlowast lowast lowast lowast lowast

Enterotoxin E (see) Enterotoxin E TGCTTTAGGCGGGGTCTTACAGCATATTATCGTAGAAGATGCACAAGCTGCACGTAAAGCS pasteuri TGCTTTAGGCGCATCGTTACAACATGTCATCGTAGAAGATGAAAAAGATGGTCGTCAGGC

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast lowastlowastlowastlowast lowastlowastlowast lowast lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast lowast lowastlowastlowast lowastlowast lowastlowastlowast lowast lowastlowastlowast

Enterotoxin E ATTAGAATTCTTGAAATCAAAACA-AGCAGGACGTGCTACATTTCTACCATTAAATGTCAS pasteuri

Enterotoxin ES pasteuri

GATACAATT-TTTAAAACAACGTAGTTTAGGACGTGCCACATTCCTACCTTTAAATGTTA lowastlowast lowastlowastlowastlowast lowastlowast lowastlowastlowast lowastlowastlowast lowast lowastlowastlowastlowastlowastlowastlowastlowastlowast lowastlowastlowastlowastlowast lowastlowastlowastlowastlowast lowastlowastlowastlowastlowastlowastlowastlowast lowast

TCAAACCTAGAAATATTTCAACCAAGATATATlowast lowastlowastlowastlowast lowastlowastlowast lowastlowastlowastlowastlowast

Enterotoxin G (seg) Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

---------------------------attaagaaatactt--ccattttaatactcttaGGGGATATTTCCGGGAAACCGGGGCTAATACTGGATAACTTTTCTCTCCGAACGAAGAGA

gaatcaacaactttattatctccg--taaat-----atttaaaaacttatatggaacaaaGATTCAACGATGGTCTTGGTTTTCAATTAAGATGAACCCGCGGCGCTTAGCTGGTGGGAC

aggtactagtt-----------------------------------------cttttttaAGGTCACGGCTCACCCAAGCCCCCATGCGTATACGACCTGGAAGGGTGATCGGCCACATT

ggaaataagtcaaaccaaaaacttgtattgttcttttcagtaaat---------------GGGACTGAGACACAGCACAAACTCCTACGGGAGGCAGCAATAGGGAATCTTCCGCAATGG

------------------------------------------------------------CCGAAAGTCTGACGGAGCAACGCCGCGAGACTGAAGAATGTCTTCGGATCGTAAAACTCT

------------------------tttatatatccagattcaaa-----tgcagaaccatGTTGTTAGGGAAGAACAAGTATCGTTCGAATAGGGCGGTACCTTGACGGTACCTAACCAG

caaactcgtatagctttttttct---------ttagtgagccagtgtcttgctttg----AAAGCTTTGCTAACTACCTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCG

-taatctagttcctgaatagtaaccatattcttatttgtagttattgta--aatccaagtGAATTATTGGGCGTAAAGCGCGCGCATGCGGTCTTTTAAATCTGATGTCAAAGCCCACGG

gattgtct----------attgtcgattgttacctgtacagt------------aattaaGCTCAACCGTGGAGGGTCATTGGAAACTGTAAGACTTGGAGTGCAGAAGAGGATAGTGGA

Figure 3 Continued

BioMed Research International 11

lowast lowast lowast lowastlowast lowastlowast lowastlowastlowast lowast lowastlowast lowast lowast lowast lowast

lowast lowast lowastlowastlowast lowastlowast lowast lowastlowastlowast lowast lowastlowast lowastlowastlowast lowast lowastlowast lowastlowast lowast lowast lowastlowast lowast

lowastlowast lowast lowast lowastlowastlowastlowastlowast lowast lowast lowastlowast lowast lowast lowast lowast lowast lowastlowastlowast

lowastlowast lowast lowastlowast lowastlowastlowast lowastlowast lowastlowast lowast lowast lowast lowast lowastlowast lowastlowast lowast lowast lowast lowastlowast

lowastlowast lowastlowastlowastlowast

lowast lowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowast lowast lowastlowastlowastlowast lowast lowastlowast lowastlowast lowast lowastlowastlowastlowast lowast lowastlowast lowast lowast lowastlowastlowastlowastlowastlowastlowast

lowastlowast lowast lowast lowast lowast lowastlowastlowast lowastlowast lowastlowast lowast lowastlowastlowastlowastlowastlowastlowastlowast lowastlowastlowast

lowast lowast lowast lowast lowast lowast lowastlowast lowastlowast lowast lowast lowastlowastlowast lowast lowast lowastlowastlowast lowast lowast

lowastlowast lowastlowastlowastlowastlowastlowast lowastlowast lowastlowastlowast lowastlowast lowast lowastlowast lowastlowast lowastlowastlowast lowast lowast

lowastlowast lowast lowastlowastlowast lowast lowastlowastlowastlowastlowast lowast lowast lowast lowastlowastlowast lowast lowastlowast lowast

lowastlowast lowast lowast lowast lowast lowastlowast lowast lowastlowast lowast lowastlowast lowast lowastlowast lowastlowastlowastlowast lowast lowast lowastlowast

lowast lowastlowastlowast lowast lowastlowast lowast lowast lowastlowast lowast lowastlowast lowast lowastlowastlowastlowastlowastlowast lowast

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

tttatctctttcattttctgaactattaaa----tgtaagaccacc---atacatacaacATTCCACGTGTAACGGTGAAAATGAGTAGAGATGTGGAGGAACACCAGTGGAGAAACGCG

aacctccaaaattttggtttatatccggttc-----------agatttaggtattatacaACTCTCTGGTCTGTTACTGACGCTGAGGCGCGAAAGCGTGGGAGCAAACAGGATTATATA

tgtataaaaatatggaacgccaaaaatgtctacttttttatctttataattgttagctaaCCCCTGGTAGTCCAAGCCGTAAACGATGAGTGCTAGTGTTACAGGGT------------T

ttctgtattttctaattcagttttaacctcattataactcttatactcaattggaaaaatTCCGCCCTTTACTGCTGCAGCTAACG---CATTAG-GCACTCCGCCTGGGGAGTACAACT

taaatcatgagataaaaactgtctagaattaataactcctcttccttcaacaggtggagaGCAA------GGTTGAAACTCAAAGCA----TTGACCGGGGGCC---CGATAAGCTGAGG

cgtataaagattcattacat----tacccatagttcccttattgtttttataatcacttaTACATGGTGTTTAATTCCAAACCACGCCAAGAACCCTCACGAGGTCTTGACATCCTCTGG

ctttattt----------------------------------------------------ACCTCCCTAGAGAATAAGGGCTTCCCCGTCGGGGATGAAATGAACAGGTGGTGCATGGAT

-------agttcgtc---AGTCTTCAGCTCGTGCAT

Enterotoxin H (seh)

------------------------------------------------------------ATCTATGAATATAAATCTTTTAATATTGGTTTAATATAAATATTCCATTTCTAGAAACCT

------------------------------------------------------------ATATTTTATGTTTGTATAAATTCATAAATTGTTCGAAATTTATATATTAAAAGTTGTTTT

------------------------------------------------------------TGTTGTTTTATAATTAAGTTAATGAAATATATTGAGGAGTTTTAAAATGATTAATAAAAT

------------------------------------------------------------TAAAATATTATTTTCGTTTTTAGCATTATTACTTTCATTCACATCATATGCGAAAGCAGA

------------ -----------TTACGATTTAGCTTTAGCTAATGCATATGGTCAATAAGATTTACACGATAAAAGTGAGTTAACAGATTTAGCTTTAGCTAATGCATATGGTCAATA

TAATCACCCATTCATTAAAGAAAATATTAAGAGTGATGAAATAAGTGGAGAAAAAGATTTTAATCACCCATTCATTAAAGAAAATATTAAGAGTGATGAAATAAGTGGAGAAAAAGATTT

AATATTTAGAAATCAAGGTGATAGTGGCAATGATTTGAGAGTAAAGTTTGCAACTGCTGAAATATTTAGAAATCAAGGTGATAGTGGCAATGATTTGAGAGTAAAGTTTGCAACTGCTGA

TTTAGCTCAGAAGTTTAAAAATAAAAATGTAGATATATATGGGGCATCTTTTTATTATAATTTAGCTCAGAAGTTTAAAAATAAAAATGTAGATATATATGGGGCATCTTTTTATTATAA

GTGTGAAAAAATAAATGAAAATATTTCTGAATGTCTATATGGAGGTACAACACTAAATAAGTGTGAAAAAATAAGTGAAAATATTTCTGAATGTCTATATGGAGGTACAACACTAAATAG

TGAAAAATTGGCACAGGAAAGGGTGATTGGTGCTAATGTTTGGGTAGATGGTATTCAAAATGAAAAATTGGCACAGGAAAGGGTGATTGGTGCTAATGTTTGGGTAGATGGTATTCAAAA

AGAAACAGAATTAATACGAACAAATAAGAAAAATGTGACATTGCAAGAATTAGATATAAAAGAAACAGAATTAATACGAACAAATAAGAAAAATGTGACATTGCAAGAATTAGATATAAA

Enterotoxin H CNS GATCAGAAAAATATTGTCCGATAAATATAAAATTTATTATAAAGACAGCGAAATAAGGG-Enterotoxin H

Enterotoxin H CNSEnterotoxin H

Enterotoxin H CNSEnterotoxin HEnterotoxin H CNSEnterotoxin HEnterotoxin H CNSEnterotoxin HEnterotoxin H CNSEnterotoxin HEnterotoxin H CNSEnterotoxin H

Enterotoxin H CNSEnterotoxin H

Enterotoxin H CNSEnterotoxin H

Enterotoxin H CNSEnterotoxin H

Enterotoxin H CNSEnterotoxin H

Enterotoxin H CNSEnterotoxin H

GATCAGAAAAATATTGTCCGATAAATATAAAATTTATTATAAAGACAGCGAAATAAGTAA

Enterotoxin N (seln) TGATTTAGCTTTAGCTAATGCATATGGTCAATAT-AATCACCCATTCATTAAAGAAAA-----TAGATATTTATGGACTGTATTATGGAAATAAATGTGTAGGCTTAACTGAAGAAAAAA

TATTAAGAGTGATGAAATAAGTGGAGAAAAAGATTTAATATTTAGAAATCAAGGTGATAGCATCATGCTTATACGGAGGAGTTAC------GA--TATATGATGGAAATCAATTAGATGA

TGGCAATGATTTGAGAGTAAAG--------------TTTGCAA-CTGCTGATTTAGCTCAAGAGAGAGTTATAGGCGTTAATGTATTTAAAGATGGTATCCAACAAGAAGGTTTTGTTAT

GAAGTTTAAAAATAAAAATGTAGATATATATGGGGCATCTTTTTATTATAAGTGTGAAAAAAAAACTAAAAAGGCTAAAGTAACAGTACAA------GAATTAGATACTAAAGTTCGATT

AATAAGTGAAAATATTTCTGAATGTCTATATGGAGGTACAACACTAAATAATGAAAAATT------------TAAATTAGAAAATTTATATAAAATATACAATAAAGATACCGGTAACAT

Enterotoxin N CNSEnterotoxin N

Enterotoxin N CNSEnterotoxin N

Enterotoxin N CNSEnterotoxin N

Enterotoxin N CNSEnterotoxin N

Enterotoxin N CNSEnterotoxin N

Figure 3 Continued

12 BioMed Research International

lowastlowastlowast lowastlowast lowastlowast lowastlowast lowast lowastlowast lowastlowastlowast lowastlowast lowast lowastlowastlowastlowast

lowastlowast lowast lowastlowast lowast lowast lowastlowast lowastlowastlowastlowastlowast lowast lowast lowast lowastlowast lowast lowast lowastlowast lowast lowast

lowast lowast

lowast lowastlowast lowast lowastlowast lowast lowast lowast lowast lowast lowast lowast lowastlowast lowast lowastlowastlowastlowast lowast

lowast lowastlowast lowast lowast lowastlowast lowast lowastlowastlowast lowast lowastlowast lowast lowastlowast lowastlowast lowast lowast

lowastlowastlowast lowast lowast lowastlowast lowastlowast lowast lowast lowast

lowast lowast lowast lowastlowastlowast lowastlowastlowast lowastlowast lowast lowastlowastlowastlowastlowast lowastlowastlowast lowast

lowastlowast lowast lowast lowastlowast lowastlowastlowast lowastlowast lowast lowast lowastlowastlowastlowast lowastlowast

lowast lowast lowast lowastlowast lowast lowastlowast lowastlowastlowastlowastlowastlowast lowastlowast lowast lowastlowast lowastlowastlowast

lowast lowast lowast lowast lowastlowast lowastlowastlowastlowast lowast lowast lowastlowastlowast lowast lowast lowast lowast lowast

lowast lowast lowast lowast lowastlowastlowast lowast lowast lowastlowast lowastlowast lowast lowastlowastlowast lowast lowastlowastlowast lowast

lowast lowast lowastlowastlowast lowast lowastlowast lowastlowastlowast lowastlowastlowastlowastlowast lowastlowast lowast lowastlowastlowast lowast lowast lowastlowastlowast

lowastlowastlowast lowast lowastlowast lowastlowast lowastlowast lowast lowast lowast lowast lowastlowast lowastlowast lowastlowast lowast lowast

lowast lowast lowast lowast lowastlowast lowast lowast lowastlowast lowastlowast lowast lowastlowastlowast lowastlowastlowastlowast lowast lowastlowastlowastlowastlowast lowast

lowastlowast lowast lowast lowastlowastlowast lowastlowast lowast lowastlowast lowast lowastlowast lowastlowast lowastlowast lowast lowast lowast

lowastlowastlowastlowastlowast lowastlowast lowastlowast lowast lowastlowast lowast lowast lowast lowast lowastlowast lowastlowast

lowast lowast lowastlowast lowastlowast lowastlowastlowastlowastlowastlowast lowastlowast lowast lowast lowast lowast lowast lowast lowast lowast lowast lowast lowast

lowastlowast lowastlowast lowast lowastlowastlowastlowast lowast lowast lowastlowastlowast lowastlowast lowast lowastlowastlowast lowast

lowastlowastlowastlowast lowastlowast lowast lowast lowast lowast lowastlowast lowast lowast lowast lowastlowastlowastlowastlowast lowastlowast lowastlowastlowast lowast

lowast lowastlowastlowast lowastlowastlowastlowastlowast lowast lowast lowast lowastlowast lowast lowast lowastlowast lowast lowastlowastlowast lowastlowast lowast

lowastlowast lowast lowast lowast lowastlowast lowast lowastlowast lowastlowast lowastlowast lowast lowastlowastlowastlowast

ATTAATACGAACAAATAAGAAAAATGTGACATTGCAAGAATTAGATATAAAGATCAGAAA-TTAT----------TATGATTTATATAAC--------------ATAAAAGGTTCAG---

AATATTGTCCGATAAATATAAAATTTATTATAAAGACAGCGAAATAAGGTAAAAGG------TAGGAGCAGAGTTTTTTCAATTTTATAGTGATAATAGAACAGTTAGCTCATCTAATTA

Enterotoxin N CNS -TAAA---Enterotoxin N

lowast lowastlowastlowastlowastlowast lowastlowast lowast lowast lowast lowastlowast lowast lowastlowastlowastlowastlowastlowast lowastlowast lowastlowast

GGCACAGGAAAGGGTGATTGGTGCTAATGTTTGGGTAGATGGTATTCAAAAAGAAACAGAACAA----AAAGGATGCATTTTCTTT-----CATTCTAATAATCATCAAAATCAATCATT

Enterotoxin N CNSEnterotoxin N

Enterotoxin N CNSEnterotoxin N

Enterotoxin N CNSEnterotoxin N

TCATATCG

Enterotoxin M (selm)

ttgctattcgcaaaatcatatcgcaaccgctgatgtcggagtt--ttgaatcttaggaacEnterotoxin M CNSEnterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

--------------AGTATGTTTAAATCCCCGCTCACAGTTATGTTTAACTCTTCCTTTC

tattatggta-gctatccaattgaagaccaccaaagtattaatcctgaaaataatcatctTTTTCACACCGCCCCGTCGGGTGTTGCGCACGGGGTTGTTTAACCCCCCCGGAAAAAATT

ttcgcatcaattagttttttctatggataattcgacagtaacagctgaatttaagaacgtEnterotoxin M CNS

Enterotoxin M CNS

TTCCCTCCCTCCTCCTTACTC---------------CCT----------------CCCCT

tgatgatgtaaaggaattcaaaaatcatgctgtagatgtatatggtctaagttatagtggEnterotoxin M CNS TTTTCCTACAAATCCTTTCTCCCCTCCCGGAGTAGAAGTAAA------------------

atattgtttgaaaaacaaatatatatacggtggagttacattagcaggtgat-tatttagEnterotoxin M CNS -------------------TAAAACAATACCCCTACCACCTTAACAATTTTTTTATTGAG

agaaatctagacgtattcct-attaatctttgggttaatggagaacatcaaactatatctEnterotoxin M CNS CCCCTCCCCCCCCTTCTCATTTTTTCTCTTTGAC-----------GTTCCCCCCATTTCT

actgacaaagtatcaactaataaaa-agttagtaacagctcaagaaattgatactaaattEnterotoxin M CNS GCCAATGCTTAACCCACCCTTAAAGGCCATCGGCACATATTAGACACTCC-----CCACC

aagaagatatctacaagaagaatataatatttatggctttaatgatac---aaataaaggEnterotoxin M CNS CGCCCCCTCTTTTTATTAAGGACACAACTCTTTTTATTTTCCCCCAAGAACTAATTCTGA

a-agaaattatggtaataagtcaaaatttagttctgga---------tttaatgcaggaaEnterotoxin M CNS AAAAAAACGTTCCGCTTAGTTCAGAATTTCCTTTGAGCCTTTTATCCTTTTGTACGCGAA

aaatattatttca--------tttgaatgatggttcatcattttcttatgacttatttga

Enterotoxin M CNS AAACCATCAGCCATTGAATCAGTTATCCCATTGCCCTAACTAACCCAATATCTAATCTAA

tactggaacaggacaagctgaaagtttctt-aaaaatatataatgacaacaaaactgtcgEnterotoxin M CNS TTTTCGCCTACGAACGAATAATAGCTTGTGGCGAAAAATATCCAGGATTATCTACTGTAG

aaact--gaaaaattccatttagatgtagaaatatcttataaggacgaaagttgaagtatEnterotoxin M CNS CGCCTCGCCAGACGTCCTTTGAATTGA--ATATGACTCCTACTCCCCCCCGCACCGTCAA

ttcaatcataacttagtaaaggaaatgccatgaaaaaatttaaatatagtttt---atatEnterotoxin M CNS TTCAAACA---------------GGTGGAAGTTAATACTGAATAGTAGGTCTTCTTCCCG

tagtttttatattactttttaacattaaagatcttacgtatgct---caaggtgatattgEnterotoxin M CNS TGCTATTCTTAGAACTTTTAAATTATGTAAACCCAATTTTTAGTGCACTCTTTCTCCCTC

gtg-taggtaacttaagaaatttctatacaaaacatgattatatagatttaaaaggcgtcEnterotoxin M CNS GTTACTCTTAGATGGAGAAGCGTTAAGACAGAAGCTTTGTAT-------------TCCCA

acagataaaaa-cctacctattgcaaatcaacttgaattttcaac-----aggt----acEnterotoxin M CNS GCAGAGTGCAACCTTTCACAATGAAGTTTTTATCTAATTTACACGCCTTTAGGCTCCAAA

caatgatttgatctcagaatctaataattgggacgaaataagtaaatttaaaggaaagaaEnterotoxin M CNS TACGTATTAAATCTCCTCCTGT--TTATCTGACCCTAAG-ATAAAACACGAAAAATTATG

actggatatttttggcattgattataatggtccttgtaaatctaaatacatgtatggaggEnterotoxin M CNS CGTGCCCACTCATCCTAT-------TACTTTCATTATATCTATAAAA-------------

Figure 3 Alignment of the enterotoxin gene sequences found in coagulase-negative staphylococci (CNS) and in coagulase-positivestaphylococci (CPS)

BioMed Research International 13

Enterotoxin (seg) S aureus AY1587031

Enterotoxin (sec) CNS

Enterotoxin (see) S pasteuri NC0227371

Enterotoxin (see) CNS

Enterotoxin (seh) CNS

Enterotoxin (selm) S aureus AF2857601

Enterotoxin (seln) CNS

Enterotoxin (seh) S aureus AJ9375481

Enterotoxin (seln) S aureus AY1587031

Enterotoxin (sec) S aureus X516611

Enterotoxin (selm) CNS

Enterotoxin (seg) CNS

150 100

100

100

100

100

99

98

96

97

72

50 0

Figure 4 Phylogenetic tree generated from the multiple alignments of the enterotoxin sequences of CNS and CPS strains using the ClustalX20 software package The phylogenetic tree was constructed using the Mega 60 software and the unweighted pair group method (UPGMA)

MAR indices varying from 066 to 046 and the remaining04 strains showed MAR indices le026 (Table 3)

The 14 MRCNS strains were resistant to 120573-lactams(oxacillin penicillin andor cefoxitin) and to vancomycincorresponding to 73 of the total CNS strains identifiedwhile 09 strains (64) showed resistance to tetracycline andgentamicin 08 strains (57) were resistant to neomycinerythromycin and chloramphenicol 07 strains (50) wereresistant to sulfamethoprim 05 strains (36) were resistantto linezolid 03 strains (21) were resistant to rifampicin 02strains (14) were resistant to ciprofloxacin and cefepimeand 01 strain (7) was resistant to clindamycin (Table 3)

The multiresistance of CNS strains demonstrated hereinis consistent with previous studies on coagulase-negativeand coagulase-positive staphylococci that have found severalresistant and multiresistant Staphylococcus aureus strains inraw milk meat and fermented meat products [43]

Surprisingly the resistance to chloramphenicol is verysimilar to that estimated for MRCNS strains isolated fromhuman clinical samples Indeed there is no direct correla-tion between the researched features and the origin of thestaphylococci strains since different virulence factors arewidespread such as antibiotic resistance [44] reinforcing thefact that the ability of foodmatrices strains to produce SE andSEl and their multiresistance character must be consideredwhen evaluating the safety hazards of food poisoning

S epidermidis KF6005891 and S hominis JX5199881showed the highestMAR indexes (093 and 080 resp)Thesestrains found in artisanal salami are homologous to strainsisolated from human skin microbiota and should be carefullyconsidered among the potential pathogenic staphylococcifound in food from animal origins which can be caused bycontamination by poor hygienic conditions during salamimanufacturing

Depending on the conditions some species of coagulase-negative staphylococci can present health risks since theyhave shown resistance to antibiotics of therapeutic impor-tance such as beta lactams [15] However multiresistantstrains like S carnosusKJ18620021 and S xylosusCP0072081and CP0087241 were intentionally introduced in the foodmatrix since they are part of the culture starter used inItalian-salami manufacturing As previously discussed foodcan be a reservoir of multiresistant microorganisms that canspread by the consumption of an apparently safe food Dueto the intensive and indiscriminate use of antibiotics forhuman and veterinarian therapeutic purposes multiresistantstaphylococci strains are being selected and reproduced infood matrices [45]

The cefoxitin and oxacillin disk diffusion tests are rec-ommended for determining the resistance and susceptibilitybreakpoint for MRSA surveillance cultures [46] In thisstudy there was a good correlation between the disc testzone diameters for oxacillin and cefoxitin (Table 3) TheMICs for ampicillin andmethicillinoxacillincefoxitin for allsusceptible CNS strainswere determined by the diffusion disctest using 003 to 2mgmLminus1 of each antimicrobial agent

S saprophyticus HF9372521 showed an MIC of003mgmLminus1 formethicillin strain S carnosusKJ8620021 Sxylosus strains CP0087241 and CP0072081 S saprophyticusJX4901221 S hominis JX5199881 and S succinusKC3298241 were resistant to 006mgmLminus1 and two strainsS saprophyticus KJ6991511 and S saprophyticus KJ0046231showed an MIC of 05mgmLminus1 (Table 4) Two of themethicillin-resistant strains S saprophyticus KJ0046231 andS xylosus CP0072081 harbor no enterotoxin gene

Five strains S saprophyticus JX4901221 S carnosusKJ8620021 S xylosusCP0087241 S xylosusKF1980801 andS saprophyticus KJ0046231 showed an MIC for ampicillin

14 BioMed Research International

Table 3 Multiple resistance to antimicrobial as found in CNS strains from salami

Salami origin CNS strains Antimicrobial agent resistance Multiple antimicrobialresistance (MAR) indexlowast

Commercial Staphylococcus spp KF1354451 CIP CLO CPM GEN NEO OXA PENSXT TET and VAN 066

Commercial S carnosus KJ8620021 CIP GEN LZD NEO OXA SXT andTET 046

Commercial S saprophyticus AB6977171 CFO CLO ERI PEN OXA TET andVAN 046

Commercial S saprophyticus EU4309921 CFO CLO ERI GEN LZD NEO OXAPEN SXT TET and VAN 080

Commercial S saprophyticus JX4901221 CFO CLO ERI GEN NEO PEN OXASXT TET and VAN 066

Commercial S saprophyticus KJ0046231 CFO OXA and PEN 020

Commercial S sciuri JX9664361 CFO CLO GEN NEO OXA PEN andTET 046

Commercial S xylosus CP0072081 OXA LZD PEN and VAN 026

Commercial S xylosus CP0087241 CFO CLO ERI GEN NEO OXA PENTET and VAN 060

Artisanal S epidermidis KF6005891 CFO CLI CLO CPM ERI GEN LZDNEO OXA PEN RIF SXT and TET 093

Artisanal S hominis JX5199881 CIP CPM CPO ERI GEN LZD NEOOXA PEN RIF SXT and VAN 080

Artisanal S saprophyticusHF9372521 CFO OXA PEN and SXT 026Artisanal S saprophyticus KJ6991511 OXA PEN and VAN 020

Artisanal S succinus KC3298241 CFO CLO ERI GEN OXA PEN RIFSXT TET and VAN 066

Artisanal S xylosus KF1980801 CFO CLO ERI GEN NEO OXA PENTET and VAN 060

lowastTheMAR index of an isolate is defined as 119886119887 where 119886 represents the number of antimicrobials to which the isolate was resistant and 119887 represents the numberof antimicrobials to which the isolate was subjectedS aureus strains ATCC WB81 (sea) ATCC 13563 (sed) and ATCC 27664 (see) showing a MAR index of 05 and S aureus strains ATCC14458 (seb) andATCCWB72 (sec) and S xylosus ATCC 29971 showing a MAR index of 03 were used as reference strainsCPM cefepime CFO cefoxitin CLO chloramphenicol CIP ciprofloxacin CLI clindamycin ERI erythromycin GEN gentamycin NEO neomycin LZDlinezolid RIF rifampicin TET tetracycline OXA oxacillin PEN penicillin SXT sulfamethoprim and VAN vancomycin

Table 4 Minimal inhibitory concentration (MIC) of compounds used in antimicrobial therapy against staphylococci infections

Salami origin GenBank accession number and similarity () MIC mgmLminus1

Methicillin Ampicillin Vancomycin Linezolid

Commercial

S carnosus KJ8620021 006 003 mdash mdashS xylosus CP0087241 006 003 006 mdash

S saprophyticus JX4901221 006 003 05 mdashS saprophyticus AB6977171 mdash mdash 05 mdashS saprophyticus EU4309921 mdash mdash 025 0125

S succinus KC3298241 006 025 003 mdash

Artisanal

S hominis JX5199881 006 05 mdash mdashS saprophyticus HF9372521 003 mdash mdash mdash

S xylosus KF1980801 003 05 mdashS saprophyticus KJ6991511 05 025 003 mdashS saprophyticus KJ0046231 05 003 003 mdash

S xylosus CP0072081 006 mdash mdash 025Strains S epidermidis KF6005891 and Staphylococcus spp KF1354451 were not susceptible to the antimicrobial concentrations tested in the present study

BioMed Research International 15

of 003mgmLminus1 two strains S succinus KC3298241 and Ssaprophyticus KJ6991511 showed an MIC of 025mgmLminus1and S hominis JX5199881 showed an MIC of 05mgmLminus1(Table 4) S saprophyticus KJ0046231 also showed an MICof 003mgmLminus1 but does not harbor enterotoxin genes It isimportant to note that most of the CNS strains that showedresistance to ampicillin were also resistant to methicillin ashighlighted by the MIC tests

Five strains S saprophyticus HF9372521 S saprophyti-cus KJ6991511 S saprophyticus JX4901221 S saprophyticusKJ0046231 and S succinus KC3298241 showed an MICfor vancomycin of 003mgmLminus1 one strain S xylosusCP0087241 showed an MIC of 006mgmLminus1 one strain Ssaprophyticus EU4309921 showed an MIC of 025mgmLminus1and two strains S xylosus KF1980801 and S saprophyticusAB6977171 showed an MIC of 05mgmLminus1 (Table 4)

Additionally five CNS strains S xylosus CP0087241S saprophyticus JX 4901221 S succinus KC3298241 Ssaprophyticus KJ6991511 and S saprophyticus KJ0046231showed resistance tomethicillin as demonstrated by theMICdeterminations (Table 4)

Previous studies have demonstrated that linezolid isactive against Gram-positive bacteria including methicillin-resistant staphylococci [47] In the present study 05 strainsshowed linezolid resistance in the disc diffusion test but itwas impossible to establish MIC values for S epidermidisKF6005891 Staphylococcus hominis JX5199881 and Staphy-lococcus carnosus KJ8620021The remaining strains S sapro-phyticus EU4309921 and S xylosus CP0072081 presentedan MIC for linezolid of 0125mgmLminus1 and 025mgmLminus1respectively

The MIC of 003mgmLminus1 for penicillin is in accordancewith the previous values estimated for resistant S aureusstrains found in several food matrices such as meat dairyproducts and ready-to-eat food [48]

There is still a lack of information on the antimicro-bial resistance of staphylococci strains from food matricesalthough they are the most worrisome vehicles of dissemi-nation of antibiotic-resistant pathogens [49]

The high resistance found for salami staphylococci strainscan be ascribed to their inappropriate use as growth promot-ers of antimicrobial agents like oxacillin vancomycin chlo-ramphenicol neomycin and erythromycin which are com-monly used in veterinary medicine to treat infections [50]Antimicrobial therapy of infections staphylococci is basedon results of susceptibility tests in vitro [51] Methicillin-resistant coagulase-negative Staphylococcus spp found inready-to-eat products such as meats fish and dairy productsalso offer risks and although they are not classical foodpoisoning bacteria their presence in food offers significantrisks to public health due to the possible spread of antibioticresistance [52]

The standardization of salami quality should includediagnostic methods to screen and quantify the presence ofclassical and newly described enterotoxins directly in thefood matrices as a routine procedure to be conducted by themeat product industry in Brazil

The safety of salami consumption could also be enhancedby the inclusion of amicrobial barrier such as the inclusion ofprobiotic strains producing natural antibiotics or competitiveflora or even the addition of natural bioagents against spoilageor pathogenic microorganisms

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors acknowledge the financial support fromFundacao Carlos Chagas Filho de Amparo a Pesquisa doEstado do Rio de Janeiro (FAPERJ Rio de Janeiro Brazil)Conselho Nacional de Desenvolvimento Cientıfico eTecnologico (CNPq Brasılia Brazil) and Coordenacao deAperfeicoamento de Pessoal de Nıvel Superior (CAPESBrasılia Brazil)

References

[1] ICMSF International Comission on Microbiological Specifica-tions for FoodsMicroorganisms in FoodsTheir Significance andMethods of Enumerations University of Toronto Press TorontoCanada 3rd edition 1983

[2] J F Veras L S Carmo L C Tong et al ldquoA study of theenterotoxigenicity of coagulase-negative and coagulase-positivestaphylococcal isolates from food poisoning outbreaks inMinasGerais Brazilrdquo International Journal of Infectious Diseases vol12 no 4 pp 410ndash415 2008

[3] K Omoe D-L Hu H Takahashi-Omoe A Nakane and KShinagawa ldquoComprehensive analysis of classical and newlydescribed staphylococcal superantigenic toxin genes in Staphy-lococcus aureus isolatesrdquo FEMS Microbiology Letters vol 246no 2 pp 191ndash198 2005

[4] J Kadariya T C Smith and D Thapaliya ldquoStaphylococcusaureus and staphylococcal food-borne disease an ongoing chal-lenge in public healthrdquoBioMedResearch International vol 2014Article ID 827965 9 pages 2014

[5] KOmoeD-LHuH KOno et al ldquoEmetic potentials of newlyidentified staphylococcal enterotoxin-like toxinsrdquo Infection andImmunity vol 81 no 10 pp 3627ndash3631 2013

[6] J K McCormick J M Yarwood and P M SchlievertldquoToxic shock syndrome and bacterial superantigens an updaterdquoAnnual Review of Microbiology vol 55 pp 77ndash104 2001

[7] T Baba F Takeuchi M Kuroda et al ldquoGenome and virulencedeterminants of high virulence community-acquired MRSArdquoThe Lancet vol 359 no 9320 pp 1819ndash1827 2002

[8] Associacao Brasileira de Industria Produtora e Exportadorada Carne Suınas (ABIPECS) ldquoStatistics slaughter ranking2000ndash2014rdquo 2014 httpabpa-brcombrsetoressuinoculturapublicacoesrelatorios-anuais

[9] N Zdolec M Hadziosmanovic L Kozacinski et al ldquoMicrobialand physicochemical succession in fermented sausages pro-duced with bacteriocinogenic culture of Lactobacillus sakei andsemi-purified bacteriocin mesenterocin Yrdquo Meat Science vol80 no 2 pp 480ndash487 2008

16 BioMed Research International

[10] F Ravyts L D Vuyst and F Leroy ldquoBacterial diversity andfunctionalities in food fermentationsrdquo Engineering in Life Sci-ences vol 12 no 4 pp 356ndash367 2012

[11] TOhlsson andN BengtssonTheHurdle ConceptMinimal Pro-cessing Technologies in the Food IndustryWoodhead Publishing2002

[12] S Bernardi B B Golineli and C J Contreras-CastilloldquoRevisao aspectos da aplicacao de culturas starter na producaode embutidos carneos fermentadosrdquo Brazilian Journal of FoodTechnology vol 13 no 2 pp 133ndash140 2010

[13] F Irlinger ldquoSafety assessment of dairy microorganismscoagulase-negative staphylococcirdquo International Journal of FoodMicrobiology vol 126 no 3 pp 302ndash310 2008

[14] B Ghebremedhin F Layer W Konig and B Konig ldquoGeneticclassification and distinguishing of Staphylococcus species basedon different partial gap 16S rRNA hsp60 rpoB sodA and tufgene sequencesrdquo Journal of Clinical Microbiology vol 46 no 3pp 1019ndash1025 2008

[15] I Babic K Markov D Kovacevic et al ldquoIdentification andcharacterization of potential autochthonous starter culturesfrom aCroatian lsquobrandrsquo product lsquoslavonski kulenrsquordquoMeat Sciencevol 88 no 3 pp 517ndash524 2011

[16] MMehrotra GWang andWM Johnson ldquoMultiplex PCR forthe detection of genes for Staphylococcus aureus enterotoxinsexfoliative toxins toxic shock syndrome toxin and methicillinresistancerdquo Journal of Clinical Microbiology vol 38 no 3 pp1032ndash1041 2000

[17] WM Johnson SD Tyler E P Ewan F EAshtonDR Pollardand K R Rozee ldquoDetection of genes for enterotoxins exfolia-tive toxins and toxic shock syndrome toxin 1 in Staphylococcusaureus by the polymerase chain reactionrdquo Journal of ClinicalMicrobiology vol 29 no 3 pp 426ndash430 1991

[18] J Mclauchlin G L Narayanan VMithani andG OrsquoNeill ldquoThedetection of enterotoxins and toxic shock syndrome toxin genesin Staphylococcus aureus by polymerase chain reactionrdquo Journalof Food Protection vol 63 no 4 pp 479ndash488 2000

[19] J P Rosec and O Gigaud ldquoStaphylococcal enterotoxin genes ofclassical and new types detected by PCR in Francerdquo Interna-tional Journal of Food Microbiology vol 77 no 1-2 pp 61ndash702002

[20] K Omoe D-L Hu H Takahashi-Omoe A Nakane andK Shinagawa ldquoIdentification and characterization of a newstaphylococcal enterotoxin-related putative toxin encoded bytwo kinds of plasmidsrdquo Infection and Immunity vol 71 no 10pp 6088ndash6094 2003

[21] N Sergeev D Volokhov V Chizhikov and A Rasooly ldquoSimul-taneous analysis of multiple staphylococcal enterotoxin genesby an oligonucleotide microarray assayrdquo Journal of ClinicalMicrobiology vol 42 no 5 pp 2134ndash2143 2004

[22] S Holtfreter D Grumann M Schmudde et al ldquoClonal distri-bution of superantigen genes in clinical Staphylococcus aureusisolatesrdquo Journal of Clinical Microbiology vol 45 no 8 pp2669ndash2680 2007

[23] W J Mason J S Blevins K Beenken NWibowo N Ojha andM S Smeltzer ldquoMultiplex PCR protocol for the diagnosis ofstaphylococcal infectionrdquo Journal of Clinical Microbiology vol39 no 9 pp 3332ndash3338 2001

[24] E Rahimi and H G Safai ldquoDetection of classical enterotoxinsof Staphylococcus aureus strains isolated frombovine subclinicalmastitis in Isfahan Iranrdquo Veterinary Microbiology vol 141 no3-4 pp 393ndash394 2010

[25] M A Larkin G Blackshields N P Brown et al ldquoClustalW andClustal X version 20rdquo Bioinformatics vol 23 no 21 pp 2947ndash2948 2007

[26] P H A Sneath and R R Sokal Unweighted Pair Group Methodwith Arithmetic Mean Numerical Taxonomy W H FreemanSan Francisco Calif USA 1973

[27] NCCLS ldquoPerformance standards for antimicrobial susceptibil-ity testing 14th informational supplementrdquo Tech Rep M 100-S14 NCCLS 2004

[28] S Corbiere Morot-Bizot S Leroy and R Talon ldquoMonitoringof staphylococcal starters in two French processing plantsmanufacturing dry fermented sausagesrdquo Journal of AppliedMicrobiology vol 102 no 1 pp 238ndash244 2007

[29] R Talon S Leroy I Lebert et al ldquoSafety improvement andpreservation of typical sensory qualities of traditional dryfermented sausages using autochthonous starter culturesrdquo Inter-national Journal of Food Microbiology vol 126 no 1-2 pp 227ndash234 2008

[30] G Mauriello A Casaburi G Blaiotta and F Villani ldquoIsolationand technological properties of coagulase negative staphylo-cocci from fermented sausages of Southern ItalyrdquoMeat Sciencevol 67 no 1 pp 149ndash158 2004

[31] M Janssens N Myter L De Vuyst and F Leroy ldquoSpeciesdiversity and metabolic impact of the microbiota are low inspontaneously acidified Belgian sausages with an added starterculture of Staphylococcus carnosusrdquo Food Microbiology vol 29no 2 pp 167ndash177 2012

[32] G Blaiotta D Ercolini G Mauriello G Salzano and F VillanildquoRapid and reliable identification of Staphylococcus equorum bya species-specific PCR assay targeting the sodA generdquo Systematicand Applied Microbiology vol 27 no 6 pp 696ndash702 2004

[33] L Kozacinski E Drosinos F Caklovica L Cocolin J Gasparik-Reichardt and S Veskovic ldquoInvestigation of microbial associa-tion of traditionally fermented sausagesrdquo Food Technology andBiotechnology vol 46 no 1 pp 93ndash106 2008

[34] S Leroy P Giammarinaro J-P Chacornac I Lebert and RTalon ldquoBiodiversity of indigenous staphylococci of naturallyfermented dry sausages and manufacturing environments ofsmall-scale processing unitsrdquo Food Microbiology vol 27 no 2pp 294ndash301 2010

[35] E Coton M-H Desmonts S Leroy et al ldquoBiodiversity ofCoagulase-Negative Staphylococci in French cheeses dry fer-mented sausages processing environments and clinical sam-plesrdquo International Journal of Food Microbiology vol 137 no 2-3 pp 221ndash229 2010

[36] S Hosseinzadeh and H D Saei ldquoStaphylococcal species associ-ated with bovinemastitis in the NorthWest of Iran emerging ofcoagulase-negative staphylococcirdquo International Journal of Vet-erinary Science and Medicine vol 2 no 1 pp 27ndash34 2014

[37] A Piette and G Verschraegen ldquoRole of coagulase-negativestaphylococci in human diseaserdquo Veterinary Microbiology vol134 no 1-2 pp 45ndash54 2009

[38] T M Moura F S Campos P A DrsquoAzevedo et al ldquoPrevalenceof enterotoxin-encoding genes and antimicrobial resistancein coagulase-negative and coagulase-positive Staphylococcusisolates from black pudding in Southern Brazilrdquo Revista daSociedade Brasileira deMedicina Tropical vol 45 no 5 pp 579ndash585 2012

[39] H I Suzuki T Lefebure P P Bitar and M J StanhopeldquoComparative genomic analysis of the genus Staphylococcusincluding Staphylococcus aureus and its newly described sister

BioMed Research International 17

species Staphylococcus simiaerdquo BMC Genomics vol 13 article38 2012

[40] R P Lamers G Muthukrishnan T A Castoe S Tafur A MCole and C L Parkinson ldquoPhylogenetic relationships amongStaphylococcus species and refinement of cluster groups basedon multilocus datardquo BMC Evolutionary Biology vol 12 article171 2012

[41] I V Pinchuk E J Beswick and V E Reyes ldquoStaphylococcalenterotoxinsrdquo Toxins vol 2 no 8 pp 2177ndash2197 2010

[42] S R Monday and G A Bohach ldquoGenes encoding staphylo-coccal enterotoxins G and I are linked and separated by DNArelated to other staphylococcal enterotoxinsrdquo Journal of NaturalToxins vol 10 no 1 pp 1ndash8 2001

[43] D Y Thomas S Jarraud B Lemercier et al ldquoStaphylococcalenterotoxin-like toxins U2 and V two new staphylococcalsuperantigens arising from recombination within the entero-toxin gene clusterrdquo Infection and Immunity vol 74 pp 4724ndash4734 2006

[44] N Indrawattana O Sungkhachat N Sookrung et al ldquoStaphylo-coccus aureus clinical isolates antibiotic susceptibility molecu-lar characteristics and ability to form biofilmrdquo BioMed ResearchInternational vol 2013 Article ID 314654 11 pages 2013

[45] V Pereira C Lopes A Castro J Silva P Gibbs and P TeixeiraldquoCharacterization for enterotoxin production virulence factorsand antibiotic susceptibility of Staphylococcus aureus isolatesfrom various foods in Portugalrdquo Food Microbiology vol 26 no3 pp 278ndash282 2009

[46] I Anacarso C Condo C Sabia and et al ldquoAntimicrobialresistance and other related virulence factors in staphylococcusspp isolated from food environmental and humans in ItalyrdquoUniversal Journal Microbiology Research vol 1 no 1 pp 1ndash92013

[47] B Martın M Garriga M Hugas S Bover-Cid M T Veciana-Nogues and T Aymerich ldquoMolecular technological and safetycharacterization of Gram-positive catalase-positive cocci fromslightly fermented sausagesrdquo International Journal of FoodMicrobiology vol 107 no 2 pp 148ndash158 2006

[48] NM Broekema T Van Tam T AMonson S AMarshall andD M Warshauer ldquoComparison of cefoxitin and oxacillin diskdiffusionmethods for detection ofmecA-mediated resistance inStaphylococcus aureus in a large-scale studyrdquo Journal of ClinicalMicrobiology vol 47 no 1 pp 217ndash219 2009

[49] E Cercenado F Garcıa-Garrote and E Bouza ldquoIn vitro activityof linezolid against multiply resistant Gram-positive clinicalisolatesrdquo Journal of Antimicrobial Chemotherapy vol 47 no 1pp 77ndash81 2001

[50] A Aydin K Muratoglu M Sudagidan K Bostan B Okukluand SHarsa ldquoPrevalence and antibiotic resistance of foodborneStaphylococcus aureus isolates in Turkeyrdquo Foodborne Pathogensand Disease vol 8 no 1 pp 63ndash69 2011

[51] D Sergelidis A Abrahim T Papadopoulos et al ldquoIsolation ofmethicillin-resistant Staphylococcus spp from ready-to-eat fishproductsrdquo Letters in Applied Microbiology vol 59 no 5 pp500ndash506 2014

[52] I PhillipsM Casewell T Cox et al ldquoDoes the use of antibioticsin food animals pose a risk to human health A critical reviewof published datardquo Journal of Antimicrobial Chemotherapy vol53 no 1 pp 28ndash52 2004

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Page 3: Research Article Safety Evaluation of the Coagulase ...downloads.hindawi.com/journals/bmri/2015/483548.pdf · Research Article Safety Evaluation of the Coagulase-Negative Staphylococci

BioMed Research International 3

Table 1 Primer set for V3 16S rDNA sequencing and PCRreal time RT-PCR tests targeting the classical and newly described staphylococcalenterotoxin genes

Primers set and sequences (51015840-31015840) Gene Amplicon (bp) ReferencesSEAf TTGGAAACGGTTAAAACGAA sea 120 [17]SEA

rGAACCTTCCCATCAAAAACA

SEBf TCGCATCAAACTGACAAACG seb 478 [17]SEBr GCAGGTACTCTATAAGTGCCSECf GACATAAAAGCTAGGAATTT sec 257 [17]SECr AAATCGGATTAACATTATCCSEDf CTAGTTTGGTAATATCTCCT sed 317 [17]SEDr TAATGCTATATCTTATAGGGSEEf TAGATAAAGTTAAAACAAGC see 170 [17]SEEr TAACTTACCGTGGACCCTTCSEGf TGCTATCGACACACTACAACC seg 704 [18]SEGr CCAGATTCAAATGCAGAACCSEHf CGAAAGCAGAAGATTTACACG seh 495 [18]SEHr GACCTTTACTTATTTCGCTGTCSEIf GACAACAAAACTGTCGAAACTG sei 630 [18]SEIr CCATATTCTTTGCCTTTACCAGSElJf CAGCGATAGCAAAAATGAAACA selj 426 [19]SElJr TCTAGCGGAACAACAGTTCTGASElMf CCAATTGAAGACCACCAAAG selm 517 [20]SElMr CTTGTCCTGTTCCAGTATCASElNf ATTGTTCTACATAGCTGCAA seln 682 [20]SElNr TTGAAAAAACTCTGCTCCCASElOf AGTCAAGTGTAGACCCTATT selo 534 [20]SElOr TATGCTCCGAATGAGAATGASElKf ATGAATCTTATGATTTAATTTCAGAATCAA selk 545 [21]SElKr ATTTATATCGTTTCTTTATAAGAAATATCGSElQf GGAAAATACACTTTATATTCACAGTTTCA selq 539 [21]SElQr ATTTATTCAGTTTTCTCATATGAAATCTCSElRf AATGGCTCTAAAATTGATGG selr 363 [22]SElRr TCTTGTACCGTAACCGTTTTSElUf AATGGCTCTAAAATTGATGG selu 215 [22]SElUr ATTTGATTTCCATCATGCTCTSST-1f ATGGCAGCATCAGCTTGATA tstH1 350 [17]TSST-1r TTTCCAATAACCACCCGTTT16S rDNAf ATA AGA CTG GGA TAA CTT CGG G 16SrDNA 500 [23]16S rDNAr CTT TGA GTT TCA ACC TTG CGG TCGf forward r reverse

Hercules CAUSA)The amplified fragments were visualizedon 10 agarose gels (Sigma) stained with GelRed (dilution1 1000) (BioAmerica Tel Aviv Israel) and documented on atransilluminator (MiniLumi Imaging Bio-Systems BioAmer-ica Tel Aviv Israel)

233 Multiplex-PCR Tests Multiplex-PCR assays were per-formed by the simultaneous amplification of the see seg sehsei selj selm seln selo selk selq selr and selu sequences usingthe primer sets listed in Table 1 Each reaction contained50120583L of a mix containing 05U Taq DNA polymerase 10xPCR buffer 100mMdNTP 02120583Mof each primer and 100 ng

of DNA template DNA amplification of see seg seh andsei was carried out as follows 95∘C for 5min 35 cycles of95∘C for 30 s 53∘C for 90s and 72∘C for 90 s and a finalextension at 72∘C for 10min The DNA amplifications of theselj selm seln and selo group and the selk selq selr andselu group were carried out in the same conditions [3] PCRproducts were visualized by electrophoresis on 12 agarosegels (Uniscience do Brasil Sao Paulo Brazil) in 1x TAE (Tris-boric acid-EDTA) buffer stained by 05 120583gmLminus1 of GelRed(BioAmerica Tel Aviv Israel) and documented on a transil-luminator (MiniLumi Imaging Bio-Systems BioAmerica TelAviv Israel)

4 BioMed Research International

DNA templates from the following reference strains wereused S aureusATCC 29231 (sea) S aureusATCC 14458 (sebtstH selk selq selr and selu) S aureus ATCC 19095 (sec segseh and sei) S aureus ATCC 13563 (sed) S aureus ATCC27664 (see) and S aureus ATCC 27154 (selj selm seln andselo) and S xylosus ATCC 29971

24 Enterotoxin ExpressionAssays Theobserved strainswerecultured aerobically overnight in 10mL Brain Heart InfusionBroth (BD BBL Le Pont de Claix France) at 37∘C for 72 hBacteria supernatants were collected by centrifugation at4000timesg for 10min and used for the detection of sea seb secsed and see by an ELISA assay using a commercial detectionkit (RIDASCREEN SET A B C D E Art number R4101 R-Biopharm AG Germany) The assay was performed accord-ing to the manufacturerrsquos recommendation and as describedelsewhere [16]Themean lower limit of detection of the assaywas 025 ngmLminus1 The threshold is defined as the average ODof two negative controls plus 015 a constant established bythe kit Samples containing SEs showed absorbance valuesequal to or greater than the threshold value All experimentswere performed in duplicate

25 Real Time RT-PCR Assays Total RNA was extracted byusing the QIAGEN RiboPure Bacteria kit (Life TechnologiesCarlsbad California USA) following the manufacturerrsquosinstructions and quantified using the Qubit fluorometer(Invitrogen Grand Island New York USA) and Qubit assaykits The cDNA synthesis was performed by using the HighCapacity cDNA Reverse Transcription Kit (Applied Biosys-tems California USA) and the ABI PRISM 7500 Fast RT-PCR system (Applied Biosystems California USA) Sampleswere plated in triplicate in 96-well plates as follows 12 120583Lof the SYBR Green PCR Master Mix 1 120583L of primer mix(sea seb sec sed and see) and 45 120583L of the cDNA ultrapurewater in each well Amplification was performed under thefollowing conditions 95∘C for 15min 40 cycles at 95∘Cfor 15 s 54∘C for 30s and 72∘C for 30 s The dissociationcurve was performed at 95∘C for 15 sec 54∘C for 30 sec and95∘C for 15 sec CT means the standard deviations and thecDNA semiquantification were calculated using the Graph-Pad Prism 5 software package Calibration curves based onfive points were constructed in triplicate corresponding toserial dilutions (1 1 10 1 100 1 1000 and 1 10000) from100 ng of a DNA template stock solution

26 16S rDNA Sequencing Amplification of the V5 regionof 16S rDNA fragment was performed using 50 ng of DNAtemplates from the 65 strains found in the salami samplesPCR was performed under the following conditions 95∘Cfor 10min followed by 30 cycles at 95∘C for 30 s 60∘Cfor 30 s and 72∘C for 45 s and a final extension at 72∘Cfor 10min PCR products were purified using the PCRDNA Purification Kit (Applied Biosystems California USA)and sequenced using 20 ng purified DNA and 13 120583L ofprimer sets in a final volume of 20120583L After amplifica-tion products were purified according to the protocol ofthe BigDye Terminator Purification X Kit (Applied Biosys-tems California USA) and sequenced on a 3130 sequencer

GeneticAnalyzer (Applied Biosystems California USA)Thesequences were compared to the 16S rDNA gene sequencesof Staphylococcus species available at the GenBank database(httpwwwncbinlmnihgovGenbankindexhtml) Multi-ple sequence alignments were performed using ClustalW (Kyoto University Bioinformatics Center httpwwwgenomejptoolsclustalw)

27 Sequencing of Enterotoxin PCR Products PCR productswere purified using the PCR DNA Purification Kit (AppliedBiosystems California USA) and sequenced using 10 ngof purified DNA and 32 pmoles of each primer set in afinal volume of 20 120583L After amplification in the sameconditions as the PCR step (Section 233) products werepurified according to the BigDye Terminator PurificationX Kit protocol (Applied Biosystems California USA)and sequenced on a 3130 sequencer Genetic Analyzer(Applied Biosystems California USA) The sequences werecompared to Staphylococcus aureus and Staphylococcuspasteuri gene sequences available at the GenBank database(httpwwwncbinlmnihgovGenbankindexhtml) Multi-ple sequence alignments were performed using ClustalW (Kyoto University Bioinformatics Center httpwwwgenomejptoolsclustalw)

28 Phylogenetic Analyses Phylogenetic relationshipsbetween the CNS strains were performed by sequencealignments using the Clustal X 20 software package [24]The phylogenetic trees were constructed using the softwareMega 60 and UPGMAmethods [25]

29 Antibiotic Susceptibility Tests An inoculumof each strainequivalent to a 05 McFarland scale was swabbed onto aMueller-Hinton agar plate (BD BBL Franklin Lakes NewJersey USA) and the antibiotic disc was then placed on theplate followed by overnight incubation at 37∘CThe inhibitionzone was interpreted according to the Clinical LaboratoryStandard (CLSI) Guidelines formerly known as the NationalCommittee for Clinical Laboratory Standards The testedantibioticswere penicillinG (10U) oxacillin (1120583g) neomycin(30 120583g) sulfamethoprim (5 120583g) clindamycin (2120583g) gen-tamicin (10 120583g) cefoxitin (30 120583g) rifampicin (5 120583g) ery-thromycin (15 120583g) tetracycline (30 120583g) vancomycin (30120583g)ciprofloxacin (5120583g) sulfazothrim (23120583g) cefepime (30 120583g)linezolid (30 120583g) and chloramphenicol (30 120583g)

210 Minimal Inhibitory Concentration (MIC) Determina-tions The MICs of vancomycin linezolid methicillin andampicillin were determined by the macrodilution brothmethod based on CLSI recommendations using in-house-prepared panels [26] Antibiotic concentrations of 003 0060125 025 05 10 and 20mgmLminus1 were tested One mLof broth was transferred to the tubes and 100 120583L of thebacteria suspension was adjusted to 106 CFUmL in saline085 according to a 05 McFarland scale and transferredto tubes containing 1mL of each antimicrobial Strains weregrown in Mueller-Hinton broth (BD BBL Franklin LakesNew Jersey USA) and the MIC was estimated as the lowest

BioMed Research International 5

antibiotic concentration that inhibits visible growth after 24 h[26]

3 Results and Discussion

31 Isolation and Identification of Coagulase-Negative Strainsfrom Salami Sixty-five presumable coagulase-negativestaphylococci microorganisms from salami were isolated bycolony morphology coagulase slide test subsequent tubetest and biochemical tests The sequencing of the V5 regionof the 16S rDNA fragment of the strains was discriminativeenough to differentiate the Staphylococcus isolated fromsalami at the subspecies level with the exception of 1 strainidentified up to the Staphylococcus spp genus

Nineteen distinct strains were identified as CNS in salamiwith 08 of 19 (42) identified as S saprophyticus thepredominant species followed by 05 strains of S xylosus(26) 02 strains of S carnosus (11) and 01 strain of eachof the following species S succinus S sciuri S epidermidisand S hominis (5 each) (Table 2)

The 08 S saprophyticus strains were identified asKJ6991511 AB6977171 JX4901221 KJ0046231 andHQ6995101 EU4309921 HF9372521 and KJ9496061 withthe latter two and S sciuri JX9664361 being homologous(96ndash98) to strains from the environment Five S xylosusstrains CP0072081 KF1980801 CP0087241 AM8827001and KC4565901 and a single S succinus strain KC3298241were identified The last three S xylosus strains and theS succinus strain are homologous (97-98) to strainsfound in fermented meat or meat starter cultures Sxylosus CP0072081 showed homology (98) to potentialopportunistic pathogenic strains from mammal species

The S carnosus strains identified were KJ8620021 andNR1164341 and the latter as well as the single S hominisJX5199881 (96-97) identified were homologous (97) tospecies from human microbiota

S carnosus and S xylosus are commonly used as com-mercial starter cultures for sausage manufacturing [26] butS succinus has also been observed in dry fermented sausagesand its use as a starter culture has been already proposed [27]

The diversity of CNS microbiota found in the samplesanalyzed in the present study could be related to the originof the salami The microbiota from the artisanal salamishowed greater biodiversity when compared to the commer-cial salami In commercial salami S saprophyticus was thepredominant species but S xylosus and S carnosus alsoobserved in commercial salami are commonly isolated fromstarter cultures [26] The predominance of S saprophyticusfollowed by S xylosus has also been reported in salamifrom South Italy [28] similar to Belgian sausages where Ssaprophyticus was the most frequently detected species [29]

S carnosus was not observed in artisanal salami but Ssuccinus S epidermidis and S hominis were detected

The biodiversity of the CNS staphylococci species foundin microbiota depends on the kind of meat-fermented prod-uct but CNS strains such as S saprophyticus S auricularisS xylosus S capitis S hominis S carnosus S haemolyticusS warneri S equorum S cohnii S capitis and S intermediushave been described inNapoli-type salami Sremska sausages

dry sausages raw meat and naturally fermented meat [30ndash33]

During the last decades S epidermidis and S sapro-phyticus have been described as emerging pathogens [34]S saprophyticus is considered a frequent contaminant offermented sausages and raw meats and has been isolatedfrom rectal swabs of cattle carcasses and pigs In humans themain reservoir of S saprophyticus is the gastrointestinal tract[35] S saprophyticus and S epidermidis can be opportunisticpathogens isolated from the human urinary tract and thepresence of these species in food should be taken into accountconcerning possible contamination of the starter inoculumandor improvements in the salami manufacturing process[13]

Other CNS species observed in the present study aremainly associated with ordinary food contaminants withS epidermidis and S hominis being the dominant speciesin human skin and occasionally isolated from the skin ofdomestic animals [36]

32 Phylogenetic Relationships of the CNS Identified in SalamiCNS strains can be grouped into four species groupssaprophyticus simulans epidermidis and haemolyticus Fre-quently the saprophyticus species group includes S xylosusand S saprophyticus while the simulans species group iscomprised of S carnosus and S piscifermentans the epi-dermidis species group is composed of S epidermidis Scapitis S caprae and S saccharolyticus and the haemolyticusspecies group encompasses S haemolyticus S hominis andS devriesei [37 38] In the present study the main clustergrouped several S saprophyticus strains namely KJ6991511AB6975171 KJ9496061 JX4901221 and KJ0046231 four Sxylosus strains CP0087241 CP0072081 AM8827001 andKC4565901 and the S succinusKC3298241 strain (Figure 1)These strains are homologous to those from fermented meatmicrobiota

The subclusters of those clusters showed mismatches ofspecies belonging to the four species groups The first sub-cluster grouped S epidermidis HF0882111 S saprophyticusHQ6995101 and S carnosusNR1164341 and the second sub-cluster grouped S hominis JX5199881 and the S saprophyticusEU4309921 all of them originally isolated from animalsand human beings The third subcluster includes speciespreviously isolated from the marine environment such as SxylosusKF1980801 while S carnosusKJ8620021 is utilized asa probiotic organism in foodsThe fourth cluster grouped theS sciuri JX9664361 and S saprophyticus HF9372521 strainswhich are homologous to species found in soil

The CNS strains clustered into groups near the bottomof the phylogenetic tree are mostly strains found in artisanalsalami whereas the species at the top of the tree are mainlyCNS strains found in commercial salami (Figure 1)

The close similarities between the S saprophyticusAB6975171 and KJ9496061 strains the S succinusKC3298241 S xylosus AM8827001 KC4565901 andCP0087241 strains the S saprophyticus KJ0046231 andKJ6991511 strains and S xylosus CP0072081 are supportedby a bootstrap value of 100 The interspecies similaritieswere over 90 which demonstrates close phylogenies

6 BioMed Research International

Table2Genotypicandph

enotypiccharacteriz

ationof

CNSstrainsfrom

salami

Strainsidentificatio

nandcharacteriz

ation

Salamiorig

inStaphylococcus

species

Genotypic

Phenotypic

GenBa

nkaccessionnu

mbera

ndsim

ilarity(

)Presence

ofenterotoxingenes

mRN

Adetection

Enterotoxinprod

uctio

n(ngm

Lminus1)

Com

mercial

Staphylococcus

sppKF

1354451(96)

seaseb

mdashmdash

Scarnosus

KJ8620021(96)

seasebsedsehseiand

selj

mdash12plusmn01

Scarnosus

NR1164341(98)

sebsecsedandsee

see

03

Ssaprophyticus

AB6

977171

(98)

sehseiandselm

seiselm

03

Ssaprophyticus

EU4309921(99)

seasebselm

seln

selo

and

tstH1

sebselm

and

selo

13plusmn01

Ssaprophyticus

HQ6995101(97)

mdashmdash

mdashSsaprophyticus

JX4901221(99)

seaseh

seh

05plusmn01

Ssaprophyticus

KJ00

46231(96)

sec

mdash04

Ssaprophyticus

KJ9496061(98)

seasebseca

ndseh

seasebandseh

mdashSsciuriJX9664361(98)

sedsei

sei

10plusmn01

SxylosusA

M8827001(97)

mdashmdash

mdashSxylosusCP

0072081(99)

mdashmdash

mdashSxylosusC

P0087241(98)

seatstH1

sea

14SxylosusK

C4565901(98)

mdashmdash

mdash

Artisa

nal

SepidermidisKF

6005891(97)

secsedseesegsehandsei

seesei

07plusmn01

ShominisJX5199881(97)

seasebsecsedseljselm

seln

andselo

selnselo

09plusmn01

Ssaprophyticus

HF9

372521(97)

seb

seb

05

Ssaprophyticus

subspbovisK

J6991511

(98)

secselm

seln

andtstH1

seln

09plusmn01

Ssuccinus

KC3298241(99)

seasebsecseljandseln

sebsec

13SxylosusK

F1980801(97)

secselm

andtstH1

mdash05plusmn01

Thep

resenceo

fenterotoxin

genesseasebsecsedseesegsehsei

seljselkselm

seln

selo

selq

selrselu

and

tstH1w

asteste

dby

PCRusingthes

pecific

seto

fprim

ers

sea-seee

nterotoxin

prod

uctio

nwas

evaluatedby

immun

e-sorbentassays(EL

ISA)u

singthed

etectio

nkitR

IDASC

REEN

SETAB

CD

EV

aluesa

redisplay

edas

them

eansplusmnSD

ofassays

perfo

rmed

indu

plicate

mRN

Atranscrip

tsfora

llenterotoxingenesw

eree

valuated

byrealtim

eRT-PC

Rtests

CT

values

ared

isplayedas

meansplusmnSD

ofRT

-PCR

tests

perfo

rmed

indu

plicateseamdash

SxylosusC

P00872413

0plusmn04sebmdash

SxylosusC

P008724133plusmn20Ssaprophyticu

sJX4901221302plusmn04Ssaprophyticus

subspbovisK

J6991511

32plusmn01SxylosusK

F19808013

6plusmn10

and

ShominisJX519988134plusmn10secmdashSsaprophyticus

subspbovisK

J6991511

31plusmn11seemdashSsaprophyticus

subspbovisK

J6991511306plusmn05

Ssuccinus

KC3298241285plusmn01andSsaprophyticus

HF9

3725213

2plusmn04sehmdash

SxylosusC

P0087241 31plusmn10Ssaprophyticu

sJX9

6643613

32plusmn06seimdash

Ssaprophyticus

AB6

977171

30plusmn10

Ssaprophyticu

sJX9664361306plusmn10

seln

mdashShominisJX5199881303plusmn10

Ssaprophyticu

sEU430992131plusmn03andselomdashShominisJX519988132plusmn10

BioMed Research International 7

O S succinus KC3298241Δ S xylosus AM8827001

O S saprophyticus KJ6991511Δ S saprophyticus AB6977171

Δ S xylosus CP0087241Δ S xylosus KC4565901Δ S saprophyticus KJ9496061Δ S saprophyticus JX4901221Δ S saprophyticus KJ0046231

Δ S xylosus CP0072081Δ S carnosus NR1164341Δ S saprophyticus HQ6995101

O S epidermidis KF0882111O S hominis JX5199881Δ S saprophyticus EU4309921

O S xylosus KF1980801Δ S carnosus KJ8620021Δ S sciuri JX9664361O S saprophyticus HF9372521

04 03 02 01 00

Figure 1 Phylogenetic tree generated from the multiple alignments of the 16S rDNA sequences of CNS strains found in salami usingthe ClustalX 20 software The phylogenetic tree was constructed by using the Mega 60 software and the unweighted pair group method(UPGMA) Bootstrap values ranged from 00 to 04 Strains found in commercial (Δ) or artisanal salami (O)

between these CNS strains Suzuki et al 2012 demonstrateda bootstrap value higher than 90 for interspecies similaritiesbetween S saprophyticus S epidermidis S hominis and Scarnosus

33 Genotypic and Phenotypic Characterization of CNSStrains Twelve distinct combinations of staphylococcalenterotoxins geneswere found in the 15CNS strains compris-ing SEs AndashE GndashJ and also the enterotoxin-like toxins (SElL)KndashR and U (Table 2)

Fifteen strains (79) carried at least one gene encodingenterotoxins in their genomes (Figure 2) The seb and secgenes were the most predominant harbored by 57 ofthe strains followed by sea carried by 50 whereas thesedsehselm genes showed intermediate incidence harboredby 33 of the strains while seiseln and tsH1 were found in27 of strains Finally see selj and selo showed low incidence(13) and seg was carried by only 1 of strains (Table 2)

The relationships between superantigenic toxin geno-types and toxin gene-encoding mobile genetic elementsin CNS strains were evaluated Distinct combinations ofSaPI and plasmids or plasmids and genes of egc operonenterotoxins were found in the CNS strains obtained fromsalami

Four strains presented only the classical enterotoxingenes namely S saprophyticus KJ0046231 and HF9372521S xylosus CP0087241 and S carnosus NR1164341 Anotherstrain S saprophyticus AB6977171 presented only the newlydescribed enterotoxins seh sei and selm and finally 10 strainspresented a combination of classical and newly described

enterotoxin genes in their genomes with at least one of eachenterotoxin type (Table 2)

Previous studies have shown that sea is the most com-mon toxin associated with Staphylococcus food poisoningfollowed by sed and see with SEHand SEI being considered asplaying only a minor role [39] Among the 15 enterotoxigenicstrains found in salami 73 of them harbored at least the seagene or combinations of these 05 genes Strains S carnosusKJ8620021 found in commercial salami and S epidermidisKF6005891 found in artisanal salami carry 04 of these genesThe sea and seb enterotoxin genes are known to occupy thesame locus on the chromosome which may explain whythese enterotoxins are commonly found together in foodpoisoning outbreaks [36] The combination of sea and sebgenes was found in 05 strains S saprophyticus EU4309921 Scarnosus KJ8620021 S hominis JX5199881 S saprophyticusKJ9496061 and S succinus KC3298241 A single strain Scarnosus KJ8620021 showed the combination of sea and seigenes

Additionally some CNS are able to produce TSST-1 aloneor in combination with other enterotoxins Herein 04 of the19 strains (21) S saprophyticus EU4309921 S saprophyti-cus subsp bovis KJ6991511 and S xylosus CP0087241 andKF1980801 were shown to harbor the tstH1 combined withse andor sel enterotoxin genes (Table 2)

The staphylococci enterotoxins genes seg and sei [40] arepart of a chromosomal operon gene cluster (egc) comprisingfive genes designated as selo selm sei seln and seg Twoof the CNS strains determined in the present study Ssaprophyticus EU4309921 and S hominis JX5199881 were

8 BioMed Research International

sed

seb

sec

see

sea

M 2 3 4 51

500bp

(a)

sea

sebsebseb

M 1 2 3 4 5

(b)

sei

sehseg

1M

(c)

seh sehseh

M 1 2 3

(d)

1 2 4M 3 5

(e)

selm

seljseln

selo

21M

(f)

seln

selm

M 1 2

(g)

Figure 2 Uniplex duplex and multiplex PCR screening for the detection of enterotoxin genes in CNS strains from salami (a) Lane M100 bp DNA ladder plus (Fermentas Foster City CA USA) lane 1 S aureus ATCC 29231 harboring sea gene lane 2 S aureus NCTC 10654harboring seb gene lane 3 S aureus ATCC19095 harboring the sec gene lane 4 S aureus ATCC 13563 harboring the sed gene and lane 5 Saureus ATCC 27664 harboring the see gene (b) Lane M 100 bp DNA ladder plus lane 1 Staphylococcus spp lane 2 S carnosus NR116434lane 3 S carnosus KJ862002 lane 4 S carnosus KJ862002 and lane 5 S carnosus KJ8620021 (c) Lane M 100 bp DNA ladder plus lane1 S aureus ATCC 19095 harboring seg seh and sei genes (d) Lane M 100 bp DNA ladder plus lane 1 S saprophyticus AB6977171 lane 2S epidermidis KF 6005891 and lane 3 S sciuri JX9664361 (e) Lane M 100 bp DNA ladder plus lane 1 S xylosus KF1980801 lane 2 SsaprophyticusKJ6991511 (f) LaneM 100 bp DNA ladder plus lane 1 S aureusATCC 27154 harboring selj slem seln and selo genes (g) LaneM 100 bp DNA ladder plus lane 1 S xylosus KF1980801 and lane 2 S saprophyticus subsp bovis KJ6991511

BioMed Research International 9

shown to carry the selm seln and selo genes and the selmand seln sequences were detected in the genome of a singlestrain S saprophyticus subsp bovisKJ6991511 S epidermidisKF6005891 showed a combination of seg and sei genesand 03 strains harbored a single gene sei or selm or selnperhaps due to the high degree of genetic polymorphism inthe chromosomal assembly [41] Indrawattana et al 2013detected the seg-sei-selm-seln-selo of the highly prevalent egclocus was 261 in contrast to this study where 13 CNSpresents the combination of selm seln and selo genes [42]

The CNS strains clustered into groups near the bottom ofthe phylogenetic tree carried the classical enterotoxin geneswhile the species at the top of the tree showed high diversityamong the enterotoxin genes combining the classical and thenewly described genes in their genomes (Figure 1)

To assess the risk of staphylococcal food poisoning theability of the identified strains in harboring the se andsel genes and in expressing and producing enterotoxins invitro was evaluated The mRNA for each enterotoxin genewas evaluated by real time RT-PCR assays and enterotoxincontent was estimated by a sandwich enzyme immunoassayfor the combined detection of Staphylococcus enterotoxins(SET) A B C D and E

The fifteen enterotoxigenic CNS strains were able toexpress the classical enterotoxins SEA seb sec sed and seein concentrations ranging from 03 ngmLminus1 to 14 ngmLminus1as assessed by the in vitro assays (Table 2) The S sapro-phyticus AB6977171 KJ6991511 JX4901221 KJ0046231 andHF9372521 strains S xylosus KF1980801 and S carnosusNR1164341 produced low amount of enterotoxins lower than05 ngmLminus1 S epidermidisKF6005891 S saprophyticus bovisKJ6991511 and S hominis JX5199881 produced intermediateamount of enterotoxins ranging from 07 to 090 ngmLminus1while S sciuri JX9664361 S saprophyticus EU4309921 Scarnosus KJ8620021 S xylosus CP0087241 and S succi-nus KC3298241 produced enterotoxins in concentrationsge10 ngmLminus1

Although the sandwich enzyme immunoassay is consid-ered themost sensitivemethod to detect seandashsee enterotoxinsable to detect 0125 ngmLminus1 differences in the specificity andsensitivity of the assays for the detection of staphylococcalenterotoxins from foods are expected [43] A single strainStaphylococcus spp KF1354451 which harbors both the seaand seb genes was unable to produce seandashsee enterotoxins

The mRNA levels evaluated by the real time RT-PCRassays for enterotoxins were detected in 12 of 15 strains(86) (Table 2) Transcripts for the classical enterotoxinsgenes were detected when S saprophyticus HF9372521 Scarnosus NR1164341 S xylosus CP0087241 and S succinusKC3298241 were assayed Transcripts for newly describedenterotoxins were observed in S saprophyticus JX4901221and AB6977171 S sciuri JX9664361 S saprophyticus subspbovis KJ6991511 and S hominis JX5199881 Transcripts fromboth the classical and newly described enterotoxins weredetected in S saprophyticus EU4309921 and KJ9496061 andS epidermidis KF6005891

No mRNA transcripts were obtained for S sapro-phyticus KJ00462321 and S carnosus KJ8620021 although

enterotoxin production was detected by the enzyme-linkedimmunosorbent tests (Table 2)

The 04 strains that carry tstH1 in their genome Ssaprophyticus EU4309921 and KJ6991511 and S xylosusCP0087241 and KF980801 were not able to produce mRNAfor TSST-1 in the assay conditions

The production of classical enterotoxins in vitro(immunologic test) matched the results shown by real timeRT-PCR assays for the following strains S saprophyticusJX4901221 S sciuri JX9664361 S saprophyticusEU4309921S saprophyticus HF9372521 S saprophyticus subsp bovisKJ6991511 S hominis JX5199881 S epidermidis KF6005891and S succinus KC3298241

Ten strains namely S saprophyticusAB6977171 S sapro-phyticus EU4309921 S saprophyticus HF9372521 S sapro-phyticus KJ6991511 S saprophyticus KJ9496061 S succinusKC3298241 S hominis JX5199881 S xylosus KF1980801and S xylosus CP0087241 and S epidermidis KF6005891expressed mRNA for multiple se andor sel genes There isa differential transcription among these genes where themost frequent among the classical ones were seb and seeseatranscribed by 04 and 02 strains respectively and the mostfrequent among the newly described genes were sei andsehselnselo transcribed by 03 and 02 strains respectively

No mRNA for the sec gene was detected although SsaprophyticusAB6977171 was able to produce the enterotoxinin vitro as detected by the ELISA assays

34 Enterotoxin Gene Homologies between Salami CNSand CPS Strains The nucleotide sequencing of six entero-toxin genesmdashtwo of them sec and see encoding classi-cal enterotoxinsmdashand four of them encoding the newlydescribed genes seg seh selm and seln fromCNS strains wascompared to the enterotoxin genes from S aureus or S pas-teuri two coagulase-positive staphylococci strains (Figure 3)The homology between the CNS and CPS enterotoxin genesvaried from 65 to 98 The homology of sec seg sehselm and seln between CNS from salami and S aureus was98 60 98 65 and 70 respectively The homologybetween see from CNS found in salami and in S pasteuri was98

Although further studies should be performed it seemsthat the sequences encoding enterotoxins can be conservedamong coagulase-negative and coagulase-positive staphy-lococci as shown in the phylogenetic analysis of the 06enterotoxin genes (Figure 4)

35 Antimicrobial Multiresistance of CNS Strains (MRCNS)Another safety hazard associated with CNS strains besidesthe ability of producing enterotoxins in food matrices is theantimicrobial resistance to antimicrobial agents commonlyused to treat staphylococci infections The antimicrobialresistance carried by CNS strains from food matrices can bespread to the population by the consumption of an apparentlysafe food

Among the 19 CNS strains identified in salami 14 showedmultiresistance to antimicrobial agentsThree strains showedthe highest MAR indices 093 and 080 07 strains presented

10 BioMed Research International

lowast lowastlowastlowastlowastlowast lowast lowast lowastlowast lowast lowast lowastlowast lowastlowastlowastlowast lowastlowastlowast lowast

lowastlowast lowast lowast lowastlowastlowastlowast lowast lowast lowastlowast lowast

lowastlowastlowastlowast lowast lowast lowast

lowastlowast lowast lowast lowastlowast lowastlowast lowast lowastlowast lowastlowastlowastlowastlowast lowastlowast lowast lowastlowast lowastlowast

lowastlowast lowastlowastlowast lowast lowast lowast lowast lowast lowastlowastlowastlowastlowast

lowastlowast lowastlowast lowastlowast lowastlowast lowast lowast lowastlowast lowast lowast lowastlowastlowast lowast lowastlowast lowastlowastlowast

lowast lowast lowast lowast lowast lowast lowastlowast lowast lowastlowastlowast lowast lowastlowastlowast lowast lowast lowastlowastlowast lowast lowastlowastlowast lowast

lowast lowast lowast lowastlowastlowastlowast lowast lowastlowastlowast lowast lowast lowastlowastlowast lowast lowast lowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast lowast lowast lowast lowast lowastlowast lowast lowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

Enterotoxin C (sec) GATAAAAATGAATAAGAGTCGATTTATTTCATGCGTAATTTTGATATTCGCACTTATACT------------------------------------------------------------

AGTTCTTTTTACACCCAACGTATTAGCAGAGAGCCAACCAGACCCTACGCCAGATGAGTT------------------------------------------------------------

GCACAAAGCGAGTAAATTCACTGGTTTGATGGAAAATATGAAAGTTTTATATGATGATCA------------------------------------------------------------

TTATGTATCAGCAACTAAAGTTAAGTCTGTAGATAAATTTTTGGCACATGATTTAATTTA------------------------------------------------------------

TAACATTAGTGATAAAAAACTGAAAAATTATGACAAAGTGAAAACAGAGTTATTAAATGA------------------------------------------------------------

AGGTTTAGCAAAGAAGTACAAAGATGAAGTAGTTGATGTGTATGGATCAAATTACTATGT------------------------------------------------------------

AAACTGCTATTTTTCATCCAAAGATAATGTAGGTAAAGTTACAGGTGGCAAAACTTGTAT------------------------------------------------------------

GTATGGAGGAATAACAAAACATGAAGGAAACCACTTTGATAATGGGAACTTACAAAATGT------------------------------------------------------------

ACTTATAAGAGTTTATGAAAATAAAAGAAACACAATTTCTTTTGAAGTGCAAACTGATAA------------------------------------------------------------

GAAAAGTGTAACAGCTCAAGAACTAGACATAAAAGCTAGGAATTTTTTAATTAATAAAAA------------------------------------------------------------

AAATTTGTATGAGTTTAACAGTTCACCATATGAAACAGGATATATAAAATTTATTGAAAA----TTGTATGAGTTTAACAGTTCACCATATGAAACAGGATATATAAAATTTATTGAAAA

TAACGGCAATACTTTTTGGTATGATATGATGCCTGCACCAGGCGATAAGTTTGACCAATCTAACGGCAATACTTTTTGGTATGATATGATGCCTGCACCAGGCGATAAGTTTGACCAATC

TAAATATTTAATGATGTACAACGACAATAAAACGGTTGATTCTAAAAGTGTGAAGATAGATAAATATTTAATGATGTACAACGACAATAAAACAGTTGATTCTAAAAGTGTGAAGATAGA

AGTCCACCTTACAACAAAGAATGGATAATGTTAATCCGATTTTGATATAAAAAGTGAAAGAGTCCACCTTACAACAAAGAATGGATAATGTAATCCGATTATGGACAAGTGTGCTTTTTT

Enterotoxin C TATTAGATATATTTGAAAGGTAAGTACTTCGGTGCTTGCCTTTTTAGGATGCATATATATEnterotoxin C CNS

Enterotoxin CEnterotoxin C CNS

Enterotoxin CEnterotoxin C CNSEnterotoxin CEnterotoxin C CNSEnterotoxin CEnterotoxin C CNSEnterotoxin CEnterotoxin C CNSEnterotoxin CEnterotoxin C CNSEnterotoxin CEnterotoxin C CNSEnterotoxin CEnterotoxin C CNSEnterotoxin CEnterotoxin C CNS

Enterotoxin CEnterotoxin C CNSEnterotoxin CEnterotoxin C CNS

Enterotoxin CEnterotoxin C CNS

Enterotoxin CEnterotoxin C CNS

Enterotoxin CEnterotoxin C CNS

TTTGTGATCGTCGGCCA-----ACCGATACCGTCGTATTCTTGTACCGCGCAAGGCGAAGlowast lowast lowastlowastlowast lowast lowast lowast lowast lowastlowast lowast lowastlowastlowast lowast lowast lowast lowast

Enterotoxin E (see) Enterotoxin E TGCTTTAGGCGGGGTCTTACAGCATATTATCGTAGAAGATGCACAAGCTGCACGTAAAGCS pasteuri TGCTTTAGGCGCATCGTTACAACATGTCATCGTAGAAGATGAAAAAGATGGTCGTCAGGC

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast lowastlowastlowastlowast lowastlowastlowast lowast lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast lowast lowastlowastlowast lowastlowast lowastlowastlowast lowast lowastlowastlowast

Enterotoxin E ATTAGAATTCTTGAAATCAAAACA-AGCAGGACGTGCTACATTTCTACCATTAAATGTCAS pasteuri

Enterotoxin ES pasteuri

GATACAATT-TTTAAAACAACGTAGTTTAGGACGTGCCACATTCCTACCTTTAAATGTTA lowastlowast lowastlowastlowastlowast lowastlowast lowastlowastlowast lowastlowastlowast lowast lowastlowastlowastlowastlowastlowastlowastlowastlowast lowastlowastlowastlowastlowast lowastlowastlowastlowastlowast lowastlowastlowastlowastlowastlowastlowastlowast lowast

TCAAACCTAGAAATATTTCAACCAAGATATATlowast lowastlowastlowastlowast lowastlowastlowast lowastlowastlowastlowastlowast

Enterotoxin G (seg) Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

---------------------------attaagaaatactt--ccattttaatactcttaGGGGATATTTCCGGGAAACCGGGGCTAATACTGGATAACTTTTCTCTCCGAACGAAGAGA

gaatcaacaactttattatctccg--taaat-----atttaaaaacttatatggaacaaaGATTCAACGATGGTCTTGGTTTTCAATTAAGATGAACCCGCGGCGCTTAGCTGGTGGGAC

aggtactagtt-----------------------------------------cttttttaAGGTCACGGCTCACCCAAGCCCCCATGCGTATACGACCTGGAAGGGTGATCGGCCACATT

ggaaataagtcaaaccaaaaacttgtattgttcttttcagtaaat---------------GGGACTGAGACACAGCACAAACTCCTACGGGAGGCAGCAATAGGGAATCTTCCGCAATGG

------------------------------------------------------------CCGAAAGTCTGACGGAGCAACGCCGCGAGACTGAAGAATGTCTTCGGATCGTAAAACTCT

------------------------tttatatatccagattcaaa-----tgcagaaccatGTTGTTAGGGAAGAACAAGTATCGTTCGAATAGGGCGGTACCTTGACGGTACCTAACCAG

caaactcgtatagctttttttct---------ttagtgagccagtgtcttgctttg----AAAGCTTTGCTAACTACCTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCG

-taatctagttcctgaatagtaaccatattcttatttgtagttattgta--aatccaagtGAATTATTGGGCGTAAAGCGCGCGCATGCGGTCTTTTAAATCTGATGTCAAAGCCCACGG

gattgtct----------attgtcgattgttacctgtacagt------------aattaaGCTCAACCGTGGAGGGTCATTGGAAACTGTAAGACTTGGAGTGCAGAAGAGGATAGTGGA

Figure 3 Continued

BioMed Research International 11

lowast lowast lowast lowastlowast lowastlowast lowastlowastlowast lowast lowastlowast lowast lowast lowast lowast

lowast lowast lowastlowastlowast lowastlowast lowast lowastlowastlowast lowast lowastlowast lowastlowastlowast lowast lowastlowast lowastlowast lowast lowast lowastlowast lowast

lowastlowast lowast lowast lowastlowastlowastlowastlowast lowast lowast lowastlowast lowast lowast lowast lowast lowast lowastlowastlowast

lowastlowast lowast lowastlowast lowastlowastlowast lowastlowast lowastlowast lowast lowast lowast lowast lowastlowast lowastlowast lowast lowast lowast lowastlowast

lowastlowast lowastlowastlowastlowast

lowast lowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowast lowast lowastlowastlowastlowast lowast lowastlowast lowastlowast lowast lowastlowastlowastlowast lowast lowastlowast lowast lowast lowastlowastlowastlowastlowastlowastlowast

lowastlowast lowast lowast lowast lowast lowastlowastlowast lowastlowast lowastlowast lowast lowastlowastlowastlowastlowastlowastlowastlowast lowastlowastlowast

lowast lowast lowast lowast lowast lowast lowastlowast lowastlowast lowast lowast lowastlowastlowast lowast lowast lowastlowastlowast lowast lowast

lowastlowast lowastlowastlowastlowastlowastlowast lowastlowast lowastlowastlowast lowastlowast lowast lowastlowast lowastlowast lowastlowastlowast lowast lowast

lowastlowast lowast lowastlowastlowast lowast lowastlowastlowastlowastlowast lowast lowast lowast lowastlowastlowast lowast lowastlowast lowast

lowastlowast lowast lowast lowast lowast lowastlowast lowast lowastlowast lowast lowastlowast lowast lowastlowast lowastlowastlowastlowast lowast lowast lowastlowast

lowast lowastlowastlowast lowast lowastlowast lowast lowast lowastlowast lowast lowastlowast lowast lowastlowastlowastlowastlowastlowast lowast

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

tttatctctttcattttctgaactattaaa----tgtaagaccacc---atacatacaacATTCCACGTGTAACGGTGAAAATGAGTAGAGATGTGGAGGAACACCAGTGGAGAAACGCG

aacctccaaaattttggtttatatccggttc-----------agatttaggtattatacaACTCTCTGGTCTGTTACTGACGCTGAGGCGCGAAAGCGTGGGAGCAAACAGGATTATATA

tgtataaaaatatggaacgccaaaaatgtctacttttttatctttataattgttagctaaCCCCTGGTAGTCCAAGCCGTAAACGATGAGTGCTAGTGTTACAGGGT------------T

ttctgtattttctaattcagttttaacctcattataactcttatactcaattggaaaaatTCCGCCCTTTACTGCTGCAGCTAACG---CATTAG-GCACTCCGCCTGGGGAGTACAACT

taaatcatgagataaaaactgtctagaattaataactcctcttccttcaacaggtggagaGCAA------GGTTGAAACTCAAAGCA----TTGACCGGGGGCC---CGATAAGCTGAGG

cgtataaagattcattacat----tacccatagttcccttattgtttttataatcacttaTACATGGTGTTTAATTCCAAACCACGCCAAGAACCCTCACGAGGTCTTGACATCCTCTGG

ctttattt----------------------------------------------------ACCTCCCTAGAGAATAAGGGCTTCCCCGTCGGGGATGAAATGAACAGGTGGTGCATGGAT

-------agttcgtc---AGTCTTCAGCTCGTGCAT

Enterotoxin H (seh)

------------------------------------------------------------ATCTATGAATATAAATCTTTTAATATTGGTTTAATATAAATATTCCATTTCTAGAAACCT

------------------------------------------------------------ATATTTTATGTTTGTATAAATTCATAAATTGTTCGAAATTTATATATTAAAAGTTGTTTT

------------------------------------------------------------TGTTGTTTTATAATTAAGTTAATGAAATATATTGAGGAGTTTTAAAATGATTAATAAAAT

------------------------------------------------------------TAAAATATTATTTTCGTTTTTAGCATTATTACTTTCATTCACATCATATGCGAAAGCAGA

------------ -----------TTACGATTTAGCTTTAGCTAATGCATATGGTCAATAAGATTTACACGATAAAAGTGAGTTAACAGATTTAGCTTTAGCTAATGCATATGGTCAATA

TAATCACCCATTCATTAAAGAAAATATTAAGAGTGATGAAATAAGTGGAGAAAAAGATTTTAATCACCCATTCATTAAAGAAAATATTAAGAGTGATGAAATAAGTGGAGAAAAAGATTT

AATATTTAGAAATCAAGGTGATAGTGGCAATGATTTGAGAGTAAAGTTTGCAACTGCTGAAATATTTAGAAATCAAGGTGATAGTGGCAATGATTTGAGAGTAAAGTTTGCAACTGCTGA

TTTAGCTCAGAAGTTTAAAAATAAAAATGTAGATATATATGGGGCATCTTTTTATTATAATTTAGCTCAGAAGTTTAAAAATAAAAATGTAGATATATATGGGGCATCTTTTTATTATAA

GTGTGAAAAAATAAATGAAAATATTTCTGAATGTCTATATGGAGGTACAACACTAAATAAGTGTGAAAAAATAAGTGAAAATATTTCTGAATGTCTATATGGAGGTACAACACTAAATAG

TGAAAAATTGGCACAGGAAAGGGTGATTGGTGCTAATGTTTGGGTAGATGGTATTCAAAATGAAAAATTGGCACAGGAAAGGGTGATTGGTGCTAATGTTTGGGTAGATGGTATTCAAAA

AGAAACAGAATTAATACGAACAAATAAGAAAAATGTGACATTGCAAGAATTAGATATAAAAGAAACAGAATTAATACGAACAAATAAGAAAAATGTGACATTGCAAGAATTAGATATAAA

Enterotoxin H CNS GATCAGAAAAATATTGTCCGATAAATATAAAATTTATTATAAAGACAGCGAAATAAGGG-Enterotoxin H

Enterotoxin H CNSEnterotoxin H

Enterotoxin H CNSEnterotoxin HEnterotoxin H CNSEnterotoxin HEnterotoxin H CNSEnterotoxin HEnterotoxin H CNSEnterotoxin HEnterotoxin H CNSEnterotoxin H

Enterotoxin H CNSEnterotoxin H

Enterotoxin H CNSEnterotoxin H

Enterotoxin H CNSEnterotoxin H

Enterotoxin H CNSEnterotoxin H

Enterotoxin H CNSEnterotoxin H

GATCAGAAAAATATTGTCCGATAAATATAAAATTTATTATAAAGACAGCGAAATAAGTAA

Enterotoxin N (seln) TGATTTAGCTTTAGCTAATGCATATGGTCAATAT-AATCACCCATTCATTAAAGAAAA-----TAGATATTTATGGACTGTATTATGGAAATAAATGTGTAGGCTTAACTGAAGAAAAAA

TATTAAGAGTGATGAAATAAGTGGAGAAAAAGATTTAATATTTAGAAATCAAGGTGATAGCATCATGCTTATACGGAGGAGTTAC------GA--TATATGATGGAAATCAATTAGATGA

TGGCAATGATTTGAGAGTAAAG--------------TTTGCAA-CTGCTGATTTAGCTCAAGAGAGAGTTATAGGCGTTAATGTATTTAAAGATGGTATCCAACAAGAAGGTTTTGTTAT

GAAGTTTAAAAATAAAAATGTAGATATATATGGGGCATCTTTTTATTATAAGTGTGAAAAAAAAACTAAAAAGGCTAAAGTAACAGTACAA------GAATTAGATACTAAAGTTCGATT

AATAAGTGAAAATATTTCTGAATGTCTATATGGAGGTACAACACTAAATAATGAAAAATT------------TAAATTAGAAAATTTATATAAAATATACAATAAAGATACCGGTAACAT

Enterotoxin N CNSEnterotoxin N

Enterotoxin N CNSEnterotoxin N

Enterotoxin N CNSEnterotoxin N

Enterotoxin N CNSEnterotoxin N

Enterotoxin N CNSEnterotoxin N

Figure 3 Continued

12 BioMed Research International

lowastlowastlowast lowastlowast lowastlowast lowastlowast lowast lowastlowast lowastlowastlowast lowastlowast lowast lowastlowastlowastlowast

lowastlowast lowast lowastlowast lowast lowast lowastlowast lowastlowastlowastlowastlowast lowast lowast lowast lowastlowast lowast lowast lowastlowast lowast lowast

lowast lowast

lowast lowastlowast lowast lowastlowast lowast lowast lowast lowast lowast lowast lowast lowastlowast lowast lowastlowastlowastlowast lowast

lowast lowastlowast lowast lowast lowastlowast lowast lowastlowastlowast lowast lowastlowast lowast lowastlowast lowastlowast lowast lowast

lowastlowastlowast lowast lowast lowastlowast lowastlowast lowast lowast lowast

lowast lowast lowast lowastlowastlowast lowastlowastlowast lowastlowast lowast lowastlowastlowastlowastlowast lowastlowastlowast lowast

lowastlowast lowast lowast lowastlowast lowastlowastlowast lowastlowast lowast lowast lowastlowastlowastlowast lowastlowast

lowast lowast lowast lowastlowast lowast lowastlowast lowastlowastlowastlowastlowastlowast lowastlowast lowast lowastlowast lowastlowastlowast

lowast lowast lowast lowast lowastlowast lowastlowastlowastlowast lowast lowast lowastlowastlowast lowast lowast lowast lowast lowast

lowast lowast lowast lowast lowastlowastlowast lowast lowast lowastlowast lowastlowast lowast lowastlowastlowast lowast lowastlowastlowast lowast

lowast lowast lowastlowastlowast lowast lowastlowast lowastlowastlowast lowastlowastlowastlowastlowast lowastlowast lowast lowastlowastlowast lowast lowast lowastlowastlowast

lowastlowastlowast lowast lowastlowast lowastlowast lowastlowast lowast lowast lowast lowast lowastlowast lowastlowast lowastlowast lowast lowast

lowast lowast lowast lowast lowastlowast lowast lowast lowastlowast lowastlowast lowast lowastlowastlowast lowastlowastlowastlowast lowast lowastlowastlowastlowastlowast lowast

lowastlowast lowast lowast lowastlowastlowast lowastlowast lowast lowastlowast lowast lowastlowast lowastlowast lowastlowast lowast lowast lowast

lowastlowastlowastlowastlowast lowastlowast lowastlowast lowast lowastlowast lowast lowast lowast lowast lowastlowast lowastlowast

lowast lowast lowastlowast lowastlowast lowastlowastlowastlowastlowastlowast lowastlowast lowast lowast lowast lowast lowast lowast lowast lowast lowast lowast lowast

lowastlowast lowastlowast lowast lowastlowastlowastlowast lowast lowast lowastlowastlowast lowastlowast lowast lowastlowastlowast lowast

lowastlowastlowastlowast lowastlowast lowast lowast lowast lowast lowastlowast lowast lowast lowast lowastlowastlowastlowastlowast lowastlowast lowastlowastlowast lowast

lowast lowastlowastlowast lowastlowastlowastlowastlowast lowast lowast lowast lowastlowast lowast lowast lowastlowast lowast lowastlowastlowast lowastlowast lowast

lowastlowast lowast lowast lowast lowastlowast lowast lowastlowast lowastlowast lowastlowast lowast lowastlowastlowastlowast

ATTAATACGAACAAATAAGAAAAATGTGACATTGCAAGAATTAGATATAAAGATCAGAAA-TTAT----------TATGATTTATATAAC--------------ATAAAAGGTTCAG---

AATATTGTCCGATAAATATAAAATTTATTATAAAGACAGCGAAATAAGGTAAAAGG------TAGGAGCAGAGTTTTTTCAATTTTATAGTGATAATAGAACAGTTAGCTCATCTAATTA

Enterotoxin N CNS -TAAA---Enterotoxin N

lowast lowastlowastlowastlowastlowast lowastlowast lowast lowast lowast lowastlowast lowast lowastlowastlowastlowastlowastlowast lowastlowast lowastlowast

GGCACAGGAAAGGGTGATTGGTGCTAATGTTTGGGTAGATGGTATTCAAAAAGAAACAGAACAA----AAAGGATGCATTTTCTTT-----CATTCTAATAATCATCAAAATCAATCATT

Enterotoxin N CNSEnterotoxin N

Enterotoxin N CNSEnterotoxin N

Enterotoxin N CNSEnterotoxin N

TCATATCG

Enterotoxin M (selm)

ttgctattcgcaaaatcatatcgcaaccgctgatgtcggagtt--ttgaatcttaggaacEnterotoxin M CNSEnterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

--------------AGTATGTTTAAATCCCCGCTCACAGTTATGTTTAACTCTTCCTTTC

tattatggta-gctatccaattgaagaccaccaaagtattaatcctgaaaataatcatctTTTTCACACCGCCCCGTCGGGTGTTGCGCACGGGGTTGTTTAACCCCCCCGGAAAAAATT

ttcgcatcaattagttttttctatggataattcgacagtaacagctgaatttaagaacgtEnterotoxin M CNS

Enterotoxin M CNS

TTCCCTCCCTCCTCCTTACTC---------------CCT----------------CCCCT

tgatgatgtaaaggaattcaaaaatcatgctgtagatgtatatggtctaagttatagtggEnterotoxin M CNS TTTTCCTACAAATCCTTTCTCCCCTCCCGGAGTAGAAGTAAA------------------

atattgtttgaaaaacaaatatatatacggtggagttacattagcaggtgat-tatttagEnterotoxin M CNS -------------------TAAAACAATACCCCTACCACCTTAACAATTTTTTTATTGAG

agaaatctagacgtattcct-attaatctttgggttaatggagaacatcaaactatatctEnterotoxin M CNS CCCCTCCCCCCCCTTCTCATTTTTTCTCTTTGAC-----------GTTCCCCCCATTTCT

actgacaaagtatcaactaataaaa-agttagtaacagctcaagaaattgatactaaattEnterotoxin M CNS GCCAATGCTTAACCCACCCTTAAAGGCCATCGGCACATATTAGACACTCC-----CCACC

aagaagatatctacaagaagaatataatatttatggctttaatgatac---aaataaaggEnterotoxin M CNS CGCCCCCTCTTTTTATTAAGGACACAACTCTTTTTATTTTCCCCCAAGAACTAATTCTGA

a-agaaattatggtaataagtcaaaatttagttctgga---------tttaatgcaggaaEnterotoxin M CNS AAAAAAACGTTCCGCTTAGTTCAGAATTTCCTTTGAGCCTTTTATCCTTTTGTACGCGAA

aaatattatttca--------tttgaatgatggttcatcattttcttatgacttatttga

Enterotoxin M CNS AAACCATCAGCCATTGAATCAGTTATCCCATTGCCCTAACTAACCCAATATCTAATCTAA

tactggaacaggacaagctgaaagtttctt-aaaaatatataatgacaacaaaactgtcgEnterotoxin M CNS TTTTCGCCTACGAACGAATAATAGCTTGTGGCGAAAAATATCCAGGATTATCTACTGTAG

aaact--gaaaaattccatttagatgtagaaatatcttataaggacgaaagttgaagtatEnterotoxin M CNS CGCCTCGCCAGACGTCCTTTGAATTGA--ATATGACTCCTACTCCCCCCCGCACCGTCAA

ttcaatcataacttagtaaaggaaatgccatgaaaaaatttaaatatagtttt---atatEnterotoxin M CNS TTCAAACA---------------GGTGGAAGTTAATACTGAATAGTAGGTCTTCTTCCCG

tagtttttatattactttttaacattaaagatcttacgtatgct---caaggtgatattgEnterotoxin M CNS TGCTATTCTTAGAACTTTTAAATTATGTAAACCCAATTTTTAGTGCACTCTTTCTCCCTC

gtg-taggtaacttaagaaatttctatacaaaacatgattatatagatttaaaaggcgtcEnterotoxin M CNS GTTACTCTTAGATGGAGAAGCGTTAAGACAGAAGCTTTGTAT-------------TCCCA

acagataaaaa-cctacctattgcaaatcaacttgaattttcaac-----aggt----acEnterotoxin M CNS GCAGAGTGCAACCTTTCACAATGAAGTTTTTATCTAATTTACACGCCTTTAGGCTCCAAA

caatgatttgatctcagaatctaataattgggacgaaataagtaaatttaaaggaaagaaEnterotoxin M CNS TACGTATTAAATCTCCTCCTGT--TTATCTGACCCTAAG-ATAAAACACGAAAAATTATG

actggatatttttggcattgattataatggtccttgtaaatctaaatacatgtatggaggEnterotoxin M CNS CGTGCCCACTCATCCTAT-------TACTTTCATTATATCTATAAAA-------------

Figure 3 Alignment of the enterotoxin gene sequences found in coagulase-negative staphylococci (CNS) and in coagulase-positivestaphylococci (CPS)

BioMed Research International 13

Enterotoxin (seg) S aureus AY1587031

Enterotoxin (sec) CNS

Enterotoxin (see) S pasteuri NC0227371

Enterotoxin (see) CNS

Enterotoxin (seh) CNS

Enterotoxin (selm) S aureus AF2857601

Enterotoxin (seln) CNS

Enterotoxin (seh) S aureus AJ9375481

Enterotoxin (seln) S aureus AY1587031

Enterotoxin (sec) S aureus X516611

Enterotoxin (selm) CNS

Enterotoxin (seg) CNS

150 100

100

100

100

100

99

98

96

97

72

50 0

Figure 4 Phylogenetic tree generated from the multiple alignments of the enterotoxin sequences of CNS and CPS strains using the ClustalX20 software package The phylogenetic tree was constructed using the Mega 60 software and the unweighted pair group method (UPGMA)

MAR indices varying from 066 to 046 and the remaining04 strains showed MAR indices le026 (Table 3)

The 14 MRCNS strains were resistant to 120573-lactams(oxacillin penicillin andor cefoxitin) and to vancomycincorresponding to 73 of the total CNS strains identifiedwhile 09 strains (64) showed resistance to tetracycline andgentamicin 08 strains (57) were resistant to neomycinerythromycin and chloramphenicol 07 strains (50) wereresistant to sulfamethoprim 05 strains (36) were resistantto linezolid 03 strains (21) were resistant to rifampicin 02strains (14) were resistant to ciprofloxacin and cefepimeand 01 strain (7) was resistant to clindamycin (Table 3)

The multiresistance of CNS strains demonstrated hereinis consistent with previous studies on coagulase-negativeand coagulase-positive staphylococci that have found severalresistant and multiresistant Staphylococcus aureus strains inraw milk meat and fermented meat products [43]

Surprisingly the resistance to chloramphenicol is verysimilar to that estimated for MRCNS strains isolated fromhuman clinical samples Indeed there is no direct correla-tion between the researched features and the origin of thestaphylococci strains since different virulence factors arewidespread such as antibiotic resistance [44] reinforcing thefact that the ability of foodmatrices strains to produce SE andSEl and their multiresistance character must be consideredwhen evaluating the safety hazards of food poisoning

S epidermidis KF6005891 and S hominis JX5199881showed the highestMAR indexes (093 and 080 resp)Thesestrains found in artisanal salami are homologous to strainsisolated from human skin microbiota and should be carefullyconsidered among the potential pathogenic staphylococcifound in food from animal origins which can be caused bycontamination by poor hygienic conditions during salamimanufacturing

Depending on the conditions some species of coagulase-negative staphylococci can present health risks since theyhave shown resistance to antibiotics of therapeutic impor-tance such as beta lactams [15] However multiresistantstrains like S carnosusKJ18620021 and S xylosusCP0072081and CP0087241 were intentionally introduced in the foodmatrix since they are part of the culture starter used inItalian-salami manufacturing As previously discussed foodcan be a reservoir of multiresistant microorganisms that canspread by the consumption of an apparently safe food Dueto the intensive and indiscriminate use of antibiotics forhuman and veterinarian therapeutic purposes multiresistantstaphylococci strains are being selected and reproduced infood matrices [45]

The cefoxitin and oxacillin disk diffusion tests are rec-ommended for determining the resistance and susceptibilitybreakpoint for MRSA surveillance cultures [46] In thisstudy there was a good correlation between the disc testzone diameters for oxacillin and cefoxitin (Table 3) TheMICs for ampicillin andmethicillinoxacillincefoxitin for allsusceptible CNS strainswere determined by the diffusion disctest using 003 to 2mgmLminus1 of each antimicrobial agent

S saprophyticus HF9372521 showed an MIC of003mgmLminus1 formethicillin strain S carnosusKJ8620021 Sxylosus strains CP0087241 and CP0072081 S saprophyticusJX4901221 S hominis JX5199881 and S succinusKC3298241 were resistant to 006mgmLminus1 and two strainsS saprophyticus KJ6991511 and S saprophyticus KJ0046231showed an MIC of 05mgmLminus1 (Table 4) Two of themethicillin-resistant strains S saprophyticus KJ0046231 andS xylosus CP0072081 harbor no enterotoxin gene

Five strains S saprophyticus JX4901221 S carnosusKJ8620021 S xylosusCP0087241 S xylosusKF1980801 andS saprophyticus KJ0046231 showed an MIC for ampicillin

14 BioMed Research International

Table 3 Multiple resistance to antimicrobial as found in CNS strains from salami

Salami origin CNS strains Antimicrobial agent resistance Multiple antimicrobialresistance (MAR) indexlowast

Commercial Staphylococcus spp KF1354451 CIP CLO CPM GEN NEO OXA PENSXT TET and VAN 066

Commercial S carnosus KJ8620021 CIP GEN LZD NEO OXA SXT andTET 046

Commercial S saprophyticus AB6977171 CFO CLO ERI PEN OXA TET andVAN 046

Commercial S saprophyticus EU4309921 CFO CLO ERI GEN LZD NEO OXAPEN SXT TET and VAN 080

Commercial S saprophyticus JX4901221 CFO CLO ERI GEN NEO PEN OXASXT TET and VAN 066

Commercial S saprophyticus KJ0046231 CFO OXA and PEN 020

Commercial S sciuri JX9664361 CFO CLO GEN NEO OXA PEN andTET 046

Commercial S xylosus CP0072081 OXA LZD PEN and VAN 026

Commercial S xylosus CP0087241 CFO CLO ERI GEN NEO OXA PENTET and VAN 060

Artisanal S epidermidis KF6005891 CFO CLI CLO CPM ERI GEN LZDNEO OXA PEN RIF SXT and TET 093

Artisanal S hominis JX5199881 CIP CPM CPO ERI GEN LZD NEOOXA PEN RIF SXT and VAN 080

Artisanal S saprophyticusHF9372521 CFO OXA PEN and SXT 026Artisanal S saprophyticus KJ6991511 OXA PEN and VAN 020

Artisanal S succinus KC3298241 CFO CLO ERI GEN OXA PEN RIFSXT TET and VAN 066

Artisanal S xylosus KF1980801 CFO CLO ERI GEN NEO OXA PENTET and VAN 060

lowastTheMAR index of an isolate is defined as 119886119887 where 119886 represents the number of antimicrobials to which the isolate was resistant and 119887 represents the numberof antimicrobials to which the isolate was subjectedS aureus strains ATCC WB81 (sea) ATCC 13563 (sed) and ATCC 27664 (see) showing a MAR index of 05 and S aureus strains ATCC14458 (seb) andATCCWB72 (sec) and S xylosus ATCC 29971 showing a MAR index of 03 were used as reference strainsCPM cefepime CFO cefoxitin CLO chloramphenicol CIP ciprofloxacin CLI clindamycin ERI erythromycin GEN gentamycin NEO neomycin LZDlinezolid RIF rifampicin TET tetracycline OXA oxacillin PEN penicillin SXT sulfamethoprim and VAN vancomycin

Table 4 Minimal inhibitory concentration (MIC) of compounds used in antimicrobial therapy against staphylococci infections

Salami origin GenBank accession number and similarity () MIC mgmLminus1

Methicillin Ampicillin Vancomycin Linezolid

Commercial

S carnosus KJ8620021 006 003 mdash mdashS xylosus CP0087241 006 003 006 mdash

S saprophyticus JX4901221 006 003 05 mdashS saprophyticus AB6977171 mdash mdash 05 mdashS saprophyticus EU4309921 mdash mdash 025 0125

S succinus KC3298241 006 025 003 mdash

Artisanal

S hominis JX5199881 006 05 mdash mdashS saprophyticus HF9372521 003 mdash mdash mdash

S xylosus KF1980801 003 05 mdashS saprophyticus KJ6991511 05 025 003 mdashS saprophyticus KJ0046231 05 003 003 mdash

S xylosus CP0072081 006 mdash mdash 025Strains S epidermidis KF6005891 and Staphylococcus spp KF1354451 were not susceptible to the antimicrobial concentrations tested in the present study

BioMed Research International 15

of 003mgmLminus1 two strains S succinus KC3298241 and Ssaprophyticus KJ6991511 showed an MIC of 025mgmLminus1and S hominis JX5199881 showed an MIC of 05mgmLminus1(Table 4) S saprophyticus KJ0046231 also showed an MICof 003mgmLminus1 but does not harbor enterotoxin genes It isimportant to note that most of the CNS strains that showedresistance to ampicillin were also resistant to methicillin ashighlighted by the MIC tests

Five strains S saprophyticus HF9372521 S saprophyti-cus KJ6991511 S saprophyticus JX4901221 S saprophyticusKJ0046231 and S succinus KC3298241 showed an MICfor vancomycin of 003mgmLminus1 one strain S xylosusCP0087241 showed an MIC of 006mgmLminus1 one strain Ssaprophyticus EU4309921 showed an MIC of 025mgmLminus1and two strains S xylosus KF1980801 and S saprophyticusAB6977171 showed an MIC of 05mgmLminus1 (Table 4)

Additionally five CNS strains S xylosus CP0087241S saprophyticus JX 4901221 S succinus KC3298241 Ssaprophyticus KJ6991511 and S saprophyticus KJ0046231showed resistance tomethicillin as demonstrated by theMICdeterminations (Table 4)

Previous studies have demonstrated that linezolid isactive against Gram-positive bacteria including methicillin-resistant staphylococci [47] In the present study 05 strainsshowed linezolid resistance in the disc diffusion test but itwas impossible to establish MIC values for S epidermidisKF6005891 Staphylococcus hominis JX5199881 and Staphy-lococcus carnosus KJ8620021The remaining strains S sapro-phyticus EU4309921 and S xylosus CP0072081 presentedan MIC for linezolid of 0125mgmLminus1 and 025mgmLminus1respectively

The MIC of 003mgmLminus1 for penicillin is in accordancewith the previous values estimated for resistant S aureusstrains found in several food matrices such as meat dairyproducts and ready-to-eat food [48]

There is still a lack of information on the antimicro-bial resistance of staphylococci strains from food matricesalthough they are the most worrisome vehicles of dissemi-nation of antibiotic-resistant pathogens [49]

The high resistance found for salami staphylococci strainscan be ascribed to their inappropriate use as growth promot-ers of antimicrobial agents like oxacillin vancomycin chlo-ramphenicol neomycin and erythromycin which are com-monly used in veterinary medicine to treat infections [50]Antimicrobial therapy of infections staphylococci is basedon results of susceptibility tests in vitro [51] Methicillin-resistant coagulase-negative Staphylococcus spp found inready-to-eat products such as meats fish and dairy productsalso offer risks and although they are not classical foodpoisoning bacteria their presence in food offers significantrisks to public health due to the possible spread of antibioticresistance [52]

The standardization of salami quality should includediagnostic methods to screen and quantify the presence ofclassical and newly described enterotoxins directly in thefood matrices as a routine procedure to be conducted by themeat product industry in Brazil

The safety of salami consumption could also be enhancedby the inclusion of amicrobial barrier such as the inclusion ofprobiotic strains producing natural antibiotics or competitiveflora or even the addition of natural bioagents against spoilageor pathogenic microorganisms

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors acknowledge the financial support fromFundacao Carlos Chagas Filho de Amparo a Pesquisa doEstado do Rio de Janeiro (FAPERJ Rio de Janeiro Brazil)Conselho Nacional de Desenvolvimento Cientıfico eTecnologico (CNPq Brasılia Brazil) and Coordenacao deAperfeicoamento de Pessoal de Nıvel Superior (CAPESBrasılia Brazil)

References

[1] ICMSF International Comission on Microbiological Specifica-tions for FoodsMicroorganisms in FoodsTheir Significance andMethods of Enumerations University of Toronto Press TorontoCanada 3rd edition 1983

[2] J F Veras L S Carmo L C Tong et al ldquoA study of theenterotoxigenicity of coagulase-negative and coagulase-positivestaphylococcal isolates from food poisoning outbreaks inMinasGerais Brazilrdquo International Journal of Infectious Diseases vol12 no 4 pp 410ndash415 2008

[3] K Omoe D-L Hu H Takahashi-Omoe A Nakane and KShinagawa ldquoComprehensive analysis of classical and newlydescribed staphylococcal superantigenic toxin genes in Staphy-lococcus aureus isolatesrdquo FEMS Microbiology Letters vol 246no 2 pp 191ndash198 2005

[4] J Kadariya T C Smith and D Thapaliya ldquoStaphylococcusaureus and staphylococcal food-borne disease an ongoing chal-lenge in public healthrdquoBioMedResearch International vol 2014Article ID 827965 9 pages 2014

[5] KOmoeD-LHuH KOno et al ldquoEmetic potentials of newlyidentified staphylococcal enterotoxin-like toxinsrdquo Infection andImmunity vol 81 no 10 pp 3627ndash3631 2013

[6] J K McCormick J M Yarwood and P M SchlievertldquoToxic shock syndrome and bacterial superantigens an updaterdquoAnnual Review of Microbiology vol 55 pp 77ndash104 2001

[7] T Baba F Takeuchi M Kuroda et al ldquoGenome and virulencedeterminants of high virulence community-acquired MRSArdquoThe Lancet vol 359 no 9320 pp 1819ndash1827 2002

[8] Associacao Brasileira de Industria Produtora e Exportadorada Carne Suınas (ABIPECS) ldquoStatistics slaughter ranking2000ndash2014rdquo 2014 httpabpa-brcombrsetoressuinoculturapublicacoesrelatorios-anuais

[9] N Zdolec M Hadziosmanovic L Kozacinski et al ldquoMicrobialand physicochemical succession in fermented sausages pro-duced with bacteriocinogenic culture of Lactobacillus sakei andsemi-purified bacteriocin mesenterocin Yrdquo Meat Science vol80 no 2 pp 480ndash487 2008

16 BioMed Research International

[10] F Ravyts L D Vuyst and F Leroy ldquoBacterial diversity andfunctionalities in food fermentationsrdquo Engineering in Life Sci-ences vol 12 no 4 pp 356ndash367 2012

[11] TOhlsson andN BengtssonTheHurdle ConceptMinimal Pro-cessing Technologies in the Food IndustryWoodhead Publishing2002

[12] S Bernardi B B Golineli and C J Contreras-CastilloldquoRevisao aspectos da aplicacao de culturas starter na producaode embutidos carneos fermentadosrdquo Brazilian Journal of FoodTechnology vol 13 no 2 pp 133ndash140 2010

[13] F Irlinger ldquoSafety assessment of dairy microorganismscoagulase-negative staphylococcirdquo International Journal of FoodMicrobiology vol 126 no 3 pp 302ndash310 2008

[14] B Ghebremedhin F Layer W Konig and B Konig ldquoGeneticclassification and distinguishing of Staphylococcus species basedon different partial gap 16S rRNA hsp60 rpoB sodA and tufgene sequencesrdquo Journal of Clinical Microbiology vol 46 no 3pp 1019ndash1025 2008

[15] I Babic K Markov D Kovacevic et al ldquoIdentification andcharacterization of potential autochthonous starter culturesfrom aCroatian lsquobrandrsquo product lsquoslavonski kulenrsquordquoMeat Sciencevol 88 no 3 pp 517ndash524 2011

[16] MMehrotra GWang andWM Johnson ldquoMultiplex PCR forthe detection of genes for Staphylococcus aureus enterotoxinsexfoliative toxins toxic shock syndrome toxin and methicillinresistancerdquo Journal of Clinical Microbiology vol 38 no 3 pp1032ndash1041 2000

[17] WM Johnson SD Tyler E P Ewan F EAshtonDR Pollardand K R Rozee ldquoDetection of genes for enterotoxins exfolia-tive toxins and toxic shock syndrome toxin 1 in Staphylococcusaureus by the polymerase chain reactionrdquo Journal of ClinicalMicrobiology vol 29 no 3 pp 426ndash430 1991

[18] J Mclauchlin G L Narayanan VMithani andG OrsquoNeill ldquoThedetection of enterotoxins and toxic shock syndrome toxin genesin Staphylococcus aureus by polymerase chain reactionrdquo Journalof Food Protection vol 63 no 4 pp 479ndash488 2000

[19] J P Rosec and O Gigaud ldquoStaphylococcal enterotoxin genes ofclassical and new types detected by PCR in Francerdquo Interna-tional Journal of Food Microbiology vol 77 no 1-2 pp 61ndash702002

[20] K Omoe D-L Hu H Takahashi-Omoe A Nakane andK Shinagawa ldquoIdentification and characterization of a newstaphylococcal enterotoxin-related putative toxin encoded bytwo kinds of plasmidsrdquo Infection and Immunity vol 71 no 10pp 6088ndash6094 2003

[21] N Sergeev D Volokhov V Chizhikov and A Rasooly ldquoSimul-taneous analysis of multiple staphylococcal enterotoxin genesby an oligonucleotide microarray assayrdquo Journal of ClinicalMicrobiology vol 42 no 5 pp 2134ndash2143 2004

[22] S Holtfreter D Grumann M Schmudde et al ldquoClonal distri-bution of superantigen genes in clinical Staphylococcus aureusisolatesrdquo Journal of Clinical Microbiology vol 45 no 8 pp2669ndash2680 2007

[23] W J Mason J S Blevins K Beenken NWibowo N Ojha andM S Smeltzer ldquoMultiplex PCR protocol for the diagnosis ofstaphylococcal infectionrdquo Journal of Clinical Microbiology vol39 no 9 pp 3332ndash3338 2001

[24] E Rahimi and H G Safai ldquoDetection of classical enterotoxinsof Staphylococcus aureus strains isolated frombovine subclinicalmastitis in Isfahan Iranrdquo Veterinary Microbiology vol 141 no3-4 pp 393ndash394 2010

[25] M A Larkin G Blackshields N P Brown et al ldquoClustalW andClustal X version 20rdquo Bioinformatics vol 23 no 21 pp 2947ndash2948 2007

[26] P H A Sneath and R R Sokal Unweighted Pair Group Methodwith Arithmetic Mean Numerical Taxonomy W H FreemanSan Francisco Calif USA 1973

[27] NCCLS ldquoPerformance standards for antimicrobial susceptibil-ity testing 14th informational supplementrdquo Tech Rep M 100-S14 NCCLS 2004

[28] S Corbiere Morot-Bizot S Leroy and R Talon ldquoMonitoringof staphylococcal starters in two French processing plantsmanufacturing dry fermented sausagesrdquo Journal of AppliedMicrobiology vol 102 no 1 pp 238ndash244 2007

[29] R Talon S Leroy I Lebert et al ldquoSafety improvement andpreservation of typical sensory qualities of traditional dryfermented sausages using autochthonous starter culturesrdquo Inter-national Journal of Food Microbiology vol 126 no 1-2 pp 227ndash234 2008

[30] G Mauriello A Casaburi G Blaiotta and F Villani ldquoIsolationand technological properties of coagulase negative staphylo-cocci from fermented sausages of Southern ItalyrdquoMeat Sciencevol 67 no 1 pp 149ndash158 2004

[31] M Janssens N Myter L De Vuyst and F Leroy ldquoSpeciesdiversity and metabolic impact of the microbiota are low inspontaneously acidified Belgian sausages with an added starterculture of Staphylococcus carnosusrdquo Food Microbiology vol 29no 2 pp 167ndash177 2012

[32] G Blaiotta D Ercolini G Mauriello G Salzano and F VillanildquoRapid and reliable identification of Staphylococcus equorum bya species-specific PCR assay targeting the sodA generdquo Systematicand Applied Microbiology vol 27 no 6 pp 696ndash702 2004

[33] L Kozacinski E Drosinos F Caklovica L Cocolin J Gasparik-Reichardt and S Veskovic ldquoInvestigation of microbial associa-tion of traditionally fermented sausagesrdquo Food Technology andBiotechnology vol 46 no 1 pp 93ndash106 2008

[34] S Leroy P Giammarinaro J-P Chacornac I Lebert and RTalon ldquoBiodiversity of indigenous staphylococci of naturallyfermented dry sausages and manufacturing environments ofsmall-scale processing unitsrdquo Food Microbiology vol 27 no 2pp 294ndash301 2010

[35] E Coton M-H Desmonts S Leroy et al ldquoBiodiversity ofCoagulase-Negative Staphylococci in French cheeses dry fer-mented sausages processing environments and clinical sam-plesrdquo International Journal of Food Microbiology vol 137 no 2-3 pp 221ndash229 2010

[36] S Hosseinzadeh and H D Saei ldquoStaphylococcal species associ-ated with bovinemastitis in the NorthWest of Iran emerging ofcoagulase-negative staphylococcirdquo International Journal of Vet-erinary Science and Medicine vol 2 no 1 pp 27ndash34 2014

[37] A Piette and G Verschraegen ldquoRole of coagulase-negativestaphylococci in human diseaserdquo Veterinary Microbiology vol134 no 1-2 pp 45ndash54 2009

[38] T M Moura F S Campos P A DrsquoAzevedo et al ldquoPrevalenceof enterotoxin-encoding genes and antimicrobial resistancein coagulase-negative and coagulase-positive Staphylococcusisolates from black pudding in Southern Brazilrdquo Revista daSociedade Brasileira deMedicina Tropical vol 45 no 5 pp 579ndash585 2012

[39] H I Suzuki T Lefebure P P Bitar and M J StanhopeldquoComparative genomic analysis of the genus Staphylococcusincluding Staphylococcus aureus and its newly described sister

BioMed Research International 17

species Staphylococcus simiaerdquo BMC Genomics vol 13 article38 2012

[40] R P Lamers G Muthukrishnan T A Castoe S Tafur A MCole and C L Parkinson ldquoPhylogenetic relationships amongStaphylococcus species and refinement of cluster groups basedon multilocus datardquo BMC Evolutionary Biology vol 12 article171 2012

[41] I V Pinchuk E J Beswick and V E Reyes ldquoStaphylococcalenterotoxinsrdquo Toxins vol 2 no 8 pp 2177ndash2197 2010

[42] S R Monday and G A Bohach ldquoGenes encoding staphylo-coccal enterotoxins G and I are linked and separated by DNArelated to other staphylococcal enterotoxinsrdquo Journal of NaturalToxins vol 10 no 1 pp 1ndash8 2001

[43] D Y Thomas S Jarraud B Lemercier et al ldquoStaphylococcalenterotoxin-like toxins U2 and V two new staphylococcalsuperantigens arising from recombination within the entero-toxin gene clusterrdquo Infection and Immunity vol 74 pp 4724ndash4734 2006

[44] N Indrawattana O Sungkhachat N Sookrung et al ldquoStaphylo-coccus aureus clinical isolates antibiotic susceptibility molecu-lar characteristics and ability to form biofilmrdquo BioMed ResearchInternational vol 2013 Article ID 314654 11 pages 2013

[45] V Pereira C Lopes A Castro J Silva P Gibbs and P TeixeiraldquoCharacterization for enterotoxin production virulence factorsand antibiotic susceptibility of Staphylococcus aureus isolatesfrom various foods in Portugalrdquo Food Microbiology vol 26 no3 pp 278ndash282 2009

[46] I Anacarso C Condo C Sabia and et al ldquoAntimicrobialresistance and other related virulence factors in staphylococcusspp isolated from food environmental and humans in ItalyrdquoUniversal Journal Microbiology Research vol 1 no 1 pp 1ndash92013

[47] B Martın M Garriga M Hugas S Bover-Cid M T Veciana-Nogues and T Aymerich ldquoMolecular technological and safetycharacterization of Gram-positive catalase-positive cocci fromslightly fermented sausagesrdquo International Journal of FoodMicrobiology vol 107 no 2 pp 148ndash158 2006

[48] NM Broekema T Van Tam T AMonson S AMarshall andD M Warshauer ldquoComparison of cefoxitin and oxacillin diskdiffusionmethods for detection ofmecA-mediated resistance inStaphylococcus aureus in a large-scale studyrdquo Journal of ClinicalMicrobiology vol 47 no 1 pp 217ndash219 2009

[49] E Cercenado F Garcıa-Garrote and E Bouza ldquoIn vitro activityof linezolid against multiply resistant Gram-positive clinicalisolatesrdquo Journal of Antimicrobial Chemotherapy vol 47 no 1pp 77ndash81 2001

[50] A Aydin K Muratoglu M Sudagidan K Bostan B Okukluand SHarsa ldquoPrevalence and antibiotic resistance of foodborneStaphylococcus aureus isolates in Turkeyrdquo Foodborne Pathogensand Disease vol 8 no 1 pp 63ndash69 2011

[51] D Sergelidis A Abrahim T Papadopoulos et al ldquoIsolation ofmethicillin-resistant Staphylococcus spp from ready-to-eat fishproductsrdquo Letters in Applied Microbiology vol 59 no 5 pp500ndash506 2014

[52] I PhillipsM Casewell T Cox et al ldquoDoes the use of antibioticsin food animals pose a risk to human health A critical reviewof published datardquo Journal of Antimicrobial Chemotherapy vol53 no 1 pp 28ndash52 2004

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Page 4: Research Article Safety Evaluation of the Coagulase ...downloads.hindawi.com/journals/bmri/2015/483548.pdf · Research Article Safety Evaluation of the Coagulase-Negative Staphylococci

4 BioMed Research International

DNA templates from the following reference strains wereused S aureusATCC 29231 (sea) S aureusATCC 14458 (sebtstH selk selq selr and selu) S aureus ATCC 19095 (sec segseh and sei) S aureus ATCC 13563 (sed) S aureus ATCC27664 (see) and S aureus ATCC 27154 (selj selm seln andselo) and S xylosus ATCC 29971

24 Enterotoxin ExpressionAssays Theobserved strainswerecultured aerobically overnight in 10mL Brain Heart InfusionBroth (BD BBL Le Pont de Claix France) at 37∘C for 72 hBacteria supernatants were collected by centrifugation at4000timesg for 10min and used for the detection of sea seb secsed and see by an ELISA assay using a commercial detectionkit (RIDASCREEN SET A B C D E Art number R4101 R-Biopharm AG Germany) The assay was performed accord-ing to the manufacturerrsquos recommendation and as describedelsewhere [16]Themean lower limit of detection of the assaywas 025 ngmLminus1 The threshold is defined as the average ODof two negative controls plus 015 a constant established bythe kit Samples containing SEs showed absorbance valuesequal to or greater than the threshold value All experimentswere performed in duplicate

25 Real Time RT-PCR Assays Total RNA was extracted byusing the QIAGEN RiboPure Bacteria kit (Life TechnologiesCarlsbad California USA) following the manufacturerrsquosinstructions and quantified using the Qubit fluorometer(Invitrogen Grand Island New York USA) and Qubit assaykits The cDNA synthesis was performed by using the HighCapacity cDNA Reverse Transcription Kit (Applied Biosys-tems California USA) and the ABI PRISM 7500 Fast RT-PCR system (Applied Biosystems California USA) Sampleswere plated in triplicate in 96-well plates as follows 12 120583Lof the SYBR Green PCR Master Mix 1 120583L of primer mix(sea seb sec sed and see) and 45 120583L of the cDNA ultrapurewater in each well Amplification was performed under thefollowing conditions 95∘C for 15min 40 cycles at 95∘Cfor 15 s 54∘C for 30s and 72∘C for 30 s The dissociationcurve was performed at 95∘C for 15 sec 54∘C for 30 sec and95∘C for 15 sec CT means the standard deviations and thecDNA semiquantification were calculated using the Graph-Pad Prism 5 software package Calibration curves based onfive points were constructed in triplicate corresponding toserial dilutions (1 1 10 1 100 1 1000 and 1 10000) from100 ng of a DNA template stock solution

26 16S rDNA Sequencing Amplification of the V5 regionof 16S rDNA fragment was performed using 50 ng of DNAtemplates from the 65 strains found in the salami samplesPCR was performed under the following conditions 95∘Cfor 10min followed by 30 cycles at 95∘C for 30 s 60∘Cfor 30 s and 72∘C for 45 s and a final extension at 72∘Cfor 10min PCR products were purified using the PCRDNA Purification Kit (Applied Biosystems California USA)and sequenced using 20 ng purified DNA and 13 120583L ofprimer sets in a final volume of 20120583L After amplifica-tion products were purified according to the protocol ofthe BigDye Terminator Purification X Kit (Applied Biosys-tems California USA) and sequenced on a 3130 sequencer

GeneticAnalyzer (Applied Biosystems California USA)Thesequences were compared to the 16S rDNA gene sequencesof Staphylococcus species available at the GenBank database(httpwwwncbinlmnihgovGenbankindexhtml) Multi-ple sequence alignments were performed using ClustalW (Kyoto University Bioinformatics Center httpwwwgenomejptoolsclustalw)

27 Sequencing of Enterotoxin PCR Products PCR productswere purified using the PCR DNA Purification Kit (AppliedBiosystems California USA) and sequenced using 10 ngof purified DNA and 32 pmoles of each primer set in afinal volume of 20 120583L After amplification in the sameconditions as the PCR step (Section 233) products werepurified according to the BigDye Terminator PurificationX Kit protocol (Applied Biosystems California USA)and sequenced on a 3130 sequencer Genetic Analyzer(Applied Biosystems California USA) The sequences werecompared to Staphylococcus aureus and Staphylococcuspasteuri gene sequences available at the GenBank database(httpwwwncbinlmnihgovGenbankindexhtml) Multi-ple sequence alignments were performed using ClustalW (Kyoto University Bioinformatics Center httpwwwgenomejptoolsclustalw)

28 Phylogenetic Analyses Phylogenetic relationshipsbetween the CNS strains were performed by sequencealignments using the Clustal X 20 software package [24]The phylogenetic trees were constructed using the softwareMega 60 and UPGMAmethods [25]

29 Antibiotic Susceptibility Tests An inoculumof each strainequivalent to a 05 McFarland scale was swabbed onto aMueller-Hinton agar plate (BD BBL Franklin Lakes NewJersey USA) and the antibiotic disc was then placed on theplate followed by overnight incubation at 37∘CThe inhibitionzone was interpreted according to the Clinical LaboratoryStandard (CLSI) Guidelines formerly known as the NationalCommittee for Clinical Laboratory Standards The testedantibioticswere penicillinG (10U) oxacillin (1120583g) neomycin(30 120583g) sulfamethoprim (5 120583g) clindamycin (2120583g) gen-tamicin (10 120583g) cefoxitin (30 120583g) rifampicin (5 120583g) ery-thromycin (15 120583g) tetracycline (30 120583g) vancomycin (30120583g)ciprofloxacin (5120583g) sulfazothrim (23120583g) cefepime (30 120583g)linezolid (30 120583g) and chloramphenicol (30 120583g)

210 Minimal Inhibitory Concentration (MIC) Determina-tions The MICs of vancomycin linezolid methicillin andampicillin were determined by the macrodilution brothmethod based on CLSI recommendations using in-house-prepared panels [26] Antibiotic concentrations of 003 0060125 025 05 10 and 20mgmLminus1 were tested One mLof broth was transferred to the tubes and 100 120583L of thebacteria suspension was adjusted to 106 CFUmL in saline085 according to a 05 McFarland scale and transferredto tubes containing 1mL of each antimicrobial Strains weregrown in Mueller-Hinton broth (BD BBL Franklin LakesNew Jersey USA) and the MIC was estimated as the lowest

BioMed Research International 5

antibiotic concentration that inhibits visible growth after 24 h[26]

3 Results and Discussion

31 Isolation and Identification of Coagulase-Negative Strainsfrom Salami Sixty-five presumable coagulase-negativestaphylococci microorganisms from salami were isolated bycolony morphology coagulase slide test subsequent tubetest and biochemical tests The sequencing of the V5 regionof the 16S rDNA fragment of the strains was discriminativeenough to differentiate the Staphylococcus isolated fromsalami at the subspecies level with the exception of 1 strainidentified up to the Staphylococcus spp genus

Nineteen distinct strains were identified as CNS in salamiwith 08 of 19 (42) identified as S saprophyticus thepredominant species followed by 05 strains of S xylosus(26) 02 strains of S carnosus (11) and 01 strain of eachof the following species S succinus S sciuri S epidermidisand S hominis (5 each) (Table 2)

The 08 S saprophyticus strains were identified asKJ6991511 AB6977171 JX4901221 KJ0046231 andHQ6995101 EU4309921 HF9372521 and KJ9496061 withthe latter two and S sciuri JX9664361 being homologous(96ndash98) to strains from the environment Five S xylosusstrains CP0072081 KF1980801 CP0087241 AM8827001and KC4565901 and a single S succinus strain KC3298241were identified The last three S xylosus strains and theS succinus strain are homologous (97-98) to strainsfound in fermented meat or meat starter cultures Sxylosus CP0072081 showed homology (98) to potentialopportunistic pathogenic strains from mammal species

The S carnosus strains identified were KJ8620021 andNR1164341 and the latter as well as the single S hominisJX5199881 (96-97) identified were homologous (97) tospecies from human microbiota

S carnosus and S xylosus are commonly used as com-mercial starter cultures for sausage manufacturing [26] butS succinus has also been observed in dry fermented sausagesand its use as a starter culture has been already proposed [27]

The diversity of CNS microbiota found in the samplesanalyzed in the present study could be related to the originof the salami The microbiota from the artisanal salamishowed greater biodiversity when compared to the commer-cial salami In commercial salami S saprophyticus was thepredominant species but S xylosus and S carnosus alsoobserved in commercial salami are commonly isolated fromstarter cultures [26] The predominance of S saprophyticusfollowed by S xylosus has also been reported in salamifrom South Italy [28] similar to Belgian sausages where Ssaprophyticus was the most frequently detected species [29]

S carnosus was not observed in artisanal salami but Ssuccinus S epidermidis and S hominis were detected

The biodiversity of the CNS staphylococci species foundin microbiota depends on the kind of meat-fermented prod-uct but CNS strains such as S saprophyticus S auricularisS xylosus S capitis S hominis S carnosus S haemolyticusS warneri S equorum S cohnii S capitis and S intermediushave been described inNapoli-type salami Sremska sausages

dry sausages raw meat and naturally fermented meat [30ndash33]

During the last decades S epidermidis and S sapro-phyticus have been described as emerging pathogens [34]S saprophyticus is considered a frequent contaminant offermented sausages and raw meats and has been isolatedfrom rectal swabs of cattle carcasses and pigs In humans themain reservoir of S saprophyticus is the gastrointestinal tract[35] S saprophyticus and S epidermidis can be opportunisticpathogens isolated from the human urinary tract and thepresence of these species in food should be taken into accountconcerning possible contamination of the starter inoculumandor improvements in the salami manufacturing process[13]

Other CNS species observed in the present study aremainly associated with ordinary food contaminants withS epidermidis and S hominis being the dominant speciesin human skin and occasionally isolated from the skin ofdomestic animals [36]

32 Phylogenetic Relationships of the CNS Identified in SalamiCNS strains can be grouped into four species groupssaprophyticus simulans epidermidis and haemolyticus Fre-quently the saprophyticus species group includes S xylosusand S saprophyticus while the simulans species group iscomprised of S carnosus and S piscifermentans the epi-dermidis species group is composed of S epidermidis Scapitis S caprae and S saccharolyticus and the haemolyticusspecies group encompasses S haemolyticus S hominis andS devriesei [37 38] In the present study the main clustergrouped several S saprophyticus strains namely KJ6991511AB6975171 KJ9496061 JX4901221 and KJ0046231 four Sxylosus strains CP0087241 CP0072081 AM8827001 andKC4565901 and the S succinusKC3298241 strain (Figure 1)These strains are homologous to those from fermented meatmicrobiota

The subclusters of those clusters showed mismatches ofspecies belonging to the four species groups The first sub-cluster grouped S epidermidis HF0882111 S saprophyticusHQ6995101 and S carnosusNR1164341 and the second sub-cluster grouped S hominis JX5199881 and the S saprophyticusEU4309921 all of them originally isolated from animalsand human beings The third subcluster includes speciespreviously isolated from the marine environment such as SxylosusKF1980801 while S carnosusKJ8620021 is utilized asa probiotic organism in foodsThe fourth cluster grouped theS sciuri JX9664361 and S saprophyticus HF9372521 strainswhich are homologous to species found in soil

The CNS strains clustered into groups near the bottomof the phylogenetic tree are mostly strains found in artisanalsalami whereas the species at the top of the tree are mainlyCNS strains found in commercial salami (Figure 1)

The close similarities between the S saprophyticusAB6975171 and KJ9496061 strains the S succinusKC3298241 S xylosus AM8827001 KC4565901 andCP0087241 strains the S saprophyticus KJ0046231 andKJ6991511 strains and S xylosus CP0072081 are supportedby a bootstrap value of 100 The interspecies similaritieswere over 90 which demonstrates close phylogenies

6 BioMed Research International

Table2Genotypicandph

enotypiccharacteriz

ationof

CNSstrainsfrom

salami

Strainsidentificatio

nandcharacteriz

ation

Salamiorig

inStaphylococcus

species

Genotypic

Phenotypic

GenBa

nkaccessionnu

mbera

ndsim

ilarity(

)Presence

ofenterotoxingenes

mRN

Adetection

Enterotoxinprod

uctio

n(ngm

Lminus1)

Com

mercial

Staphylococcus

sppKF

1354451(96)

seaseb

mdashmdash

Scarnosus

KJ8620021(96)

seasebsedsehseiand

selj

mdash12plusmn01

Scarnosus

NR1164341(98)

sebsecsedandsee

see

03

Ssaprophyticus

AB6

977171

(98)

sehseiandselm

seiselm

03

Ssaprophyticus

EU4309921(99)

seasebselm

seln

selo

and

tstH1

sebselm

and

selo

13plusmn01

Ssaprophyticus

HQ6995101(97)

mdashmdash

mdashSsaprophyticus

JX4901221(99)

seaseh

seh

05plusmn01

Ssaprophyticus

KJ00

46231(96)

sec

mdash04

Ssaprophyticus

KJ9496061(98)

seasebseca

ndseh

seasebandseh

mdashSsciuriJX9664361(98)

sedsei

sei

10plusmn01

SxylosusA

M8827001(97)

mdashmdash

mdashSxylosusCP

0072081(99)

mdashmdash

mdashSxylosusC

P0087241(98)

seatstH1

sea

14SxylosusK

C4565901(98)

mdashmdash

mdash

Artisa

nal

SepidermidisKF

6005891(97)

secsedseesegsehandsei

seesei

07plusmn01

ShominisJX5199881(97)

seasebsecsedseljselm

seln

andselo

selnselo

09plusmn01

Ssaprophyticus

HF9

372521(97)

seb

seb

05

Ssaprophyticus

subspbovisK

J6991511

(98)

secselm

seln

andtstH1

seln

09plusmn01

Ssuccinus

KC3298241(99)

seasebsecseljandseln

sebsec

13SxylosusK

F1980801(97)

secselm

andtstH1

mdash05plusmn01

Thep

resenceo

fenterotoxin

genesseasebsecsedseesegsehsei

seljselkselm

seln

selo

selq

selrselu

and

tstH1w

asteste

dby

PCRusingthes

pecific

seto

fprim

ers

sea-seee

nterotoxin

prod

uctio

nwas

evaluatedby

immun

e-sorbentassays(EL

ISA)u

singthed

etectio

nkitR

IDASC

REEN

SETAB

CD

EV

aluesa

redisplay

edas

them

eansplusmnSD

ofassays

perfo

rmed

indu

plicate

mRN

Atranscrip

tsfora

llenterotoxingenesw

eree

valuated

byrealtim

eRT-PC

Rtests

CT

values

ared

isplayedas

meansplusmnSD

ofRT

-PCR

tests

perfo

rmed

indu

plicateseamdash

SxylosusC

P00872413

0plusmn04sebmdash

SxylosusC

P008724133plusmn20Ssaprophyticu

sJX4901221302plusmn04Ssaprophyticus

subspbovisK

J6991511

32plusmn01SxylosusK

F19808013

6plusmn10

and

ShominisJX519988134plusmn10secmdashSsaprophyticus

subspbovisK

J6991511

31plusmn11seemdashSsaprophyticus

subspbovisK

J6991511306plusmn05

Ssuccinus

KC3298241285plusmn01andSsaprophyticus

HF9

3725213

2plusmn04sehmdash

SxylosusC

P0087241 31plusmn10Ssaprophyticu

sJX9

6643613

32plusmn06seimdash

Ssaprophyticus

AB6

977171

30plusmn10

Ssaprophyticu

sJX9664361306plusmn10

seln

mdashShominisJX5199881303plusmn10

Ssaprophyticu

sEU430992131plusmn03andselomdashShominisJX519988132plusmn10

BioMed Research International 7

O S succinus KC3298241Δ S xylosus AM8827001

O S saprophyticus KJ6991511Δ S saprophyticus AB6977171

Δ S xylosus CP0087241Δ S xylosus KC4565901Δ S saprophyticus KJ9496061Δ S saprophyticus JX4901221Δ S saprophyticus KJ0046231

Δ S xylosus CP0072081Δ S carnosus NR1164341Δ S saprophyticus HQ6995101

O S epidermidis KF0882111O S hominis JX5199881Δ S saprophyticus EU4309921

O S xylosus KF1980801Δ S carnosus KJ8620021Δ S sciuri JX9664361O S saprophyticus HF9372521

04 03 02 01 00

Figure 1 Phylogenetic tree generated from the multiple alignments of the 16S rDNA sequences of CNS strains found in salami usingthe ClustalX 20 software The phylogenetic tree was constructed by using the Mega 60 software and the unweighted pair group method(UPGMA) Bootstrap values ranged from 00 to 04 Strains found in commercial (Δ) or artisanal salami (O)

between these CNS strains Suzuki et al 2012 demonstrateda bootstrap value higher than 90 for interspecies similaritiesbetween S saprophyticus S epidermidis S hominis and Scarnosus

33 Genotypic and Phenotypic Characterization of CNSStrains Twelve distinct combinations of staphylococcalenterotoxins geneswere found in the 15CNS strains compris-ing SEs AndashE GndashJ and also the enterotoxin-like toxins (SElL)KndashR and U (Table 2)

Fifteen strains (79) carried at least one gene encodingenterotoxins in their genomes (Figure 2) The seb and secgenes were the most predominant harbored by 57 ofthe strains followed by sea carried by 50 whereas thesedsehselm genes showed intermediate incidence harboredby 33 of the strains while seiseln and tsH1 were found in27 of strains Finally see selj and selo showed low incidence(13) and seg was carried by only 1 of strains (Table 2)

The relationships between superantigenic toxin geno-types and toxin gene-encoding mobile genetic elementsin CNS strains were evaluated Distinct combinations ofSaPI and plasmids or plasmids and genes of egc operonenterotoxins were found in the CNS strains obtained fromsalami

Four strains presented only the classical enterotoxingenes namely S saprophyticus KJ0046231 and HF9372521S xylosus CP0087241 and S carnosus NR1164341 Anotherstrain S saprophyticus AB6977171 presented only the newlydescribed enterotoxins seh sei and selm and finally 10 strainspresented a combination of classical and newly described

enterotoxin genes in their genomes with at least one of eachenterotoxin type (Table 2)

Previous studies have shown that sea is the most com-mon toxin associated with Staphylococcus food poisoningfollowed by sed and see with SEHand SEI being considered asplaying only a minor role [39] Among the 15 enterotoxigenicstrains found in salami 73 of them harbored at least the seagene or combinations of these 05 genes Strains S carnosusKJ8620021 found in commercial salami and S epidermidisKF6005891 found in artisanal salami carry 04 of these genesThe sea and seb enterotoxin genes are known to occupy thesame locus on the chromosome which may explain whythese enterotoxins are commonly found together in foodpoisoning outbreaks [36] The combination of sea and sebgenes was found in 05 strains S saprophyticus EU4309921 Scarnosus KJ8620021 S hominis JX5199881 S saprophyticusKJ9496061 and S succinus KC3298241 A single strain Scarnosus KJ8620021 showed the combination of sea and seigenes

Additionally some CNS are able to produce TSST-1 aloneor in combination with other enterotoxins Herein 04 of the19 strains (21) S saprophyticus EU4309921 S saprophyti-cus subsp bovis KJ6991511 and S xylosus CP0087241 andKF1980801 were shown to harbor the tstH1 combined withse andor sel enterotoxin genes (Table 2)

The staphylococci enterotoxins genes seg and sei [40] arepart of a chromosomal operon gene cluster (egc) comprisingfive genes designated as selo selm sei seln and seg Twoof the CNS strains determined in the present study Ssaprophyticus EU4309921 and S hominis JX5199881 were

8 BioMed Research International

sed

seb

sec

see

sea

M 2 3 4 51

500bp

(a)

sea

sebsebseb

M 1 2 3 4 5

(b)

sei

sehseg

1M

(c)

seh sehseh

M 1 2 3

(d)

1 2 4M 3 5

(e)

selm

seljseln

selo

21M

(f)

seln

selm

M 1 2

(g)

Figure 2 Uniplex duplex and multiplex PCR screening for the detection of enterotoxin genes in CNS strains from salami (a) Lane M100 bp DNA ladder plus (Fermentas Foster City CA USA) lane 1 S aureus ATCC 29231 harboring sea gene lane 2 S aureus NCTC 10654harboring seb gene lane 3 S aureus ATCC19095 harboring the sec gene lane 4 S aureus ATCC 13563 harboring the sed gene and lane 5 Saureus ATCC 27664 harboring the see gene (b) Lane M 100 bp DNA ladder plus lane 1 Staphylococcus spp lane 2 S carnosus NR116434lane 3 S carnosus KJ862002 lane 4 S carnosus KJ862002 and lane 5 S carnosus KJ8620021 (c) Lane M 100 bp DNA ladder plus lane1 S aureus ATCC 19095 harboring seg seh and sei genes (d) Lane M 100 bp DNA ladder plus lane 1 S saprophyticus AB6977171 lane 2S epidermidis KF 6005891 and lane 3 S sciuri JX9664361 (e) Lane M 100 bp DNA ladder plus lane 1 S xylosus KF1980801 lane 2 SsaprophyticusKJ6991511 (f) LaneM 100 bp DNA ladder plus lane 1 S aureusATCC 27154 harboring selj slem seln and selo genes (g) LaneM 100 bp DNA ladder plus lane 1 S xylosus KF1980801 and lane 2 S saprophyticus subsp bovis KJ6991511

BioMed Research International 9

shown to carry the selm seln and selo genes and the selmand seln sequences were detected in the genome of a singlestrain S saprophyticus subsp bovisKJ6991511 S epidermidisKF6005891 showed a combination of seg and sei genesand 03 strains harbored a single gene sei or selm or selnperhaps due to the high degree of genetic polymorphism inthe chromosomal assembly [41] Indrawattana et al 2013detected the seg-sei-selm-seln-selo of the highly prevalent egclocus was 261 in contrast to this study where 13 CNSpresents the combination of selm seln and selo genes [42]

The CNS strains clustered into groups near the bottom ofthe phylogenetic tree carried the classical enterotoxin geneswhile the species at the top of the tree showed high diversityamong the enterotoxin genes combining the classical and thenewly described genes in their genomes (Figure 1)

To assess the risk of staphylococcal food poisoning theability of the identified strains in harboring the se andsel genes and in expressing and producing enterotoxins invitro was evaluated The mRNA for each enterotoxin genewas evaluated by real time RT-PCR assays and enterotoxincontent was estimated by a sandwich enzyme immunoassayfor the combined detection of Staphylococcus enterotoxins(SET) A B C D and E

The fifteen enterotoxigenic CNS strains were able toexpress the classical enterotoxins SEA seb sec sed and seein concentrations ranging from 03 ngmLminus1 to 14 ngmLminus1as assessed by the in vitro assays (Table 2) The S sapro-phyticus AB6977171 KJ6991511 JX4901221 KJ0046231 andHF9372521 strains S xylosus KF1980801 and S carnosusNR1164341 produced low amount of enterotoxins lower than05 ngmLminus1 S epidermidisKF6005891 S saprophyticus bovisKJ6991511 and S hominis JX5199881 produced intermediateamount of enterotoxins ranging from 07 to 090 ngmLminus1while S sciuri JX9664361 S saprophyticus EU4309921 Scarnosus KJ8620021 S xylosus CP0087241 and S succi-nus KC3298241 produced enterotoxins in concentrationsge10 ngmLminus1

Although the sandwich enzyme immunoassay is consid-ered themost sensitivemethod to detect seandashsee enterotoxinsable to detect 0125 ngmLminus1 differences in the specificity andsensitivity of the assays for the detection of staphylococcalenterotoxins from foods are expected [43] A single strainStaphylococcus spp KF1354451 which harbors both the seaand seb genes was unable to produce seandashsee enterotoxins

The mRNA levels evaluated by the real time RT-PCRassays for enterotoxins were detected in 12 of 15 strains(86) (Table 2) Transcripts for the classical enterotoxinsgenes were detected when S saprophyticus HF9372521 Scarnosus NR1164341 S xylosus CP0087241 and S succinusKC3298241 were assayed Transcripts for newly describedenterotoxins were observed in S saprophyticus JX4901221and AB6977171 S sciuri JX9664361 S saprophyticus subspbovis KJ6991511 and S hominis JX5199881 Transcripts fromboth the classical and newly described enterotoxins weredetected in S saprophyticus EU4309921 and KJ9496061 andS epidermidis KF6005891

No mRNA transcripts were obtained for S sapro-phyticus KJ00462321 and S carnosus KJ8620021 although

enterotoxin production was detected by the enzyme-linkedimmunosorbent tests (Table 2)

The 04 strains that carry tstH1 in their genome Ssaprophyticus EU4309921 and KJ6991511 and S xylosusCP0087241 and KF980801 were not able to produce mRNAfor TSST-1 in the assay conditions

The production of classical enterotoxins in vitro(immunologic test) matched the results shown by real timeRT-PCR assays for the following strains S saprophyticusJX4901221 S sciuri JX9664361 S saprophyticusEU4309921S saprophyticus HF9372521 S saprophyticus subsp bovisKJ6991511 S hominis JX5199881 S epidermidis KF6005891and S succinus KC3298241

Ten strains namely S saprophyticusAB6977171 S sapro-phyticus EU4309921 S saprophyticus HF9372521 S sapro-phyticus KJ6991511 S saprophyticus KJ9496061 S succinusKC3298241 S hominis JX5199881 S xylosus KF1980801and S xylosus CP0087241 and S epidermidis KF6005891expressed mRNA for multiple se andor sel genes There isa differential transcription among these genes where themost frequent among the classical ones were seb and seeseatranscribed by 04 and 02 strains respectively and the mostfrequent among the newly described genes were sei andsehselnselo transcribed by 03 and 02 strains respectively

No mRNA for the sec gene was detected although SsaprophyticusAB6977171 was able to produce the enterotoxinin vitro as detected by the ELISA assays

34 Enterotoxin Gene Homologies between Salami CNSand CPS Strains The nucleotide sequencing of six entero-toxin genesmdashtwo of them sec and see encoding classi-cal enterotoxinsmdashand four of them encoding the newlydescribed genes seg seh selm and seln fromCNS strains wascompared to the enterotoxin genes from S aureus or S pas-teuri two coagulase-positive staphylococci strains (Figure 3)The homology between the CNS and CPS enterotoxin genesvaried from 65 to 98 The homology of sec seg sehselm and seln between CNS from salami and S aureus was98 60 98 65 and 70 respectively The homologybetween see from CNS found in salami and in S pasteuri was98

Although further studies should be performed it seemsthat the sequences encoding enterotoxins can be conservedamong coagulase-negative and coagulase-positive staphy-lococci as shown in the phylogenetic analysis of the 06enterotoxin genes (Figure 4)

35 Antimicrobial Multiresistance of CNS Strains (MRCNS)Another safety hazard associated with CNS strains besidesthe ability of producing enterotoxins in food matrices is theantimicrobial resistance to antimicrobial agents commonlyused to treat staphylococci infections The antimicrobialresistance carried by CNS strains from food matrices can bespread to the population by the consumption of an apparentlysafe food

Among the 19 CNS strains identified in salami 14 showedmultiresistance to antimicrobial agentsThree strains showedthe highest MAR indices 093 and 080 07 strains presented

10 BioMed Research International

lowast lowastlowastlowastlowastlowast lowast lowast lowastlowast lowast lowast lowastlowast lowastlowastlowastlowast lowastlowastlowast lowast

lowastlowast lowast lowast lowastlowastlowastlowast lowast lowast lowastlowast lowast

lowastlowastlowastlowast lowast lowast lowast

lowastlowast lowast lowast lowastlowast lowastlowast lowast lowastlowast lowastlowastlowastlowastlowast lowastlowast lowast lowastlowast lowastlowast

lowastlowast lowastlowastlowast lowast lowast lowast lowast lowast lowastlowastlowastlowastlowast

lowastlowast lowastlowast lowastlowast lowastlowast lowast lowast lowastlowast lowast lowast lowastlowastlowast lowast lowastlowast lowastlowastlowast

lowast lowast lowast lowast lowast lowast lowastlowast lowast lowastlowastlowast lowast lowastlowastlowast lowast lowast lowastlowastlowast lowast lowastlowastlowast lowast

lowast lowast lowast lowastlowastlowastlowast lowast lowastlowastlowast lowast lowast lowastlowastlowast lowast lowast lowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast lowast lowast lowast lowast lowastlowast lowast lowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

Enterotoxin C (sec) GATAAAAATGAATAAGAGTCGATTTATTTCATGCGTAATTTTGATATTCGCACTTATACT------------------------------------------------------------

AGTTCTTTTTACACCCAACGTATTAGCAGAGAGCCAACCAGACCCTACGCCAGATGAGTT------------------------------------------------------------

GCACAAAGCGAGTAAATTCACTGGTTTGATGGAAAATATGAAAGTTTTATATGATGATCA------------------------------------------------------------

TTATGTATCAGCAACTAAAGTTAAGTCTGTAGATAAATTTTTGGCACATGATTTAATTTA------------------------------------------------------------

TAACATTAGTGATAAAAAACTGAAAAATTATGACAAAGTGAAAACAGAGTTATTAAATGA------------------------------------------------------------

AGGTTTAGCAAAGAAGTACAAAGATGAAGTAGTTGATGTGTATGGATCAAATTACTATGT------------------------------------------------------------

AAACTGCTATTTTTCATCCAAAGATAATGTAGGTAAAGTTACAGGTGGCAAAACTTGTAT------------------------------------------------------------

GTATGGAGGAATAACAAAACATGAAGGAAACCACTTTGATAATGGGAACTTACAAAATGT------------------------------------------------------------

ACTTATAAGAGTTTATGAAAATAAAAGAAACACAATTTCTTTTGAAGTGCAAACTGATAA------------------------------------------------------------

GAAAAGTGTAACAGCTCAAGAACTAGACATAAAAGCTAGGAATTTTTTAATTAATAAAAA------------------------------------------------------------

AAATTTGTATGAGTTTAACAGTTCACCATATGAAACAGGATATATAAAATTTATTGAAAA----TTGTATGAGTTTAACAGTTCACCATATGAAACAGGATATATAAAATTTATTGAAAA

TAACGGCAATACTTTTTGGTATGATATGATGCCTGCACCAGGCGATAAGTTTGACCAATCTAACGGCAATACTTTTTGGTATGATATGATGCCTGCACCAGGCGATAAGTTTGACCAATC

TAAATATTTAATGATGTACAACGACAATAAAACGGTTGATTCTAAAAGTGTGAAGATAGATAAATATTTAATGATGTACAACGACAATAAAACAGTTGATTCTAAAAGTGTGAAGATAGA

AGTCCACCTTACAACAAAGAATGGATAATGTTAATCCGATTTTGATATAAAAAGTGAAAGAGTCCACCTTACAACAAAGAATGGATAATGTAATCCGATTATGGACAAGTGTGCTTTTTT

Enterotoxin C TATTAGATATATTTGAAAGGTAAGTACTTCGGTGCTTGCCTTTTTAGGATGCATATATATEnterotoxin C CNS

Enterotoxin CEnterotoxin C CNS

Enterotoxin CEnterotoxin C CNSEnterotoxin CEnterotoxin C CNSEnterotoxin CEnterotoxin C CNSEnterotoxin CEnterotoxin C CNSEnterotoxin CEnterotoxin C CNSEnterotoxin CEnterotoxin C CNSEnterotoxin CEnterotoxin C CNSEnterotoxin CEnterotoxin C CNS

Enterotoxin CEnterotoxin C CNSEnterotoxin CEnterotoxin C CNS

Enterotoxin CEnterotoxin C CNS

Enterotoxin CEnterotoxin C CNS

Enterotoxin CEnterotoxin C CNS

TTTGTGATCGTCGGCCA-----ACCGATACCGTCGTATTCTTGTACCGCGCAAGGCGAAGlowast lowast lowastlowastlowast lowast lowast lowast lowast lowastlowast lowast lowastlowastlowast lowast lowast lowast lowast

Enterotoxin E (see) Enterotoxin E TGCTTTAGGCGGGGTCTTACAGCATATTATCGTAGAAGATGCACAAGCTGCACGTAAAGCS pasteuri TGCTTTAGGCGCATCGTTACAACATGTCATCGTAGAAGATGAAAAAGATGGTCGTCAGGC

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast lowastlowastlowastlowast lowastlowastlowast lowast lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast lowast lowastlowastlowast lowastlowast lowastlowastlowast lowast lowastlowastlowast

Enterotoxin E ATTAGAATTCTTGAAATCAAAACA-AGCAGGACGTGCTACATTTCTACCATTAAATGTCAS pasteuri

Enterotoxin ES pasteuri

GATACAATT-TTTAAAACAACGTAGTTTAGGACGTGCCACATTCCTACCTTTAAATGTTA lowastlowast lowastlowastlowastlowast lowastlowast lowastlowastlowast lowastlowastlowast lowast lowastlowastlowastlowastlowastlowastlowastlowastlowast lowastlowastlowastlowastlowast lowastlowastlowastlowastlowast lowastlowastlowastlowastlowastlowastlowastlowast lowast

TCAAACCTAGAAATATTTCAACCAAGATATATlowast lowastlowastlowastlowast lowastlowastlowast lowastlowastlowastlowastlowast

Enterotoxin G (seg) Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

---------------------------attaagaaatactt--ccattttaatactcttaGGGGATATTTCCGGGAAACCGGGGCTAATACTGGATAACTTTTCTCTCCGAACGAAGAGA

gaatcaacaactttattatctccg--taaat-----atttaaaaacttatatggaacaaaGATTCAACGATGGTCTTGGTTTTCAATTAAGATGAACCCGCGGCGCTTAGCTGGTGGGAC

aggtactagtt-----------------------------------------cttttttaAGGTCACGGCTCACCCAAGCCCCCATGCGTATACGACCTGGAAGGGTGATCGGCCACATT

ggaaataagtcaaaccaaaaacttgtattgttcttttcagtaaat---------------GGGACTGAGACACAGCACAAACTCCTACGGGAGGCAGCAATAGGGAATCTTCCGCAATGG

------------------------------------------------------------CCGAAAGTCTGACGGAGCAACGCCGCGAGACTGAAGAATGTCTTCGGATCGTAAAACTCT

------------------------tttatatatccagattcaaa-----tgcagaaccatGTTGTTAGGGAAGAACAAGTATCGTTCGAATAGGGCGGTACCTTGACGGTACCTAACCAG

caaactcgtatagctttttttct---------ttagtgagccagtgtcttgctttg----AAAGCTTTGCTAACTACCTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCG

-taatctagttcctgaatagtaaccatattcttatttgtagttattgta--aatccaagtGAATTATTGGGCGTAAAGCGCGCGCATGCGGTCTTTTAAATCTGATGTCAAAGCCCACGG

gattgtct----------attgtcgattgttacctgtacagt------------aattaaGCTCAACCGTGGAGGGTCATTGGAAACTGTAAGACTTGGAGTGCAGAAGAGGATAGTGGA

Figure 3 Continued

BioMed Research International 11

lowast lowast lowast lowastlowast lowastlowast lowastlowastlowast lowast lowastlowast lowast lowast lowast lowast

lowast lowast lowastlowastlowast lowastlowast lowast lowastlowastlowast lowast lowastlowast lowastlowastlowast lowast lowastlowast lowastlowast lowast lowast lowastlowast lowast

lowastlowast lowast lowast lowastlowastlowastlowastlowast lowast lowast lowastlowast lowast lowast lowast lowast lowast lowastlowastlowast

lowastlowast lowast lowastlowast lowastlowastlowast lowastlowast lowastlowast lowast lowast lowast lowast lowastlowast lowastlowast lowast lowast lowast lowastlowast

lowastlowast lowastlowastlowastlowast

lowast lowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowast lowast lowastlowastlowastlowast lowast lowastlowast lowastlowast lowast lowastlowastlowastlowast lowast lowastlowast lowast lowast lowastlowastlowastlowastlowastlowastlowast

lowastlowast lowast lowast lowast lowast lowastlowastlowast lowastlowast lowastlowast lowast lowastlowastlowastlowastlowastlowastlowastlowast lowastlowastlowast

lowast lowast lowast lowast lowast lowast lowastlowast lowastlowast lowast lowast lowastlowastlowast lowast lowast lowastlowastlowast lowast lowast

lowastlowast lowastlowastlowastlowastlowastlowast lowastlowast lowastlowastlowast lowastlowast lowast lowastlowast lowastlowast lowastlowastlowast lowast lowast

lowastlowast lowast lowastlowastlowast lowast lowastlowastlowastlowastlowast lowast lowast lowast lowastlowastlowast lowast lowastlowast lowast

lowastlowast lowast lowast lowast lowast lowastlowast lowast lowastlowast lowast lowastlowast lowast lowastlowast lowastlowastlowastlowast lowast lowast lowastlowast

lowast lowastlowastlowast lowast lowastlowast lowast lowast lowastlowast lowast lowastlowast lowast lowastlowastlowastlowastlowastlowast lowast

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

tttatctctttcattttctgaactattaaa----tgtaagaccacc---atacatacaacATTCCACGTGTAACGGTGAAAATGAGTAGAGATGTGGAGGAACACCAGTGGAGAAACGCG

aacctccaaaattttggtttatatccggttc-----------agatttaggtattatacaACTCTCTGGTCTGTTACTGACGCTGAGGCGCGAAAGCGTGGGAGCAAACAGGATTATATA

tgtataaaaatatggaacgccaaaaatgtctacttttttatctttataattgttagctaaCCCCTGGTAGTCCAAGCCGTAAACGATGAGTGCTAGTGTTACAGGGT------------T

ttctgtattttctaattcagttttaacctcattataactcttatactcaattggaaaaatTCCGCCCTTTACTGCTGCAGCTAACG---CATTAG-GCACTCCGCCTGGGGAGTACAACT

taaatcatgagataaaaactgtctagaattaataactcctcttccttcaacaggtggagaGCAA------GGTTGAAACTCAAAGCA----TTGACCGGGGGCC---CGATAAGCTGAGG

cgtataaagattcattacat----tacccatagttcccttattgtttttataatcacttaTACATGGTGTTTAATTCCAAACCACGCCAAGAACCCTCACGAGGTCTTGACATCCTCTGG

ctttattt----------------------------------------------------ACCTCCCTAGAGAATAAGGGCTTCCCCGTCGGGGATGAAATGAACAGGTGGTGCATGGAT

-------agttcgtc---AGTCTTCAGCTCGTGCAT

Enterotoxin H (seh)

------------------------------------------------------------ATCTATGAATATAAATCTTTTAATATTGGTTTAATATAAATATTCCATTTCTAGAAACCT

------------------------------------------------------------ATATTTTATGTTTGTATAAATTCATAAATTGTTCGAAATTTATATATTAAAAGTTGTTTT

------------------------------------------------------------TGTTGTTTTATAATTAAGTTAATGAAATATATTGAGGAGTTTTAAAATGATTAATAAAAT

------------------------------------------------------------TAAAATATTATTTTCGTTTTTAGCATTATTACTTTCATTCACATCATATGCGAAAGCAGA

------------ -----------TTACGATTTAGCTTTAGCTAATGCATATGGTCAATAAGATTTACACGATAAAAGTGAGTTAACAGATTTAGCTTTAGCTAATGCATATGGTCAATA

TAATCACCCATTCATTAAAGAAAATATTAAGAGTGATGAAATAAGTGGAGAAAAAGATTTTAATCACCCATTCATTAAAGAAAATATTAAGAGTGATGAAATAAGTGGAGAAAAAGATTT

AATATTTAGAAATCAAGGTGATAGTGGCAATGATTTGAGAGTAAAGTTTGCAACTGCTGAAATATTTAGAAATCAAGGTGATAGTGGCAATGATTTGAGAGTAAAGTTTGCAACTGCTGA

TTTAGCTCAGAAGTTTAAAAATAAAAATGTAGATATATATGGGGCATCTTTTTATTATAATTTAGCTCAGAAGTTTAAAAATAAAAATGTAGATATATATGGGGCATCTTTTTATTATAA

GTGTGAAAAAATAAATGAAAATATTTCTGAATGTCTATATGGAGGTACAACACTAAATAAGTGTGAAAAAATAAGTGAAAATATTTCTGAATGTCTATATGGAGGTACAACACTAAATAG

TGAAAAATTGGCACAGGAAAGGGTGATTGGTGCTAATGTTTGGGTAGATGGTATTCAAAATGAAAAATTGGCACAGGAAAGGGTGATTGGTGCTAATGTTTGGGTAGATGGTATTCAAAA

AGAAACAGAATTAATACGAACAAATAAGAAAAATGTGACATTGCAAGAATTAGATATAAAAGAAACAGAATTAATACGAACAAATAAGAAAAATGTGACATTGCAAGAATTAGATATAAA

Enterotoxin H CNS GATCAGAAAAATATTGTCCGATAAATATAAAATTTATTATAAAGACAGCGAAATAAGGG-Enterotoxin H

Enterotoxin H CNSEnterotoxin H

Enterotoxin H CNSEnterotoxin HEnterotoxin H CNSEnterotoxin HEnterotoxin H CNSEnterotoxin HEnterotoxin H CNSEnterotoxin HEnterotoxin H CNSEnterotoxin H

Enterotoxin H CNSEnterotoxin H

Enterotoxin H CNSEnterotoxin H

Enterotoxin H CNSEnterotoxin H

Enterotoxin H CNSEnterotoxin H

Enterotoxin H CNSEnterotoxin H

GATCAGAAAAATATTGTCCGATAAATATAAAATTTATTATAAAGACAGCGAAATAAGTAA

Enterotoxin N (seln) TGATTTAGCTTTAGCTAATGCATATGGTCAATAT-AATCACCCATTCATTAAAGAAAA-----TAGATATTTATGGACTGTATTATGGAAATAAATGTGTAGGCTTAACTGAAGAAAAAA

TATTAAGAGTGATGAAATAAGTGGAGAAAAAGATTTAATATTTAGAAATCAAGGTGATAGCATCATGCTTATACGGAGGAGTTAC------GA--TATATGATGGAAATCAATTAGATGA

TGGCAATGATTTGAGAGTAAAG--------------TTTGCAA-CTGCTGATTTAGCTCAAGAGAGAGTTATAGGCGTTAATGTATTTAAAGATGGTATCCAACAAGAAGGTTTTGTTAT

GAAGTTTAAAAATAAAAATGTAGATATATATGGGGCATCTTTTTATTATAAGTGTGAAAAAAAAACTAAAAAGGCTAAAGTAACAGTACAA------GAATTAGATACTAAAGTTCGATT

AATAAGTGAAAATATTTCTGAATGTCTATATGGAGGTACAACACTAAATAATGAAAAATT------------TAAATTAGAAAATTTATATAAAATATACAATAAAGATACCGGTAACAT

Enterotoxin N CNSEnterotoxin N

Enterotoxin N CNSEnterotoxin N

Enterotoxin N CNSEnterotoxin N

Enterotoxin N CNSEnterotoxin N

Enterotoxin N CNSEnterotoxin N

Figure 3 Continued

12 BioMed Research International

lowastlowastlowast lowastlowast lowastlowast lowastlowast lowast lowastlowast lowastlowastlowast lowastlowast lowast lowastlowastlowastlowast

lowastlowast lowast lowastlowast lowast lowast lowastlowast lowastlowastlowastlowastlowast lowast lowast lowast lowastlowast lowast lowast lowastlowast lowast lowast

lowast lowast

lowast lowastlowast lowast lowastlowast lowast lowast lowast lowast lowast lowast lowast lowastlowast lowast lowastlowastlowastlowast lowast

lowast lowastlowast lowast lowast lowastlowast lowast lowastlowastlowast lowast lowastlowast lowast lowastlowast lowastlowast lowast lowast

lowastlowastlowast lowast lowast lowastlowast lowastlowast lowast lowast lowast

lowast lowast lowast lowastlowastlowast lowastlowastlowast lowastlowast lowast lowastlowastlowastlowastlowast lowastlowastlowast lowast

lowastlowast lowast lowast lowastlowast lowastlowastlowast lowastlowast lowast lowast lowastlowastlowastlowast lowastlowast

lowast lowast lowast lowastlowast lowast lowastlowast lowastlowastlowastlowastlowastlowast lowastlowast lowast lowastlowast lowastlowastlowast

lowast lowast lowast lowast lowastlowast lowastlowastlowastlowast lowast lowast lowastlowastlowast lowast lowast lowast lowast lowast

lowast lowast lowast lowast lowastlowastlowast lowast lowast lowastlowast lowastlowast lowast lowastlowastlowast lowast lowastlowastlowast lowast

lowast lowast lowastlowastlowast lowast lowastlowast lowastlowastlowast lowastlowastlowastlowastlowast lowastlowast lowast lowastlowastlowast lowast lowast lowastlowastlowast

lowastlowastlowast lowast lowastlowast lowastlowast lowastlowast lowast lowast lowast lowast lowastlowast lowastlowast lowastlowast lowast lowast

lowast lowast lowast lowast lowastlowast lowast lowast lowastlowast lowastlowast lowast lowastlowastlowast lowastlowastlowastlowast lowast lowastlowastlowastlowastlowast lowast

lowastlowast lowast lowast lowastlowastlowast lowastlowast lowast lowastlowast lowast lowastlowast lowastlowast lowastlowast lowast lowast lowast

lowastlowastlowastlowastlowast lowastlowast lowastlowast lowast lowastlowast lowast lowast lowast lowast lowastlowast lowastlowast

lowast lowast lowastlowast lowastlowast lowastlowastlowastlowastlowastlowast lowastlowast lowast lowast lowast lowast lowast lowast lowast lowast lowast lowast lowast

lowastlowast lowastlowast lowast lowastlowastlowastlowast lowast lowast lowastlowastlowast lowastlowast lowast lowastlowastlowast lowast

lowastlowastlowastlowast lowastlowast lowast lowast lowast lowast lowastlowast lowast lowast lowast lowastlowastlowastlowastlowast lowastlowast lowastlowastlowast lowast

lowast lowastlowastlowast lowastlowastlowastlowastlowast lowast lowast lowast lowastlowast lowast lowast lowastlowast lowast lowastlowastlowast lowastlowast lowast

lowastlowast lowast lowast lowast lowastlowast lowast lowastlowast lowastlowast lowastlowast lowast lowastlowastlowastlowast

ATTAATACGAACAAATAAGAAAAATGTGACATTGCAAGAATTAGATATAAAGATCAGAAA-TTAT----------TATGATTTATATAAC--------------ATAAAAGGTTCAG---

AATATTGTCCGATAAATATAAAATTTATTATAAAGACAGCGAAATAAGGTAAAAGG------TAGGAGCAGAGTTTTTTCAATTTTATAGTGATAATAGAACAGTTAGCTCATCTAATTA

Enterotoxin N CNS -TAAA---Enterotoxin N

lowast lowastlowastlowastlowastlowast lowastlowast lowast lowast lowast lowastlowast lowast lowastlowastlowastlowastlowastlowast lowastlowast lowastlowast

GGCACAGGAAAGGGTGATTGGTGCTAATGTTTGGGTAGATGGTATTCAAAAAGAAACAGAACAA----AAAGGATGCATTTTCTTT-----CATTCTAATAATCATCAAAATCAATCATT

Enterotoxin N CNSEnterotoxin N

Enterotoxin N CNSEnterotoxin N

Enterotoxin N CNSEnterotoxin N

TCATATCG

Enterotoxin M (selm)

ttgctattcgcaaaatcatatcgcaaccgctgatgtcggagtt--ttgaatcttaggaacEnterotoxin M CNSEnterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

--------------AGTATGTTTAAATCCCCGCTCACAGTTATGTTTAACTCTTCCTTTC

tattatggta-gctatccaattgaagaccaccaaagtattaatcctgaaaataatcatctTTTTCACACCGCCCCGTCGGGTGTTGCGCACGGGGTTGTTTAACCCCCCCGGAAAAAATT

ttcgcatcaattagttttttctatggataattcgacagtaacagctgaatttaagaacgtEnterotoxin M CNS

Enterotoxin M CNS

TTCCCTCCCTCCTCCTTACTC---------------CCT----------------CCCCT

tgatgatgtaaaggaattcaaaaatcatgctgtagatgtatatggtctaagttatagtggEnterotoxin M CNS TTTTCCTACAAATCCTTTCTCCCCTCCCGGAGTAGAAGTAAA------------------

atattgtttgaaaaacaaatatatatacggtggagttacattagcaggtgat-tatttagEnterotoxin M CNS -------------------TAAAACAATACCCCTACCACCTTAACAATTTTTTTATTGAG

agaaatctagacgtattcct-attaatctttgggttaatggagaacatcaaactatatctEnterotoxin M CNS CCCCTCCCCCCCCTTCTCATTTTTTCTCTTTGAC-----------GTTCCCCCCATTTCT

actgacaaagtatcaactaataaaa-agttagtaacagctcaagaaattgatactaaattEnterotoxin M CNS GCCAATGCTTAACCCACCCTTAAAGGCCATCGGCACATATTAGACACTCC-----CCACC

aagaagatatctacaagaagaatataatatttatggctttaatgatac---aaataaaggEnterotoxin M CNS CGCCCCCTCTTTTTATTAAGGACACAACTCTTTTTATTTTCCCCCAAGAACTAATTCTGA

a-agaaattatggtaataagtcaaaatttagttctgga---------tttaatgcaggaaEnterotoxin M CNS AAAAAAACGTTCCGCTTAGTTCAGAATTTCCTTTGAGCCTTTTATCCTTTTGTACGCGAA

aaatattatttca--------tttgaatgatggttcatcattttcttatgacttatttga

Enterotoxin M CNS AAACCATCAGCCATTGAATCAGTTATCCCATTGCCCTAACTAACCCAATATCTAATCTAA

tactggaacaggacaagctgaaagtttctt-aaaaatatataatgacaacaaaactgtcgEnterotoxin M CNS TTTTCGCCTACGAACGAATAATAGCTTGTGGCGAAAAATATCCAGGATTATCTACTGTAG

aaact--gaaaaattccatttagatgtagaaatatcttataaggacgaaagttgaagtatEnterotoxin M CNS CGCCTCGCCAGACGTCCTTTGAATTGA--ATATGACTCCTACTCCCCCCCGCACCGTCAA

ttcaatcataacttagtaaaggaaatgccatgaaaaaatttaaatatagtttt---atatEnterotoxin M CNS TTCAAACA---------------GGTGGAAGTTAATACTGAATAGTAGGTCTTCTTCCCG

tagtttttatattactttttaacattaaagatcttacgtatgct---caaggtgatattgEnterotoxin M CNS TGCTATTCTTAGAACTTTTAAATTATGTAAACCCAATTTTTAGTGCACTCTTTCTCCCTC

gtg-taggtaacttaagaaatttctatacaaaacatgattatatagatttaaaaggcgtcEnterotoxin M CNS GTTACTCTTAGATGGAGAAGCGTTAAGACAGAAGCTTTGTAT-------------TCCCA

acagataaaaa-cctacctattgcaaatcaacttgaattttcaac-----aggt----acEnterotoxin M CNS GCAGAGTGCAACCTTTCACAATGAAGTTTTTATCTAATTTACACGCCTTTAGGCTCCAAA

caatgatttgatctcagaatctaataattgggacgaaataagtaaatttaaaggaaagaaEnterotoxin M CNS TACGTATTAAATCTCCTCCTGT--TTATCTGACCCTAAG-ATAAAACACGAAAAATTATG

actggatatttttggcattgattataatggtccttgtaaatctaaatacatgtatggaggEnterotoxin M CNS CGTGCCCACTCATCCTAT-------TACTTTCATTATATCTATAAAA-------------

Figure 3 Alignment of the enterotoxin gene sequences found in coagulase-negative staphylococci (CNS) and in coagulase-positivestaphylococci (CPS)

BioMed Research International 13

Enterotoxin (seg) S aureus AY1587031

Enterotoxin (sec) CNS

Enterotoxin (see) S pasteuri NC0227371

Enterotoxin (see) CNS

Enterotoxin (seh) CNS

Enterotoxin (selm) S aureus AF2857601

Enterotoxin (seln) CNS

Enterotoxin (seh) S aureus AJ9375481

Enterotoxin (seln) S aureus AY1587031

Enterotoxin (sec) S aureus X516611

Enterotoxin (selm) CNS

Enterotoxin (seg) CNS

150 100

100

100

100

100

99

98

96

97

72

50 0

Figure 4 Phylogenetic tree generated from the multiple alignments of the enterotoxin sequences of CNS and CPS strains using the ClustalX20 software package The phylogenetic tree was constructed using the Mega 60 software and the unweighted pair group method (UPGMA)

MAR indices varying from 066 to 046 and the remaining04 strains showed MAR indices le026 (Table 3)

The 14 MRCNS strains were resistant to 120573-lactams(oxacillin penicillin andor cefoxitin) and to vancomycincorresponding to 73 of the total CNS strains identifiedwhile 09 strains (64) showed resistance to tetracycline andgentamicin 08 strains (57) were resistant to neomycinerythromycin and chloramphenicol 07 strains (50) wereresistant to sulfamethoprim 05 strains (36) were resistantto linezolid 03 strains (21) were resistant to rifampicin 02strains (14) were resistant to ciprofloxacin and cefepimeand 01 strain (7) was resistant to clindamycin (Table 3)

The multiresistance of CNS strains demonstrated hereinis consistent with previous studies on coagulase-negativeand coagulase-positive staphylococci that have found severalresistant and multiresistant Staphylococcus aureus strains inraw milk meat and fermented meat products [43]

Surprisingly the resistance to chloramphenicol is verysimilar to that estimated for MRCNS strains isolated fromhuman clinical samples Indeed there is no direct correla-tion between the researched features and the origin of thestaphylococci strains since different virulence factors arewidespread such as antibiotic resistance [44] reinforcing thefact that the ability of foodmatrices strains to produce SE andSEl and their multiresistance character must be consideredwhen evaluating the safety hazards of food poisoning

S epidermidis KF6005891 and S hominis JX5199881showed the highestMAR indexes (093 and 080 resp)Thesestrains found in artisanal salami are homologous to strainsisolated from human skin microbiota and should be carefullyconsidered among the potential pathogenic staphylococcifound in food from animal origins which can be caused bycontamination by poor hygienic conditions during salamimanufacturing

Depending on the conditions some species of coagulase-negative staphylococci can present health risks since theyhave shown resistance to antibiotics of therapeutic impor-tance such as beta lactams [15] However multiresistantstrains like S carnosusKJ18620021 and S xylosusCP0072081and CP0087241 were intentionally introduced in the foodmatrix since they are part of the culture starter used inItalian-salami manufacturing As previously discussed foodcan be a reservoir of multiresistant microorganisms that canspread by the consumption of an apparently safe food Dueto the intensive and indiscriminate use of antibiotics forhuman and veterinarian therapeutic purposes multiresistantstaphylococci strains are being selected and reproduced infood matrices [45]

The cefoxitin and oxacillin disk diffusion tests are rec-ommended for determining the resistance and susceptibilitybreakpoint for MRSA surveillance cultures [46] In thisstudy there was a good correlation between the disc testzone diameters for oxacillin and cefoxitin (Table 3) TheMICs for ampicillin andmethicillinoxacillincefoxitin for allsusceptible CNS strainswere determined by the diffusion disctest using 003 to 2mgmLminus1 of each antimicrobial agent

S saprophyticus HF9372521 showed an MIC of003mgmLminus1 formethicillin strain S carnosusKJ8620021 Sxylosus strains CP0087241 and CP0072081 S saprophyticusJX4901221 S hominis JX5199881 and S succinusKC3298241 were resistant to 006mgmLminus1 and two strainsS saprophyticus KJ6991511 and S saprophyticus KJ0046231showed an MIC of 05mgmLminus1 (Table 4) Two of themethicillin-resistant strains S saprophyticus KJ0046231 andS xylosus CP0072081 harbor no enterotoxin gene

Five strains S saprophyticus JX4901221 S carnosusKJ8620021 S xylosusCP0087241 S xylosusKF1980801 andS saprophyticus KJ0046231 showed an MIC for ampicillin

14 BioMed Research International

Table 3 Multiple resistance to antimicrobial as found in CNS strains from salami

Salami origin CNS strains Antimicrobial agent resistance Multiple antimicrobialresistance (MAR) indexlowast

Commercial Staphylococcus spp KF1354451 CIP CLO CPM GEN NEO OXA PENSXT TET and VAN 066

Commercial S carnosus KJ8620021 CIP GEN LZD NEO OXA SXT andTET 046

Commercial S saprophyticus AB6977171 CFO CLO ERI PEN OXA TET andVAN 046

Commercial S saprophyticus EU4309921 CFO CLO ERI GEN LZD NEO OXAPEN SXT TET and VAN 080

Commercial S saprophyticus JX4901221 CFO CLO ERI GEN NEO PEN OXASXT TET and VAN 066

Commercial S saprophyticus KJ0046231 CFO OXA and PEN 020

Commercial S sciuri JX9664361 CFO CLO GEN NEO OXA PEN andTET 046

Commercial S xylosus CP0072081 OXA LZD PEN and VAN 026

Commercial S xylosus CP0087241 CFO CLO ERI GEN NEO OXA PENTET and VAN 060

Artisanal S epidermidis KF6005891 CFO CLI CLO CPM ERI GEN LZDNEO OXA PEN RIF SXT and TET 093

Artisanal S hominis JX5199881 CIP CPM CPO ERI GEN LZD NEOOXA PEN RIF SXT and VAN 080

Artisanal S saprophyticusHF9372521 CFO OXA PEN and SXT 026Artisanal S saprophyticus KJ6991511 OXA PEN and VAN 020

Artisanal S succinus KC3298241 CFO CLO ERI GEN OXA PEN RIFSXT TET and VAN 066

Artisanal S xylosus KF1980801 CFO CLO ERI GEN NEO OXA PENTET and VAN 060

lowastTheMAR index of an isolate is defined as 119886119887 where 119886 represents the number of antimicrobials to which the isolate was resistant and 119887 represents the numberof antimicrobials to which the isolate was subjectedS aureus strains ATCC WB81 (sea) ATCC 13563 (sed) and ATCC 27664 (see) showing a MAR index of 05 and S aureus strains ATCC14458 (seb) andATCCWB72 (sec) and S xylosus ATCC 29971 showing a MAR index of 03 were used as reference strainsCPM cefepime CFO cefoxitin CLO chloramphenicol CIP ciprofloxacin CLI clindamycin ERI erythromycin GEN gentamycin NEO neomycin LZDlinezolid RIF rifampicin TET tetracycline OXA oxacillin PEN penicillin SXT sulfamethoprim and VAN vancomycin

Table 4 Minimal inhibitory concentration (MIC) of compounds used in antimicrobial therapy against staphylococci infections

Salami origin GenBank accession number and similarity () MIC mgmLminus1

Methicillin Ampicillin Vancomycin Linezolid

Commercial

S carnosus KJ8620021 006 003 mdash mdashS xylosus CP0087241 006 003 006 mdash

S saprophyticus JX4901221 006 003 05 mdashS saprophyticus AB6977171 mdash mdash 05 mdashS saprophyticus EU4309921 mdash mdash 025 0125

S succinus KC3298241 006 025 003 mdash

Artisanal

S hominis JX5199881 006 05 mdash mdashS saprophyticus HF9372521 003 mdash mdash mdash

S xylosus KF1980801 003 05 mdashS saprophyticus KJ6991511 05 025 003 mdashS saprophyticus KJ0046231 05 003 003 mdash

S xylosus CP0072081 006 mdash mdash 025Strains S epidermidis KF6005891 and Staphylococcus spp KF1354451 were not susceptible to the antimicrobial concentrations tested in the present study

BioMed Research International 15

of 003mgmLminus1 two strains S succinus KC3298241 and Ssaprophyticus KJ6991511 showed an MIC of 025mgmLminus1and S hominis JX5199881 showed an MIC of 05mgmLminus1(Table 4) S saprophyticus KJ0046231 also showed an MICof 003mgmLminus1 but does not harbor enterotoxin genes It isimportant to note that most of the CNS strains that showedresistance to ampicillin were also resistant to methicillin ashighlighted by the MIC tests

Five strains S saprophyticus HF9372521 S saprophyti-cus KJ6991511 S saprophyticus JX4901221 S saprophyticusKJ0046231 and S succinus KC3298241 showed an MICfor vancomycin of 003mgmLminus1 one strain S xylosusCP0087241 showed an MIC of 006mgmLminus1 one strain Ssaprophyticus EU4309921 showed an MIC of 025mgmLminus1and two strains S xylosus KF1980801 and S saprophyticusAB6977171 showed an MIC of 05mgmLminus1 (Table 4)

Additionally five CNS strains S xylosus CP0087241S saprophyticus JX 4901221 S succinus KC3298241 Ssaprophyticus KJ6991511 and S saprophyticus KJ0046231showed resistance tomethicillin as demonstrated by theMICdeterminations (Table 4)

Previous studies have demonstrated that linezolid isactive against Gram-positive bacteria including methicillin-resistant staphylococci [47] In the present study 05 strainsshowed linezolid resistance in the disc diffusion test but itwas impossible to establish MIC values for S epidermidisKF6005891 Staphylococcus hominis JX5199881 and Staphy-lococcus carnosus KJ8620021The remaining strains S sapro-phyticus EU4309921 and S xylosus CP0072081 presentedan MIC for linezolid of 0125mgmLminus1 and 025mgmLminus1respectively

The MIC of 003mgmLminus1 for penicillin is in accordancewith the previous values estimated for resistant S aureusstrains found in several food matrices such as meat dairyproducts and ready-to-eat food [48]

There is still a lack of information on the antimicro-bial resistance of staphylococci strains from food matricesalthough they are the most worrisome vehicles of dissemi-nation of antibiotic-resistant pathogens [49]

The high resistance found for salami staphylococci strainscan be ascribed to their inappropriate use as growth promot-ers of antimicrobial agents like oxacillin vancomycin chlo-ramphenicol neomycin and erythromycin which are com-monly used in veterinary medicine to treat infections [50]Antimicrobial therapy of infections staphylococci is basedon results of susceptibility tests in vitro [51] Methicillin-resistant coagulase-negative Staphylococcus spp found inready-to-eat products such as meats fish and dairy productsalso offer risks and although they are not classical foodpoisoning bacteria their presence in food offers significantrisks to public health due to the possible spread of antibioticresistance [52]

The standardization of salami quality should includediagnostic methods to screen and quantify the presence ofclassical and newly described enterotoxins directly in thefood matrices as a routine procedure to be conducted by themeat product industry in Brazil

The safety of salami consumption could also be enhancedby the inclusion of amicrobial barrier such as the inclusion ofprobiotic strains producing natural antibiotics or competitiveflora or even the addition of natural bioagents against spoilageor pathogenic microorganisms

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors acknowledge the financial support fromFundacao Carlos Chagas Filho de Amparo a Pesquisa doEstado do Rio de Janeiro (FAPERJ Rio de Janeiro Brazil)Conselho Nacional de Desenvolvimento Cientıfico eTecnologico (CNPq Brasılia Brazil) and Coordenacao deAperfeicoamento de Pessoal de Nıvel Superior (CAPESBrasılia Brazil)

References

[1] ICMSF International Comission on Microbiological Specifica-tions for FoodsMicroorganisms in FoodsTheir Significance andMethods of Enumerations University of Toronto Press TorontoCanada 3rd edition 1983

[2] J F Veras L S Carmo L C Tong et al ldquoA study of theenterotoxigenicity of coagulase-negative and coagulase-positivestaphylococcal isolates from food poisoning outbreaks inMinasGerais Brazilrdquo International Journal of Infectious Diseases vol12 no 4 pp 410ndash415 2008

[3] K Omoe D-L Hu H Takahashi-Omoe A Nakane and KShinagawa ldquoComprehensive analysis of classical and newlydescribed staphylococcal superantigenic toxin genes in Staphy-lococcus aureus isolatesrdquo FEMS Microbiology Letters vol 246no 2 pp 191ndash198 2005

[4] J Kadariya T C Smith and D Thapaliya ldquoStaphylococcusaureus and staphylococcal food-borne disease an ongoing chal-lenge in public healthrdquoBioMedResearch International vol 2014Article ID 827965 9 pages 2014

[5] KOmoeD-LHuH KOno et al ldquoEmetic potentials of newlyidentified staphylococcal enterotoxin-like toxinsrdquo Infection andImmunity vol 81 no 10 pp 3627ndash3631 2013

[6] J K McCormick J M Yarwood and P M SchlievertldquoToxic shock syndrome and bacterial superantigens an updaterdquoAnnual Review of Microbiology vol 55 pp 77ndash104 2001

[7] T Baba F Takeuchi M Kuroda et al ldquoGenome and virulencedeterminants of high virulence community-acquired MRSArdquoThe Lancet vol 359 no 9320 pp 1819ndash1827 2002

[8] Associacao Brasileira de Industria Produtora e Exportadorada Carne Suınas (ABIPECS) ldquoStatistics slaughter ranking2000ndash2014rdquo 2014 httpabpa-brcombrsetoressuinoculturapublicacoesrelatorios-anuais

[9] N Zdolec M Hadziosmanovic L Kozacinski et al ldquoMicrobialand physicochemical succession in fermented sausages pro-duced with bacteriocinogenic culture of Lactobacillus sakei andsemi-purified bacteriocin mesenterocin Yrdquo Meat Science vol80 no 2 pp 480ndash487 2008

16 BioMed Research International

[10] F Ravyts L D Vuyst and F Leroy ldquoBacterial diversity andfunctionalities in food fermentationsrdquo Engineering in Life Sci-ences vol 12 no 4 pp 356ndash367 2012

[11] TOhlsson andN BengtssonTheHurdle ConceptMinimal Pro-cessing Technologies in the Food IndustryWoodhead Publishing2002

[12] S Bernardi B B Golineli and C J Contreras-CastilloldquoRevisao aspectos da aplicacao de culturas starter na producaode embutidos carneos fermentadosrdquo Brazilian Journal of FoodTechnology vol 13 no 2 pp 133ndash140 2010

[13] F Irlinger ldquoSafety assessment of dairy microorganismscoagulase-negative staphylococcirdquo International Journal of FoodMicrobiology vol 126 no 3 pp 302ndash310 2008

[14] B Ghebremedhin F Layer W Konig and B Konig ldquoGeneticclassification and distinguishing of Staphylococcus species basedon different partial gap 16S rRNA hsp60 rpoB sodA and tufgene sequencesrdquo Journal of Clinical Microbiology vol 46 no 3pp 1019ndash1025 2008

[15] I Babic K Markov D Kovacevic et al ldquoIdentification andcharacterization of potential autochthonous starter culturesfrom aCroatian lsquobrandrsquo product lsquoslavonski kulenrsquordquoMeat Sciencevol 88 no 3 pp 517ndash524 2011

[16] MMehrotra GWang andWM Johnson ldquoMultiplex PCR forthe detection of genes for Staphylococcus aureus enterotoxinsexfoliative toxins toxic shock syndrome toxin and methicillinresistancerdquo Journal of Clinical Microbiology vol 38 no 3 pp1032ndash1041 2000

[17] WM Johnson SD Tyler E P Ewan F EAshtonDR Pollardand K R Rozee ldquoDetection of genes for enterotoxins exfolia-tive toxins and toxic shock syndrome toxin 1 in Staphylococcusaureus by the polymerase chain reactionrdquo Journal of ClinicalMicrobiology vol 29 no 3 pp 426ndash430 1991

[18] J Mclauchlin G L Narayanan VMithani andG OrsquoNeill ldquoThedetection of enterotoxins and toxic shock syndrome toxin genesin Staphylococcus aureus by polymerase chain reactionrdquo Journalof Food Protection vol 63 no 4 pp 479ndash488 2000

[19] J P Rosec and O Gigaud ldquoStaphylococcal enterotoxin genes ofclassical and new types detected by PCR in Francerdquo Interna-tional Journal of Food Microbiology vol 77 no 1-2 pp 61ndash702002

[20] K Omoe D-L Hu H Takahashi-Omoe A Nakane andK Shinagawa ldquoIdentification and characterization of a newstaphylococcal enterotoxin-related putative toxin encoded bytwo kinds of plasmidsrdquo Infection and Immunity vol 71 no 10pp 6088ndash6094 2003

[21] N Sergeev D Volokhov V Chizhikov and A Rasooly ldquoSimul-taneous analysis of multiple staphylococcal enterotoxin genesby an oligonucleotide microarray assayrdquo Journal of ClinicalMicrobiology vol 42 no 5 pp 2134ndash2143 2004

[22] S Holtfreter D Grumann M Schmudde et al ldquoClonal distri-bution of superantigen genes in clinical Staphylococcus aureusisolatesrdquo Journal of Clinical Microbiology vol 45 no 8 pp2669ndash2680 2007

[23] W J Mason J S Blevins K Beenken NWibowo N Ojha andM S Smeltzer ldquoMultiplex PCR protocol for the diagnosis ofstaphylococcal infectionrdquo Journal of Clinical Microbiology vol39 no 9 pp 3332ndash3338 2001

[24] E Rahimi and H G Safai ldquoDetection of classical enterotoxinsof Staphylococcus aureus strains isolated frombovine subclinicalmastitis in Isfahan Iranrdquo Veterinary Microbiology vol 141 no3-4 pp 393ndash394 2010

[25] M A Larkin G Blackshields N P Brown et al ldquoClustalW andClustal X version 20rdquo Bioinformatics vol 23 no 21 pp 2947ndash2948 2007

[26] P H A Sneath and R R Sokal Unweighted Pair Group Methodwith Arithmetic Mean Numerical Taxonomy W H FreemanSan Francisco Calif USA 1973

[27] NCCLS ldquoPerformance standards for antimicrobial susceptibil-ity testing 14th informational supplementrdquo Tech Rep M 100-S14 NCCLS 2004

[28] S Corbiere Morot-Bizot S Leroy and R Talon ldquoMonitoringof staphylococcal starters in two French processing plantsmanufacturing dry fermented sausagesrdquo Journal of AppliedMicrobiology vol 102 no 1 pp 238ndash244 2007

[29] R Talon S Leroy I Lebert et al ldquoSafety improvement andpreservation of typical sensory qualities of traditional dryfermented sausages using autochthonous starter culturesrdquo Inter-national Journal of Food Microbiology vol 126 no 1-2 pp 227ndash234 2008

[30] G Mauriello A Casaburi G Blaiotta and F Villani ldquoIsolationand technological properties of coagulase negative staphylo-cocci from fermented sausages of Southern ItalyrdquoMeat Sciencevol 67 no 1 pp 149ndash158 2004

[31] M Janssens N Myter L De Vuyst and F Leroy ldquoSpeciesdiversity and metabolic impact of the microbiota are low inspontaneously acidified Belgian sausages with an added starterculture of Staphylococcus carnosusrdquo Food Microbiology vol 29no 2 pp 167ndash177 2012

[32] G Blaiotta D Ercolini G Mauriello G Salzano and F VillanildquoRapid and reliable identification of Staphylococcus equorum bya species-specific PCR assay targeting the sodA generdquo Systematicand Applied Microbiology vol 27 no 6 pp 696ndash702 2004

[33] L Kozacinski E Drosinos F Caklovica L Cocolin J Gasparik-Reichardt and S Veskovic ldquoInvestigation of microbial associa-tion of traditionally fermented sausagesrdquo Food Technology andBiotechnology vol 46 no 1 pp 93ndash106 2008

[34] S Leroy P Giammarinaro J-P Chacornac I Lebert and RTalon ldquoBiodiversity of indigenous staphylococci of naturallyfermented dry sausages and manufacturing environments ofsmall-scale processing unitsrdquo Food Microbiology vol 27 no 2pp 294ndash301 2010

[35] E Coton M-H Desmonts S Leroy et al ldquoBiodiversity ofCoagulase-Negative Staphylococci in French cheeses dry fer-mented sausages processing environments and clinical sam-plesrdquo International Journal of Food Microbiology vol 137 no 2-3 pp 221ndash229 2010

[36] S Hosseinzadeh and H D Saei ldquoStaphylococcal species associ-ated with bovinemastitis in the NorthWest of Iran emerging ofcoagulase-negative staphylococcirdquo International Journal of Vet-erinary Science and Medicine vol 2 no 1 pp 27ndash34 2014

[37] A Piette and G Verschraegen ldquoRole of coagulase-negativestaphylococci in human diseaserdquo Veterinary Microbiology vol134 no 1-2 pp 45ndash54 2009

[38] T M Moura F S Campos P A DrsquoAzevedo et al ldquoPrevalenceof enterotoxin-encoding genes and antimicrobial resistancein coagulase-negative and coagulase-positive Staphylococcusisolates from black pudding in Southern Brazilrdquo Revista daSociedade Brasileira deMedicina Tropical vol 45 no 5 pp 579ndash585 2012

[39] H I Suzuki T Lefebure P P Bitar and M J StanhopeldquoComparative genomic analysis of the genus Staphylococcusincluding Staphylococcus aureus and its newly described sister

BioMed Research International 17

species Staphylococcus simiaerdquo BMC Genomics vol 13 article38 2012

[40] R P Lamers G Muthukrishnan T A Castoe S Tafur A MCole and C L Parkinson ldquoPhylogenetic relationships amongStaphylococcus species and refinement of cluster groups basedon multilocus datardquo BMC Evolutionary Biology vol 12 article171 2012

[41] I V Pinchuk E J Beswick and V E Reyes ldquoStaphylococcalenterotoxinsrdquo Toxins vol 2 no 8 pp 2177ndash2197 2010

[42] S R Monday and G A Bohach ldquoGenes encoding staphylo-coccal enterotoxins G and I are linked and separated by DNArelated to other staphylococcal enterotoxinsrdquo Journal of NaturalToxins vol 10 no 1 pp 1ndash8 2001

[43] D Y Thomas S Jarraud B Lemercier et al ldquoStaphylococcalenterotoxin-like toxins U2 and V two new staphylococcalsuperantigens arising from recombination within the entero-toxin gene clusterrdquo Infection and Immunity vol 74 pp 4724ndash4734 2006

[44] N Indrawattana O Sungkhachat N Sookrung et al ldquoStaphylo-coccus aureus clinical isolates antibiotic susceptibility molecu-lar characteristics and ability to form biofilmrdquo BioMed ResearchInternational vol 2013 Article ID 314654 11 pages 2013

[45] V Pereira C Lopes A Castro J Silva P Gibbs and P TeixeiraldquoCharacterization for enterotoxin production virulence factorsand antibiotic susceptibility of Staphylococcus aureus isolatesfrom various foods in Portugalrdquo Food Microbiology vol 26 no3 pp 278ndash282 2009

[46] I Anacarso C Condo C Sabia and et al ldquoAntimicrobialresistance and other related virulence factors in staphylococcusspp isolated from food environmental and humans in ItalyrdquoUniversal Journal Microbiology Research vol 1 no 1 pp 1ndash92013

[47] B Martın M Garriga M Hugas S Bover-Cid M T Veciana-Nogues and T Aymerich ldquoMolecular technological and safetycharacterization of Gram-positive catalase-positive cocci fromslightly fermented sausagesrdquo International Journal of FoodMicrobiology vol 107 no 2 pp 148ndash158 2006

[48] NM Broekema T Van Tam T AMonson S AMarshall andD M Warshauer ldquoComparison of cefoxitin and oxacillin diskdiffusionmethods for detection ofmecA-mediated resistance inStaphylococcus aureus in a large-scale studyrdquo Journal of ClinicalMicrobiology vol 47 no 1 pp 217ndash219 2009

[49] E Cercenado F Garcıa-Garrote and E Bouza ldquoIn vitro activityof linezolid against multiply resistant Gram-positive clinicalisolatesrdquo Journal of Antimicrobial Chemotherapy vol 47 no 1pp 77ndash81 2001

[50] A Aydin K Muratoglu M Sudagidan K Bostan B Okukluand SHarsa ldquoPrevalence and antibiotic resistance of foodborneStaphylococcus aureus isolates in Turkeyrdquo Foodborne Pathogensand Disease vol 8 no 1 pp 63ndash69 2011

[51] D Sergelidis A Abrahim T Papadopoulos et al ldquoIsolation ofmethicillin-resistant Staphylococcus spp from ready-to-eat fishproductsrdquo Letters in Applied Microbiology vol 59 no 5 pp500ndash506 2014

[52] I PhillipsM Casewell T Cox et al ldquoDoes the use of antibioticsin food animals pose a risk to human health A critical reviewof published datardquo Journal of Antimicrobial Chemotherapy vol53 no 1 pp 28ndash52 2004

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Page 5: Research Article Safety Evaluation of the Coagulase ...downloads.hindawi.com/journals/bmri/2015/483548.pdf · Research Article Safety Evaluation of the Coagulase-Negative Staphylococci

BioMed Research International 5

antibiotic concentration that inhibits visible growth after 24 h[26]

3 Results and Discussion

31 Isolation and Identification of Coagulase-Negative Strainsfrom Salami Sixty-five presumable coagulase-negativestaphylococci microorganisms from salami were isolated bycolony morphology coagulase slide test subsequent tubetest and biochemical tests The sequencing of the V5 regionof the 16S rDNA fragment of the strains was discriminativeenough to differentiate the Staphylococcus isolated fromsalami at the subspecies level with the exception of 1 strainidentified up to the Staphylococcus spp genus

Nineteen distinct strains were identified as CNS in salamiwith 08 of 19 (42) identified as S saprophyticus thepredominant species followed by 05 strains of S xylosus(26) 02 strains of S carnosus (11) and 01 strain of eachof the following species S succinus S sciuri S epidermidisand S hominis (5 each) (Table 2)

The 08 S saprophyticus strains were identified asKJ6991511 AB6977171 JX4901221 KJ0046231 andHQ6995101 EU4309921 HF9372521 and KJ9496061 withthe latter two and S sciuri JX9664361 being homologous(96ndash98) to strains from the environment Five S xylosusstrains CP0072081 KF1980801 CP0087241 AM8827001and KC4565901 and a single S succinus strain KC3298241were identified The last three S xylosus strains and theS succinus strain are homologous (97-98) to strainsfound in fermented meat or meat starter cultures Sxylosus CP0072081 showed homology (98) to potentialopportunistic pathogenic strains from mammal species

The S carnosus strains identified were KJ8620021 andNR1164341 and the latter as well as the single S hominisJX5199881 (96-97) identified were homologous (97) tospecies from human microbiota

S carnosus and S xylosus are commonly used as com-mercial starter cultures for sausage manufacturing [26] butS succinus has also been observed in dry fermented sausagesand its use as a starter culture has been already proposed [27]

The diversity of CNS microbiota found in the samplesanalyzed in the present study could be related to the originof the salami The microbiota from the artisanal salamishowed greater biodiversity when compared to the commer-cial salami In commercial salami S saprophyticus was thepredominant species but S xylosus and S carnosus alsoobserved in commercial salami are commonly isolated fromstarter cultures [26] The predominance of S saprophyticusfollowed by S xylosus has also been reported in salamifrom South Italy [28] similar to Belgian sausages where Ssaprophyticus was the most frequently detected species [29]

S carnosus was not observed in artisanal salami but Ssuccinus S epidermidis and S hominis were detected

The biodiversity of the CNS staphylococci species foundin microbiota depends on the kind of meat-fermented prod-uct but CNS strains such as S saprophyticus S auricularisS xylosus S capitis S hominis S carnosus S haemolyticusS warneri S equorum S cohnii S capitis and S intermediushave been described inNapoli-type salami Sremska sausages

dry sausages raw meat and naturally fermented meat [30ndash33]

During the last decades S epidermidis and S sapro-phyticus have been described as emerging pathogens [34]S saprophyticus is considered a frequent contaminant offermented sausages and raw meats and has been isolatedfrom rectal swabs of cattle carcasses and pigs In humans themain reservoir of S saprophyticus is the gastrointestinal tract[35] S saprophyticus and S epidermidis can be opportunisticpathogens isolated from the human urinary tract and thepresence of these species in food should be taken into accountconcerning possible contamination of the starter inoculumandor improvements in the salami manufacturing process[13]

Other CNS species observed in the present study aremainly associated with ordinary food contaminants withS epidermidis and S hominis being the dominant speciesin human skin and occasionally isolated from the skin ofdomestic animals [36]

32 Phylogenetic Relationships of the CNS Identified in SalamiCNS strains can be grouped into four species groupssaprophyticus simulans epidermidis and haemolyticus Fre-quently the saprophyticus species group includes S xylosusand S saprophyticus while the simulans species group iscomprised of S carnosus and S piscifermentans the epi-dermidis species group is composed of S epidermidis Scapitis S caprae and S saccharolyticus and the haemolyticusspecies group encompasses S haemolyticus S hominis andS devriesei [37 38] In the present study the main clustergrouped several S saprophyticus strains namely KJ6991511AB6975171 KJ9496061 JX4901221 and KJ0046231 four Sxylosus strains CP0087241 CP0072081 AM8827001 andKC4565901 and the S succinusKC3298241 strain (Figure 1)These strains are homologous to those from fermented meatmicrobiota

The subclusters of those clusters showed mismatches ofspecies belonging to the four species groups The first sub-cluster grouped S epidermidis HF0882111 S saprophyticusHQ6995101 and S carnosusNR1164341 and the second sub-cluster grouped S hominis JX5199881 and the S saprophyticusEU4309921 all of them originally isolated from animalsand human beings The third subcluster includes speciespreviously isolated from the marine environment such as SxylosusKF1980801 while S carnosusKJ8620021 is utilized asa probiotic organism in foodsThe fourth cluster grouped theS sciuri JX9664361 and S saprophyticus HF9372521 strainswhich are homologous to species found in soil

The CNS strains clustered into groups near the bottomof the phylogenetic tree are mostly strains found in artisanalsalami whereas the species at the top of the tree are mainlyCNS strains found in commercial salami (Figure 1)

The close similarities between the S saprophyticusAB6975171 and KJ9496061 strains the S succinusKC3298241 S xylosus AM8827001 KC4565901 andCP0087241 strains the S saprophyticus KJ0046231 andKJ6991511 strains and S xylosus CP0072081 are supportedby a bootstrap value of 100 The interspecies similaritieswere over 90 which demonstrates close phylogenies

6 BioMed Research International

Table2Genotypicandph

enotypiccharacteriz

ationof

CNSstrainsfrom

salami

Strainsidentificatio

nandcharacteriz

ation

Salamiorig

inStaphylococcus

species

Genotypic

Phenotypic

GenBa

nkaccessionnu

mbera

ndsim

ilarity(

)Presence

ofenterotoxingenes

mRN

Adetection

Enterotoxinprod

uctio

n(ngm

Lminus1)

Com

mercial

Staphylococcus

sppKF

1354451(96)

seaseb

mdashmdash

Scarnosus

KJ8620021(96)

seasebsedsehseiand

selj

mdash12plusmn01

Scarnosus

NR1164341(98)

sebsecsedandsee

see

03

Ssaprophyticus

AB6

977171

(98)

sehseiandselm

seiselm

03

Ssaprophyticus

EU4309921(99)

seasebselm

seln

selo

and

tstH1

sebselm

and

selo

13plusmn01

Ssaprophyticus

HQ6995101(97)

mdashmdash

mdashSsaprophyticus

JX4901221(99)

seaseh

seh

05plusmn01

Ssaprophyticus

KJ00

46231(96)

sec

mdash04

Ssaprophyticus

KJ9496061(98)

seasebseca

ndseh

seasebandseh

mdashSsciuriJX9664361(98)

sedsei

sei

10plusmn01

SxylosusA

M8827001(97)

mdashmdash

mdashSxylosusCP

0072081(99)

mdashmdash

mdashSxylosusC

P0087241(98)

seatstH1

sea

14SxylosusK

C4565901(98)

mdashmdash

mdash

Artisa

nal

SepidermidisKF

6005891(97)

secsedseesegsehandsei

seesei

07plusmn01

ShominisJX5199881(97)

seasebsecsedseljselm

seln

andselo

selnselo

09plusmn01

Ssaprophyticus

HF9

372521(97)

seb

seb

05

Ssaprophyticus

subspbovisK

J6991511

(98)

secselm

seln

andtstH1

seln

09plusmn01

Ssuccinus

KC3298241(99)

seasebsecseljandseln

sebsec

13SxylosusK

F1980801(97)

secselm

andtstH1

mdash05plusmn01

Thep

resenceo

fenterotoxin

genesseasebsecsedseesegsehsei

seljselkselm

seln

selo

selq

selrselu

and

tstH1w

asteste

dby

PCRusingthes

pecific

seto

fprim

ers

sea-seee

nterotoxin

prod

uctio

nwas

evaluatedby

immun

e-sorbentassays(EL

ISA)u

singthed

etectio

nkitR

IDASC

REEN

SETAB

CD

EV

aluesa

redisplay

edas

them

eansplusmnSD

ofassays

perfo

rmed

indu

plicate

mRN

Atranscrip

tsfora

llenterotoxingenesw

eree

valuated

byrealtim

eRT-PC

Rtests

CT

values

ared

isplayedas

meansplusmnSD

ofRT

-PCR

tests

perfo

rmed

indu

plicateseamdash

SxylosusC

P00872413

0plusmn04sebmdash

SxylosusC

P008724133plusmn20Ssaprophyticu

sJX4901221302plusmn04Ssaprophyticus

subspbovisK

J6991511

32plusmn01SxylosusK

F19808013

6plusmn10

and

ShominisJX519988134plusmn10secmdashSsaprophyticus

subspbovisK

J6991511

31plusmn11seemdashSsaprophyticus

subspbovisK

J6991511306plusmn05

Ssuccinus

KC3298241285plusmn01andSsaprophyticus

HF9

3725213

2plusmn04sehmdash

SxylosusC

P0087241 31plusmn10Ssaprophyticu

sJX9

6643613

32plusmn06seimdash

Ssaprophyticus

AB6

977171

30plusmn10

Ssaprophyticu

sJX9664361306plusmn10

seln

mdashShominisJX5199881303plusmn10

Ssaprophyticu

sEU430992131plusmn03andselomdashShominisJX519988132plusmn10

BioMed Research International 7

O S succinus KC3298241Δ S xylosus AM8827001

O S saprophyticus KJ6991511Δ S saprophyticus AB6977171

Δ S xylosus CP0087241Δ S xylosus KC4565901Δ S saprophyticus KJ9496061Δ S saprophyticus JX4901221Δ S saprophyticus KJ0046231

Δ S xylosus CP0072081Δ S carnosus NR1164341Δ S saprophyticus HQ6995101

O S epidermidis KF0882111O S hominis JX5199881Δ S saprophyticus EU4309921

O S xylosus KF1980801Δ S carnosus KJ8620021Δ S sciuri JX9664361O S saprophyticus HF9372521

04 03 02 01 00

Figure 1 Phylogenetic tree generated from the multiple alignments of the 16S rDNA sequences of CNS strains found in salami usingthe ClustalX 20 software The phylogenetic tree was constructed by using the Mega 60 software and the unweighted pair group method(UPGMA) Bootstrap values ranged from 00 to 04 Strains found in commercial (Δ) or artisanal salami (O)

between these CNS strains Suzuki et al 2012 demonstrateda bootstrap value higher than 90 for interspecies similaritiesbetween S saprophyticus S epidermidis S hominis and Scarnosus

33 Genotypic and Phenotypic Characterization of CNSStrains Twelve distinct combinations of staphylococcalenterotoxins geneswere found in the 15CNS strains compris-ing SEs AndashE GndashJ and also the enterotoxin-like toxins (SElL)KndashR and U (Table 2)

Fifteen strains (79) carried at least one gene encodingenterotoxins in their genomes (Figure 2) The seb and secgenes were the most predominant harbored by 57 ofthe strains followed by sea carried by 50 whereas thesedsehselm genes showed intermediate incidence harboredby 33 of the strains while seiseln and tsH1 were found in27 of strains Finally see selj and selo showed low incidence(13) and seg was carried by only 1 of strains (Table 2)

The relationships between superantigenic toxin geno-types and toxin gene-encoding mobile genetic elementsin CNS strains were evaluated Distinct combinations ofSaPI and plasmids or plasmids and genes of egc operonenterotoxins were found in the CNS strains obtained fromsalami

Four strains presented only the classical enterotoxingenes namely S saprophyticus KJ0046231 and HF9372521S xylosus CP0087241 and S carnosus NR1164341 Anotherstrain S saprophyticus AB6977171 presented only the newlydescribed enterotoxins seh sei and selm and finally 10 strainspresented a combination of classical and newly described

enterotoxin genes in their genomes with at least one of eachenterotoxin type (Table 2)

Previous studies have shown that sea is the most com-mon toxin associated with Staphylococcus food poisoningfollowed by sed and see with SEHand SEI being considered asplaying only a minor role [39] Among the 15 enterotoxigenicstrains found in salami 73 of them harbored at least the seagene or combinations of these 05 genes Strains S carnosusKJ8620021 found in commercial salami and S epidermidisKF6005891 found in artisanal salami carry 04 of these genesThe sea and seb enterotoxin genes are known to occupy thesame locus on the chromosome which may explain whythese enterotoxins are commonly found together in foodpoisoning outbreaks [36] The combination of sea and sebgenes was found in 05 strains S saprophyticus EU4309921 Scarnosus KJ8620021 S hominis JX5199881 S saprophyticusKJ9496061 and S succinus KC3298241 A single strain Scarnosus KJ8620021 showed the combination of sea and seigenes

Additionally some CNS are able to produce TSST-1 aloneor in combination with other enterotoxins Herein 04 of the19 strains (21) S saprophyticus EU4309921 S saprophyti-cus subsp bovis KJ6991511 and S xylosus CP0087241 andKF1980801 were shown to harbor the tstH1 combined withse andor sel enterotoxin genes (Table 2)

The staphylococci enterotoxins genes seg and sei [40] arepart of a chromosomal operon gene cluster (egc) comprisingfive genes designated as selo selm sei seln and seg Twoof the CNS strains determined in the present study Ssaprophyticus EU4309921 and S hominis JX5199881 were

8 BioMed Research International

sed

seb

sec

see

sea

M 2 3 4 51

500bp

(a)

sea

sebsebseb

M 1 2 3 4 5

(b)

sei

sehseg

1M

(c)

seh sehseh

M 1 2 3

(d)

1 2 4M 3 5

(e)

selm

seljseln

selo

21M

(f)

seln

selm

M 1 2

(g)

Figure 2 Uniplex duplex and multiplex PCR screening for the detection of enterotoxin genes in CNS strains from salami (a) Lane M100 bp DNA ladder plus (Fermentas Foster City CA USA) lane 1 S aureus ATCC 29231 harboring sea gene lane 2 S aureus NCTC 10654harboring seb gene lane 3 S aureus ATCC19095 harboring the sec gene lane 4 S aureus ATCC 13563 harboring the sed gene and lane 5 Saureus ATCC 27664 harboring the see gene (b) Lane M 100 bp DNA ladder plus lane 1 Staphylococcus spp lane 2 S carnosus NR116434lane 3 S carnosus KJ862002 lane 4 S carnosus KJ862002 and lane 5 S carnosus KJ8620021 (c) Lane M 100 bp DNA ladder plus lane1 S aureus ATCC 19095 harboring seg seh and sei genes (d) Lane M 100 bp DNA ladder plus lane 1 S saprophyticus AB6977171 lane 2S epidermidis KF 6005891 and lane 3 S sciuri JX9664361 (e) Lane M 100 bp DNA ladder plus lane 1 S xylosus KF1980801 lane 2 SsaprophyticusKJ6991511 (f) LaneM 100 bp DNA ladder plus lane 1 S aureusATCC 27154 harboring selj slem seln and selo genes (g) LaneM 100 bp DNA ladder plus lane 1 S xylosus KF1980801 and lane 2 S saprophyticus subsp bovis KJ6991511

BioMed Research International 9

shown to carry the selm seln and selo genes and the selmand seln sequences were detected in the genome of a singlestrain S saprophyticus subsp bovisKJ6991511 S epidermidisKF6005891 showed a combination of seg and sei genesand 03 strains harbored a single gene sei or selm or selnperhaps due to the high degree of genetic polymorphism inthe chromosomal assembly [41] Indrawattana et al 2013detected the seg-sei-selm-seln-selo of the highly prevalent egclocus was 261 in contrast to this study where 13 CNSpresents the combination of selm seln and selo genes [42]

The CNS strains clustered into groups near the bottom ofthe phylogenetic tree carried the classical enterotoxin geneswhile the species at the top of the tree showed high diversityamong the enterotoxin genes combining the classical and thenewly described genes in their genomes (Figure 1)

To assess the risk of staphylococcal food poisoning theability of the identified strains in harboring the se andsel genes and in expressing and producing enterotoxins invitro was evaluated The mRNA for each enterotoxin genewas evaluated by real time RT-PCR assays and enterotoxincontent was estimated by a sandwich enzyme immunoassayfor the combined detection of Staphylococcus enterotoxins(SET) A B C D and E

The fifteen enterotoxigenic CNS strains were able toexpress the classical enterotoxins SEA seb sec sed and seein concentrations ranging from 03 ngmLminus1 to 14 ngmLminus1as assessed by the in vitro assays (Table 2) The S sapro-phyticus AB6977171 KJ6991511 JX4901221 KJ0046231 andHF9372521 strains S xylosus KF1980801 and S carnosusNR1164341 produced low amount of enterotoxins lower than05 ngmLminus1 S epidermidisKF6005891 S saprophyticus bovisKJ6991511 and S hominis JX5199881 produced intermediateamount of enterotoxins ranging from 07 to 090 ngmLminus1while S sciuri JX9664361 S saprophyticus EU4309921 Scarnosus KJ8620021 S xylosus CP0087241 and S succi-nus KC3298241 produced enterotoxins in concentrationsge10 ngmLminus1

Although the sandwich enzyme immunoassay is consid-ered themost sensitivemethod to detect seandashsee enterotoxinsable to detect 0125 ngmLminus1 differences in the specificity andsensitivity of the assays for the detection of staphylococcalenterotoxins from foods are expected [43] A single strainStaphylococcus spp KF1354451 which harbors both the seaand seb genes was unable to produce seandashsee enterotoxins

The mRNA levels evaluated by the real time RT-PCRassays for enterotoxins were detected in 12 of 15 strains(86) (Table 2) Transcripts for the classical enterotoxinsgenes were detected when S saprophyticus HF9372521 Scarnosus NR1164341 S xylosus CP0087241 and S succinusKC3298241 were assayed Transcripts for newly describedenterotoxins were observed in S saprophyticus JX4901221and AB6977171 S sciuri JX9664361 S saprophyticus subspbovis KJ6991511 and S hominis JX5199881 Transcripts fromboth the classical and newly described enterotoxins weredetected in S saprophyticus EU4309921 and KJ9496061 andS epidermidis KF6005891

No mRNA transcripts were obtained for S sapro-phyticus KJ00462321 and S carnosus KJ8620021 although

enterotoxin production was detected by the enzyme-linkedimmunosorbent tests (Table 2)

The 04 strains that carry tstH1 in their genome Ssaprophyticus EU4309921 and KJ6991511 and S xylosusCP0087241 and KF980801 were not able to produce mRNAfor TSST-1 in the assay conditions

The production of classical enterotoxins in vitro(immunologic test) matched the results shown by real timeRT-PCR assays for the following strains S saprophyticusJX4901221 S sciuri JX9664361 S saprophyticusEU4309921S saprophyticus HF9372521 S saprophyticus subsp bovisKJ6991511 S hominis JX5199881 S epidermidis KF6005891and S succinus KC3298241

Ten strains namely S saprophyticusAB6977171 S sapro-phyticus EU4309921 S saprophyticus HF9372521 S sapro-phyticus KJ6991511 S saprophyticus KJ9496061 S succinusKC3298241 S hominis JX5199881 S xylosus KF1980801and S xylosus CP0087241 and S epidermidis KF6005891expressed mRNA for multiple se andor sel genes There isa differential transcription among these genes where themost frequent among the classical ones were seb and seeseatranscribed by 04 and 02 strains respectively and the mostfrequent among the newly described genes were sei andsehselnselo transcribed by 03 and 02 strains respectively

No mRNA for the sec gene was detected although SsaprophyticusAB6977171 was able to produce the enterotoxinin vitro as detected by the ELISA assays

34 Enterotoxin Gene Homologies between Salami CNSand CPS Strains The nucleotide sequencing of six entero-toxin genesmdashtwo of them sec and see encoding classi-cal enterotoxinsmdashand four of them encoding the newlydescribed genes seg seh selm and seln fromCNS strains wascompared to the enterotoxin genes from S aureus or S pas-teuri two coagulase-positive staphylococci strains (Figure 3)The homology between the CNS and CPS enterotoxin genesvaried from 65 to 98 The homology of sec seg sehselm and seln between CNS from salami and S aureus was98 60 98 65 and 70 respectively The homologybetween see from CNS found in salami and in S pasteuri was98

Although further studies should be performed it seemsthat the sequences encoding enterotoxins can be conservedamong coagulase-negative and coagulase-positive staphy-lococci as shown in the phylogenetic analysis of the 06enterotoxin genes (Figure 4)

35 Antimicrobial Multiresistance of CNS Strains (MRCNS)Another safety hazard associated with CNS strains besidesthe ability of producing enterotoxins in food matrices is theantimicrobial resistance to antimicrobial agents commonlyused to treat staphylococci infections The antimicrobialresistance carried by CNS strains from food matrices can bespread to the population by the consumption of an apparentlysafe food

Among the 19 CNS strains identified in salami 14 showedmultiresistance to antimicrobial agentsThree strains showedthe highest MAR indices 093 and 080 07 strains presented

10 BioMed Research International

lowast lowastlowastlowastlowastlowast lowast lowast lowastlowast lowast lowast lowastlowast lowastlowastlowastlowast lowastlowastlowast lowast

lowastlowast lowast lowast lowastlowastlowastlowast lowast lowast lowastlowast lowast

lowastlowastlowastlowast lowast lowast lowast

lowastlowast lowast lowast lowastlowast lowastlowast lowast lowastlowast lowastlowastlowastlowastlowast lowastlowast lowast lowastlowast lowastlowast

lowastlowast lowastlowastlowast lowast lowast lowast lowast lowast lowastlowastlowastlowastlowast

lowastlowast lowastlowast lowastlowast lowastlowast lowast lowast lowastlowast lowast lowast lowastlowastlowast lowast lowastlowast lowastlowastlowast

lowast lowast lowast lowast lowast lowast lowastlowast lowast lowastlowastlowast lowast lowastlowastlowast lowast lowast lowastlowastlowast lowast lowastlowastlowast lowast

lowast lowast lowast lowastlowastlowastlowast lowast lowastlowastlowast lowast lowast lowastlowastlowast lowast lowast lowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast lowast lowast lowast lowast lowastlowast lowast lowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

Enterotoxin C (sec) GATAAAAATGAATAAGAGTCGATTTATTTCATGCGTAATTTTGATATTCGCACTTATACT------------------------------------------------------------

AGTTCTTTTTACACCCAACGTATTAGCAGAGAGCCAACCAGACCCTACGCCAGATGAGTT------------------------------------------------------------

GCACAAAGCGAGTAAATTCACTGGTTTGATGGAAAATATGAAAGTTTTATATGATGATCA------------------------------------------------------------

TTATGTATCAGCAACTAAAGTTAAGTCTGTAGATAAATTTTTGGCACATGATTTAATTTA------------------------------------------------------------

TAACATTAGTGATAAAAAACTGAAAAATTATGACAAAGTGAAAACAGAGTTATTAAATGA------------------------------------------------------------

AGGTTTAGCAAAGAAGTACAAAGATGAAGTAGTTGATGTGTATGGATCAAATTACTATGT------------------------------------------------------------

AAACTGCTATTTTTCATCCAAAGATAATGTAGGTAAAGTTACAGGTGGCAAAACTTGTAT------------------------------------------------------------

GTATGGAGGAATAACAAAACATGAAGGAAACCACTTTGATAATGGGAACTTACAAAATGT------------------------------------------------------------

ACTTATAAGAGTTTATGAAAATAAAAGAAACACAATTTCTTTTGAAGTGCAAACTGATAA------------------------------------------------------------

GAAAAGTGTAACAGCTCAAGAACTAGACATAAAAGCTAGGAATTTTTTAATTAATAAAAA------------------------------------------------------------

AAATTTGTATGAGTTTAACAGTTCACCATATGAAACAGGATATATAAAATTTATTGAAAA----TTGTATGAGTTTAACAGTTCACCATATGAAACAGGATATATAAAATTTATTGAAAA

TAACGGCAATACTTTTTGGTATGATATGATGCCTGCACCAGGCGATAAGTTTGACCAATCTAACGGCAATACTTTTTGGTATGATATGATGCCTGCACCAGGCGATAAGTTTGACCAATC

TAAATATTTAATGATGTACAACGACAATAAAACGGTTGATTCTAAAAGTGTGAAGATAGATAAATATTTAATGATGTACAACGACAATAAAACAGTTGATTCTAAAAGTGTGAAGATAGA

AGTCCACCTTACAACAAAGAATGGATAATGTTAATCCGATTTTGATATAAAAAGTGAAAGAGTCCACCTTACAACAAAGAATGGATAATGTAATCCGATTATGGACAAGTGTGCTTTTTT

Enterotoxin C TATTAGATATATTTGAAAGGTAAGTACTTCGGTGCTTGCCTTTTTAGGATGCATATATATEnterotoxin C CNS

Enterotoxin CEnterotoxin C CNS

Enterotoxin CEnterotoxin C CNSEnterotoxin CEnterotoxin C CNSEnterotoxin CEnterotoxin C CNSEnterotoxin CEnterotoxin C CNSEnterotoxin CEnterotoxin C CNSEnterotoxin CEnterotoxin C CNSEnterotoxin CEnterotoxin C CNSEnterotoxin CEnterotoxin C CNS

Enterotoxin CEnterotoxin C CNSEnterotoxin CEnterotoxin C CNS

Enterotoxin CEnterotoxin C CNS

Enterotoxin CEnterotoxin C CNS

Enterotoxin CEnterotoxin C CNS

TTTGTGATCGTCGGCCA-----ACCGATACCGTCGTATTCTTGTACCGCGCAAGGCGAAGlowast lowast lowastlowastlowast lowast lowast lowast lowast lowastlowast lowast lowastlowastlowast lowast lowast lowast lowast

Enterotoxin E (see) Enterotoxin E TGCTTTAGGCGGGGTCTTACAGCATATTATCGTAGAAGATGCACAAGCTGCACGTAAAGCS pasteuri TGCTTTAGGCGCATCGTTACAACATGTCATCGTAGAAGATGAAAAAGATGGTCGTCAGGC

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast lowastlowastlowastlowast lowastlowastlowast lowast lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast lowast lowastlowastlowast lowastlowast lowastlowastlowast lowast lowastlowastlowast

Enterotoxin E ATTAGAATTCTTGAAATCAAAACA-AGCAGGACGTGCTACATTTCTACCATTAAATGTCAS pasteuri

Enterotoxin ES pasteuri

GATACAATT-TTTAAAACAACGTAGTTTAGGACGTGCCACATTCCTACCTTTAAATGTTA lowastlowast lowastlowastlowastlowast lowastlowast lowastlowastlowast lowastlowastlowast lowast lowastlowastlowastlowastlowastlowastlowastlowastlowast lowastlowastlowastlowastlowast lowastlowastlowastlowastlowast lowastlowastlowastlowastlowastlowastlowastlowast lowast

TCAAACCTAGAAATATTTCAACCAAGATATATlowast lowastlowastlowastlowast lowastlowastlowast lowastlowastlowastlowastlowast

Enterotoxin G (seg) Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

---------------------------attaagaaatactt--ccattttaatactcttaGGGGATATTTCCGGGAAACCGGGGCTAATACTGGATAACTTTTCTCTCCGAACGAAGAGA

gaatcaacaactttattatctccg--taaat-----atttaaaaacttatatggaacaaaGATTCAACGATGGTCTTGGTTTTCAATTAAGATGAACCCGCGGCGCTTAGCTGGTGGGAC

aggtactagtt-----------------------------------------cttttttaAGGTCACGGCTCACCCAAGCCCCCATGCGTATACGACCTGGAAGGGTGATCGGCCACATT

ggaaataagtcaaaccaaaaacttgtattgttcttttcagtaaat---------------GGGACTGAGACACAGCACAAACTCCTACGGGAGGCAGCAATAGGGAATCTTCCGCAATGG

------------------------------------------------------------CCGAAAGTCTGACGGAGCAACGCCGCGAGACTGAAGAATGTCTTCGGATCGTAAAACTCT

------------------------tttatatatccagattcaaa-----tgcagaaccatGTTGTTAGGGAAGAACAAGTATCGTTCGAATAGGGCGGTACCTTGACGGTACCTAACCAG

caaactcgtatagctttttttct---------ttagtgagccagtgtcttgctttg----AAAGCTTTGCTAACTACCTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCG

-taatctagttcctgaatagtaaccatattcttatttgtagttattgta--aatccaagtGAATTATTGGGCGTAAAGCGCGCGCATGCGGTCTTTTAAATCTGATGTCAAAGCCCACGG

gattgtct----------attgtcgattgttacctgtacagt------------aattaaGCTCAACCGTGGAGGGTCATTGGAAACTGTAAGACTTGGAGTGCAGAAGAGGATAGTGGA

Figure 3 Continued

BioMed Research International 11

lowast lowast lowast lowastlowast lowastlowast lowastlowastlowast lowast lowastlowast lowast lowast lowast lowast

lowast lowast lowastlowastlowast lowastlowast lowast lowastlowastlowast lowast lowastlowast lowastlowastlowast lowast lowastlowast lowastlowast lowast lowast lowastlowast lowast

lowastlowast lowast lowast lowastlowastlowastlowastlowast lowast lowast lowastlowast lowast lowast lowast lowast lowast lowastlowastlowast

lowastlowast lowast lowastlowast lowastlowastlowast lowastlowast lowastlowast lowast lowast lowast lowast lowastlowast lowastlowast lowast lowast lowast lowastlowast

lowastlowast lowastlowastlowastlowast

lowast lowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowast lowast lowastlowastlowastlowast lowast lowastlowast lowastlowast lowast lowastlowastlowastlowast lowast lowastlowast lowast lowast lowastlowastlowastlowastlowastlowastlowast

lowastlowast lowast lowast lowast lowast lowastlowastlowast lowastlowast lowastlowast lowast lowastlowastlowastlowastlowastlowastlowastlowast lowastlowastlowast

lowast lowast lowast lowast lowast lowast lowastlowast lowastlowast lowast lowast lowastlowastlowast lowast lowast lowastlowastlowast lowast lowast

lowastlowast lowastlowastlowastlowastlowastlowast lowastlowast lowastlowastlowast lowastlowast lowast lowastlowast lowastlowast lowastlowastlowast lowast lowast

lowastlowast lowast lowastlowastlowast lowast lowastlowastlowastlowastlowast lowast lowast lowast lowastlowastlowast lowast lowastlowast lowast

lowastlowast lowast lowast lowast lowast lowastlowast lowast lowastlowast lowast lowastlowast lowast lowastlowast lowastlowastlowastlowast lowast lowast lowastlowast

lowast lowastlowastlowast lowast lowastlowast lowast lowast lowastlowast lowast lowastlowast lowast lowastlowastlowastlowastlowastlowast lowast

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

tttatctctttcattttctgaactattaaa----tgtaagaccacc---atacatacaacATTCCACGTGTAACGGTGAAAATGAGTAGAGATGTGGAGGAACACCAGTGGAGAAACGCG

aacctccaaaattttggtttatatccggttc-----------agatttaggtattatacaACTCTCTGGTCTGTTACTGACGCTGAGGCGCGAAAGCGTGGGAGCAAACAGGATTATATA

tgtataaaaatatggaacgccaaaaatgtctacttttttatctttataattgttagctaaCCCCTGGTAGTCCAAGCCGTAAACGATGAGTGCTAGTGTTACAGGGT------------T

ttctgtattttctaattcagttttaacctcattataactcttatactcaattggaaaaatTCCGCCCTTTACTGCTGCAGCTAACG---CATTAG-GCACTCCGCCTGGGGAGTACAACT

taaatcatgagataaaaactgtctagaattaataactcctcttccttcaacaggtggagaGCAA------GGTTGAAACTCAAAGCA----TTGACCGGGGGCC---CGATAAGCTGAGG

cgtataaagattcattacat----tacccatagttcccttattgtttttataatcacttaTACATGGTGTTTAATTCCAAACCACGCCAAGAACCCTCACGAGGTCTTGACATCCTCTGG

ctttattt----------------------------------------------------ACCTCCCTAGAGAATAAGGGCTTCCCCGTCGGGGATGAAATGAACAGGTGGTGCATGGAT

-------agttcgtc---AGTCTTCAGCTCGTGCAT

Enterotoxin H (seh)

------------------------------------------------------------ATCTATGAATATAAATCTTTTAATATTGGTTTAATATAAATATTCCATTTCTAGAAACCT

------------------------------------------------------------ATATTTTATGTTTGTATAAATTCATAAATTGTTCGAAATTTATATATTAAAAGTTGTTTT

------------------------------------------------------------TGTTGTTTTATAATTAAGTTAATGAAATATATTGAGGAGTTTTAAAATGATTAATAAAAT

------------------------------------------------------------TAAAATATTATTTTCGTTTTTAGCATTATTACTTTCATTCACATCATATGCGAAAGCAGA

------------ -----------TTACGATTTAGCTTTAGCTAATGCATATGGTCAATAAGATTTACACGATAAAAGTGAGTTAACAGATTTAGCTTTAGCTAATGCATATGGTCAATA

TAATCACCCATTCATTAAAGAAAATATTAAGAGTGATGAAATAAGTGGAGAAAAAGATTTTAATCACCCATTCATTAAAGAAAATATTAAGAGTGATGAAATAAGTGGAGAAAAAGATTT

AATATTTAGAAATCAAGGTGATAGTGGCAATGATTTGAGAGTAAAGTTTGCAACTGCTGAAATATTTAGAAATCAAGGTGATAGTGGCAATGATTTGAGAGTAAAGTTTGCAACTGCTGA

TTTAGCTCAGAAGTTTAAAAATAAAAATGTAGATATATATGGGGCATCTTTTTATTATAATTTAGCTCAGAAGTTTAAAAATAAAAATGTAGATATATATGGGGCATCTTTTTATTATAA

GTGTGAAAAAATAAATGAAAATATTTCTGAATGTCTATATGGAGGTACAACACTAAATAAGTGTGAAAAAATAAGTGAAAATATTTCTGAATGTCTATATGGAGGTACAACACTAAATAG

TGAAAAATTGGCACAGGAAAGGGTGATTGGTGCTAATGTTTGGGTAGATGGTATTCAAAATGAAAAATTGGCACAGGAAAGGGTGATTGGTGCTAATGTTTGGGTAGATGGTATTCAAAA

AGAAACAGAATTAATACGAACAAATAAGAAAAATGTGACATTGCAAGAATTAGATATAAAAGAAACAGAATTAATACGAACAAATAAGAAAAATGTGACATTGCAAGAATTAGATATAAA

Enterotoxin H CNS GATCAGAAAAATATTGTCCGATAAATATAAAATTTATTATAAAGACAGCGAAATAAGGG-Enterotoxin H

Enterotoxin H CNSEnterotoxin H

Enterotoxin H CNSEnterotoxin HEnterotoxin H CNSEnterotoxin HEnterotoxin H CNSEnterotoxin HEnterotoxin H CNSEnterotoxin HEnterotoxin H CNSEnterotoxin H

Enterotoxin H CNSEnterotoxin H

Enterotoxin H CNSEnterotoxin H

Enterotoxin H CNSEnterotoxin H

Enterotoxin H CNSEnterotoxin H

Enterotoxin H CNSEnterotoxin H

GATCAGAAAAATATTGTCCGATAAATATAAAATTTATTATAAAGACAGCGAAATAAGTAA

Enterotoxin N (seln) TGATTTAGCTTTAGCTAATGCATATGGTCAATAT-AATCACCCATTCATTAAAGAAAA-----TAGATATTTATGGACTGTATTATGGAAATAAATGTGTAGGCTTAACTGAAGAAAAAA

TATTAAGAGTGATGAAATAAGTGGAGAAAAAGATTTAATATTTAGAAATCAAGGTGATAGCATCATGCTTATACGGAGGAGTTAC------GA--TATATGATGGAAATCAATTAGATGA

TGGCAATGATTTGAGAGTAAAG--------------TTTGCAA-CTGCTGATTTAGCTCAAGAGAGAGTTATAGGCGTTAATGTATTTAAAGATGGTATCCAACAAGAAGGTTTTGTTAT

GAAGTTTAAAAATAAAAATGTAGATATATATGGGGCATCTTTTTATTATAAGTGTGAAAAAAAAACTAAAAAGGCTAAAGTAACAGTACAA------GAATTAGATACTAAAGTTCGATT

AATAAGTGAAAATATTTCTGAATGTCTATATGGAGGTACAACACTAAATAATGAAAAATT------------TAAATTAGAAAATTTATATAAAATATACAATAAAGATACCGGTAACAT

Enterotoxin N CNSEnterotoxin N

Enterotoxin N CNSEnterotoxin N

Enterotoxin N CNSEnterotoxin N

Enterotoxin N CNSEnterotoxin N

Enterotoxin N CNSEnterotoxin N

Figure 3 Continued

12 BioMed Research International

lowastlowastlowast lowastlowast lowastlowast lowastlowast lowast lowastlowast lowastlowastlowast lowastlowast lowast lowastlowastlowastlowast

lowastlowast lowast lowastlowast lowast lowast lowastlowast lowastlowastlowastlowastlowast lowast lowast lowast lowastlowast lowast lowast lowastlowast lowast lowast

lowast lowast

lowast lowastlowast lowast lowastlowast lowast lowast lowast lowast lowast lowast lowast lowastlowast lowast lowastlowastlowastlowast lowast

lowast lowastlowast lowast lowast lowastlowast lowast lowastlowastlowast lowast lowastlowast lowast lowastlowast lowastlowast lowast lowast

lowastlowastlowast lowast lowast lowastlowast lowastlowast lowast lowast lowast

lowast lowast lowast lowastlowastlowast lowastlowastlowast lowastlowast lowast lowastlowastlowastlowastlowast lowastlowastlowast lowast

lowastlowast lowast lowast lowastlowast lowastlowastlowast lowastlowast lowast lowast lowastlowastlowastlowast lowastlowast

lowast lowast lowast lowastlowast lowast lowastlowast lowastlowastlowastlowastlowastlowast lowastlowast lowast lowastlowast lowastlowastlowast

lowast lowast lowast lowast lowastlowast lowastlowastlowastlowast lowast lowast lowastlowastlowast lowast lowast lowast lowast lowast

lowast lowast lowast lowast lowastlowastlowast lowast lowast lowastlowast lowastlowast lowast lowastlowastlowast lowast lowastlowastlowast lowast

lowast lowast lowastlowastlowast lowast lowastlowast lowastlowastlowast lowastlowastlowastlowastlowast lowastlowast lowast lowastlowastlowast lowast lowast lowastlowastlowast

lowastlowastlowast lowast lowastlowast lowastlowast lowastlowast lowast lowast lowast lowast lowastlowast lowastlowast lowastlowast lowast lowast

lowast lowast lowast lowast lowastlowast lowast lowast lowastlowast lowastlowast lowast lowastlowastlowast lowastlowastlowastlowast lowast lowastlowastlowastlowastlowast lowast

lowastlowast lowast lowast lowastlowastlowast lowastlowast lowast lowastlowast lowast lowastlowast lowastlowast lowastlowast lowast lowast lowast

lowastlowastlowastlowastlowast lowastlowast lowastlowast lowast lowastlowast lowast lowast lowast lowast lowastlowast lowastlowast

lowast lowast lowastlowast lowastlowast lowastlowastlowastlowastlowastlowast lowastlowast lowast lowast lowast lowast lowast lowast lowast lowast lowast lowast lowast

lowastlowast lowastlowast lowast lowastlowastlowastlowast lowast lowast lowastlowastlowast lowastlowast lowast lowastlowastlowast lowast

lowastlowastlowastlowast lowastlowast lowast lowast lowast lowast lowastlowast lowast lowast lowast lowastlowastlowastlowastlowast lowastlowast lowastlowastlowast lowast

lowast lowastlowastlowast lowastlowastlowastlowastlowast lowast lowast lowast lowastlowast lowast lowast lowastlowast lowast lowastlowastlowast lowastlowast lowast

lowastlowast lowast lowast lowast lowastlowast lowast lowastlowast lowastlowast lowastlowast lowast lowastlowastlowastlowast

ATTAATACGAACAAATAAGAAAAATGTGACATTGCAAGAATTAGATATAAAGATCAGAAA-TTAT----------TATGATTTATATAAC--------------ATAAAAGGTTCAG---

AATATTGTCCGATAAATATAAAATTTATTATAAAGACAGCGAAATAAGGTAAAAGG------TAGGAGCAGAGTTTTTTCAATTTTATAGTGATAATAGAACAGTTAGCTCATCTAATTA

Enterotoxin N CNS -TAAA---Enterotoxin N

lowast lowastlowastlowastlowastlowast lowastlowast lowast lowast lowast lowastlowast lowast lowastlowastlowastlowastlowastlowast lowastlowast lowastlowast

GGCACAGGAAAGGGTGATTGGTGCTAATGTTTGGGTAGATGGTATTCAAAAAGAAACAGAACAA----AAAGGATGCATTTTCTTT-----CATTCTAATAATCATCAAAATCAATCATT

Enterotoxin N CNSEnterotoxin N

Enterotoxin N CNSEnterotoxin N

Enterotoxin N CNSEnterotoxin N

TCATATCG

Enterotoxin M (selm)

ttgctattcgcaaaatcatatcgcaaccgctgatgtcggagtt--ttgaatcttaggaacEnterotoxin M CNSEnterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

--------------AGTATGTTTAAATCCCCGCTCACAGTTATGTTTAACTCTTCCTTTC

tattatggta-gctatccaattgaagaccaccaaagtattaatcctgaaaataatcatctTTTTCACACCGCCCCGTCGGGTGTTGCGCACGGGGTTGTTTAACCCCCCCGGAAAAAATT

ttcgcatcaattagttttttctatggataattcgacagtaacagctgaatttaagaacgtEnterotoxin M CNS

Enterotoxin M CNS

TTCCCTCCCTCCTCCTTACTC---------------CCT----------------CCCCT

tgatgatgtaaaggaattcaaaaatcatgctgtagatgtatatggtctaagttatagtggEnterotoxin M CNS TTTTCCTACAAATCCTTTCTCCCCTCCCGGAGTAGAAGTAAA------------------

atattgtttgaaaaacaaatatatatacggtggagttacattagcaggtgat-tatttagEnterotoxin M CNS -------------------TAAAACAATACCCCTACCACCTTAACAATTTTTTTATTGAG

agaaatctagacgtattcct-attaatctttgggttaatggagaacatcaaactatatctEnterotoxin M CNS CCCCTCCCCCCCCTTCTCATTTTTTCTCTTTGAC-----------GTTCCCCCCATTTCT

actgacaaagtatcaactaataaaa-agttagtaacagctcaagaaattgatactaaattEnterotoxin M CNS GCCAATGCTTAACCCACCCTTAAAGGCCATCGGCACATATTAGACACTCC-----CCACC

aagaagatatctacaagaagaatataatatttatggctttaatgatac---aaataaaggEnterotoxin M CNS CGCCCCCTCTTTTTATTAAGGACACAACTCTTTTTATTTTCCCCCAAGAACTAATTCTGA

a-agaaattatggtaataagtcaaaatttagttctgga---------tttaatgcaggaaEnterotoxin M CNS AAAAAAACGTTCCGCTTAGTTCAGAATTTCCTTTGAGCCTTTTATCCTTTTGTACGCGAA

aaatattatttca--------tttgaatgatggttcatcattttcttatgacttatttga

Enterotoxin M CNS AAACCATCAGCCATTGAATCAGTTATCCCATTGCCCTAACTAACCCAATATCTAATCTAA

tactggaacaggacaagctgaaagtttctt-aaaaatatataatgacaacaaaactgtcgEnterotoxin M CNS TTTTCGCCTACGAACGAATAATAGCTTGTGGCGAAAAATATCCAGGATTATCTACTGTAG

aaact--gaaaaattccatttagatgtagaaatatcttataaggacgaaagttgaagtatEnterotoxin M CNS CGCCTCGCCAGACGTCCTTTGAATTGA--ATATGACTCCTACTCCCCCCCGCACCGTCAA

ttcaatcataacttagtaaaggaaatgccatgaaaaaatttaaatatagtttt---atatEnterotoxin M CNS TTCAAACA---------------GGTGGAAGTTAATACTGAATAGTAGGTCTTCTTCCCG

tagtttttatattactttttaacattaaagatcttacgtatgct---caaggtgatattgEnterotoxin M CNS TGCTATTCTTAGAACTTTTAAATTATGTAAACCCAATTTTTAGTGCACTCTTTCTCCCTC

gtg-taggtaacttaagaaatttctatacaaaacatgattatatagatttaaaaggcgtcEnterotoxin M CNS GTTACTCTTAGATGGAGAAGCGTTAAGACAGAAGCTTTGTAT-------------TCCCA

acagataaaaa-cctacctattgcaaatcaacttgaattttcaac-----aggt----acEnterotoxin M CNS GCAGAGTGCAACCTTTCACAATGAAGTTTTTATCTAATTTACACGCCTTTAGGCTCCAAA

caatgatttgatctcagaatctaataattgggacgaaataagtaaatttaaaggaaagaaEnterotoxin M CNS TACGTATTAAATCTCCTCCTGT--TTATCTGACCCTAAG-ATAAAACACGAAAAATTATG

actggatatttttggcattgattataatggtccttgtaaatctaaatacatgtatggaggEnterotoxin M CNS CGTGCCCACTCATCCTAT-------TACTTTCATTATATCTATAAAA-------------

Figure 3 Alignment of the enterotoxin gene sequences found in coagulase-negative staphylococci (CNS) and in coagulase-positivestaphylococci (CPS)

BioMed Research International 13

Enterotoxin (seg) S aureus AY1587031

Enterotoxin (sec) CNS

Enterotoxin (see) S pasteuri NC0227371

Enterotoxin (see) CNS

Enterotoxin (seh) CNS

Enterotoxin (selm) S aureus AF2857601

Enterotoxin (seln) CNS

Enterotoxin (seh) S aureus AJ9375481

Enterotoxin (seln) S aureus AY1587031

Enterotoxin (sec) S aureus X516611

Enterotoxin (selm) CNS

Enterotoxin (seg) CNS

150 100

100

100

100

100

99

98

96

97

72

50 0

Figure 4 Phylogenetic tree generated from the multiple alignments of the enterotoxin sequences of CNS and CPS strains using the ClustalX20 software package The phylogenetic tree was constructed using the Mega 60 software and the unweighted pair group method (UPGMA)

MAR indices varying from 066 to 046 and the remaining04 strains showed MAR indices le026 (Table 3)

The 14 MRCNS strains were resistant to 120573-lactams(oxacillin penicillin andor cefoxitin) and to vancomycincorresponding to 73 of the total CNS strains identifiedwhile 09 strains (64) showed resistance to tetracycline andgentamicin 08 strains (57) were resistant to neomycinerythromycin and chloramphenicol 07 strains (50) wereresistant to sulfamethoprim 05 strains (36) were resistantto linezolid 03 strains (21) were resistant to rifampicin 02strains (14) were resistant to ciprofloxacin and cefepimeand 01 strain (7) was resistant to clindamycin (Table 3)

The multiresistance of CNS strains demonstrated hereinis consistent with previous studies on coagulase-negativeand coagulase-positive staphylococci that have found severalresistant and multiresistant Staphylococcus aureus strains inraw milk meat and fermented meat products [43]

Surprisingly the resistance to chloramphenicol is verysimilar to that estimated for MRCNS strains isolated fromhuman clinical samples Indeed there is no direct correla-tion between the researched features and the origin of thestaphylococci strains since different virulence factors arewidespread such as antibiotic resistance [44] reinforcing thefact that the ability of foodmatrices strains to produce SE andSEl and their multiresistance character must be consideredwhen evaluating the safety hazards of food poisoning

S epidermidis KF6005891 and S hominis JX5199881showed the highestMAR indexes (093 and 080 resp)Thesestrains found in artisanal salami are homologous to strainsisolated from human skin microbiota and should be carefullyconsidered among the potential pathogenic staphylococcifound in food from animal origins which can be caused bycontamination by poor hygienic conditions during salamimanufacturing

Depending on the conditions some species of coagulase-negative staphylococci can present health risks since theyhave shown resistance to antibiotics of therapeutic impor-tance such as beta lactams [15] However multiresistantstrains like S carnosusKJ18620021 and S xylosusCP0072081and CP0087241 were intentionally introduced in the foodmatrix since they are part of the culture starter used inItalian-salami manufacturing As previously discussed foodcan be a reservoir of multiresistant microorganisms that canspread by the consumption of an apparently safe food Dueto the intensive and indiscriminate use of antibiotics forhuman and veterinarian therapeutic purposes multiresistantstaphylococci strains are being selected and reproduced infood matrices [45]

The cefoxitin and oxacillin disk diffusion tests are rec-ommended for determining the resistance and susceptibilitybreakpoint for MRSA surveillance cultures [46] In thisstudy there was a good correlation between the disc testzone diameters for oxacillin and cefoxitin (Table 3) TheMICs for ampicillin andmethicillinoxacillincefoxitin for allsusceptible CNS strainswere determined by the diffusion disctest using 003 to 2mgmLminus1 of each antimicrobial agent

S saprophyticus HF9372521 showed an MIC of003mgmLminus1 formethicillin strain S carnosusKJ8620021 Sxylosus strains CP0087241 and CP0072081 S saprophyticusJX4901221 S hominis JX5199881 and S succinusKC3298241 were resistant to 006mgmLminus1 and two strainsS saprophyticus KJ6991511 and S saprophyticus KJ0046231showed an MIC of 05mgmLminus1 (Table 4) Two of themethicillin-resistant strains S saprophyticus KJ0046231 andS xylosus CP0072081 harbor no enterotoxin gene

Five strains S saprophyticus JX4901221 S carnosusKJ8620021 S xylosusCP0087241 S xylosusKF1980801 andS saprophyticus KJ0046231 showed an MIC for ampicillin

14 BioMed Research International

Table 3 Multiple resistance to antimicrobial as found in CNS strains from salami

Salami origin CNS strains Antimicrobial agent resistance Multiple antimicrobialresistance (MAR) indexlowast

Commercial Staphylococcus spp KF1354451 CIP CLO CPM GEN NEO OXA PENSXT TET and VAN 066

Commercial S carnosus KJ8620021 CIP GEN LZD NEO OXA SXT andTET 046

Commercial S saprophyticus AB6977171 CFO CLO ERI PEN OXA TET andVAN 046

Commercial S saprophyticus EU4309921 CFO CLO ERI GEN LZD NEO OXAPEN SXT TET and VAN 080

Commercial S saprophyticus JX4901221 CFO CLO ERI GEN NEO PEN OXASXT TET and VAN 066

Commercial S saprophyticus KJ0046231 CFO OXA and PEN 020

Commercial S sciuri JX9664361 CFO CLO GEN NEO OXA PEN andTET 046

Commercial S xylosus CP0072081 OXA LZD PEN and VAN 026

Commercial S xylosus CP0087241 CFO CLO ERI GEN NEO OXA PENTET and VAN 060

Artisanal S epidermidis KF6005891 CFO CLI CLO CPM ERI GEN LZDNEO OXA PEN RIF SXT and TET 093

Artisanal S hominis JX5199881 CIP CPM CPO ERI GEN LZD NEOOXA PEN RIF SXT and VAN 080

Artisanal S saprophyticusHF9372521 CFO OXA PEN and SXT 026Artisanal S saprophyticus KJ6991511 OXA PEN and VAN 020

Artisanal S succinus KC3298241 CFO CLO ERI GEN OXA PEN RIFSXT TET and VAN 066

Artisanal S xylosus KF1980801 CFO CLO ERI GEN NEO OXA PENTET and VAN 060

lowastTheMAR index of an isolate is defined as 119886119887 where 119886 represents the number of antimicrobials to which the isolate was resistant and 119887 represents the numberof antimicrobials to which the isolate was subjectedS aureus strains ATCC WB81 (sea) ATCC 13563 (sed) and ATCC 27664 (see) showing a MAR index of 05 and S aureus strains ATCC14458 (seb) andATCCWB72 (sec) and S xylosus ATCC 29971 showing a MAR index of 03 were used as reference strainsCPM cefepime CFO cefoxitin CLO chloramphenicol CIP ciprofloxacin CLI clindamycin ERI erythromycin GEN gentamycin NEO neomycin LZDlinezolid RIF rifampicin TET tetracycline OXA oxacillin PEN penicillin SXT sulfamethoprim and VAN vancomycin

Table 4 Minimal inhibitory concentration (MIC) of compounds used in antimicrobial therapy against staphylococci infections

Salami origin GenBank accession number and similarity () MIC mgmLminus1

Methicillin Ampicillin Vancomycin Linezolid

Commercial

S carnosus KJ8620021 006 003 mdash mdashS xylosus CP0087241 006 003 006 mdash

S saprophyticus JX4901221 006 003 05 mdashS saprophyticus AB6977171 mdash mdash 05 mdashS saprophyticus EU4309921 mdash mdash 025 0125

S succinus KC3298241 006 025 003 mdash

Artisanal

S hominis JX5199881 006 05 mdash mdashS saprophyticus HF9372521 003 mdash mdash mdash

S xylosus KF1980801 003 05 mdashS saprophyticus KJ6991511 05 025 003 mdashS saprophyticus KJ0046231 05 003 003 mdash

S xylosus CP0072081 006 mdash mdash 025Strains S epidermidis KF6005891 and Staphylococcus spp KF1354451 were not susceptible to the antimicrobial concentrations tested in the present study

BioMed Research International 15

of 003mgmLminus1 two strains S succinus KC3298241 and Ssaprophyticus KJ6991511 showed an MIC of 025mgmLminus1and S hominis JX5199881 showed an MIC of 05mgmLminus1(Table 4) S saprophyticus KJ0046231 also showed an MICof 003mgmLminus1 but does not harbor enterotoxin genes It isimportant to note that most of the CNS strains that showedresistance to ampicillin were also resistant to methicillin ashighlighted by the MIC tests

Five strains S saprophyticus HF9372521 S saprophyti-cus KJ6991511 S saprophyticus JX4901221 S saprophyticusKJ0046231 and S succinus KC3298241 showed an MICfor vancomycin of 003mgmLminus1 one strain S xylosusCP0087241 showed an MIC of 006mgmLminus1 one strain Ssaprophyticus EU4309921 showed an MIC of 025mgmLminus1and two strains S xylosus KF1980801 and S saprophyticusAB6977171 showed an MIC of 05mgmLminus1 (Table 4)

Additionally five CNS strains S xylosus CP0087241S saprophyticus JX 4901221 S succinus KC3298241 Ssaprophyticus KJ6991511 and S saprophyticus KJ0046231showed resistance tomethicillin as demonstrated by theMICdeterminations (Table 4)

Previous studies have demonstrated that linezolid isactive against Gram-positive bacteria including methicillin-resistant staphylococci [47] In the present study 05 strainsshowed linezolid resistance in the disc diffusion test but itwas impossible to establish MIC values for S epidermidisKF6005891 Staphylococcus hominis JX5199881 and Staphy-lococcus carnosus KJ8620021The remaining strains S sapro-phyticus EU4309921 and S xylosus CP0072081 presentedan MIC for linezolid of 0125mgmLminus1 and 025mgmLminus1respectively

The MIC of 003mgmLminus1 for penicillin is in accordancewith the previous values estimated for resistant S aureusstrains found in several food matrices such as meat dairyproducts and ready-to-eat food [48]

There is still a lack of information on the antimicro-bial resistance of staphylococci strains from food matricesalthough they are the most worrisome vehicles of dissemi-nation of antibiotic-resistant pathogens [49]

The high resistance found for salami staphylococci strainscan be ascribed to their inappropriate use as growth promot-ers of antimicrobial agents like oxacillin vancomycin chlo-ramphenicol neomycin and erythromycin which are com-monly used in veterinary medicine to treat infections [50]Antimicrobial therapy of infections staphylococci is basedon results of susceptibility tests in vitro [51] Methicillin-resistant coagulase-negative Staphylococcus spp found inready-to-eat products such as meats fish and dairy productsalso offer risks and although they are not classical foodpoisoning bacteria their presence in food offers significantrisks to public health due to the possible spread of antibioticresistance [52]

The standardization of salami quality should includediagnostic methods to screen and quantify the presence ofclassical and newly described enterotoxins directly in thefood matrices as a routine procedure to be conducted by themeat product industry in Brazil

The safety of salami consumption could also be enhancedby the inclusion of amicrobial barrier such as the inclusion ofprobiotic strains producing natural antibiotics or competitiveflora or even the addition of natural bioagents against spoilageor pathogenic microorganisms

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors acknowledge the financial support fromFundacao Carlos Chagas Filho de Amparo a Pesquisa doEstado do Rio de Janeiro (FAPERJ Rio de Janeiro Brazil)Conselho Nacional de Desenvolvimento Cientıfico eTecnologico (CNPq Brasılia Brazil) and Coordenacao deAperfeicoamento de Pessoal de Nıvel Superior (CAPESBrasılia Brazil)

References

[1] ICMSF International Comission on Microbiological Specifica-tions for FoodsMicroorganisms in FoodsTheir Significance andMethods of Enumerations University of Toronto Press TorontoCanada 3rd edition 1983

[2] J F Veras L S Carmo L C Tong et al ldquoA study of theenterotoxigenicity of coagulase-negative and coagulase-positivestaphylococcal isolates from food poisoning outbreaks inMinasGerais Brazilrdquo International Journal of Infectious Diseases vol12 no 4 pp 410ndash415 2008

[3] K Omoe D-L Hu H Takahashi-Omoe A Nakane and KShinagawa ldquoComprehensive analysis of classical and newlydescribed staphylococcal superantigenic toxin genes in Staphy-lococcus aureus isolatesrdquo FEMS Microbiology Letters vol 246no 2 pp 191ndash198 2005

[4] J Kadariya T C Smith and D Thapaliya ldquoStaphylococcusaureus and staphylococcal food-borne disease an ongoing chal-lenge in public healthrdquoBioMedResearch International vol 2014Article ID 827965 9 pages 2014

[5] KOmoeD-LHuH KOno et al ldquoEmetic potentials of newlyidentified staphylococcal enterotoxin-like toxinsrdquo Infection andImmunity vol 81 no 10 pp 3627ndash3631 2013

[6] J K McCormick J M Yarwood and P M SchlievertldquoToxic shock syndrome and bacterial superantigens an updaterdquoAnnual Review of Microbiology vol 55 pp 77ndash104 2001

[7] T Baba F Takeuchi M Kuroda et al ldquoGenome and virulencedeterminants of high virulence community-acquired MRSArdquoThe Lancet vol 359 no 9320 pp 1819ndash1827 2002

[8] Associacao Brasileira de Industria Produtora e Exportadorada Carne Suınas (ABIPECS) ldquoStatistics slaughter ranking2000ndash2014rdquo 2014 httpabpa-brcombrsetoressuinoculturapublicacoesrelatorios-anuais

[9] N Zdolec M Hadziosmanovic L Kozacinski et al ldquoMicrobialand physicochemical succession in fermented sausages pro-duced with bacteriocinogenic culture of Lactobacillus sakei andsemi-purified bacteriocin mesenterocin Yrdquo Meat Science vol80 no 2 pp 480ndash487 2008

16 BioMed Research International

[10] F Ravyts L D Vuyst and F Leroy ldquoBacterial diversity andfunctionalities in food fermentationsrdquo Engineering in Life Sci-ences vol 12 no 4 pp 356ndash367 2012

[11] TOhlsson andN BengtssonTheHurdle ConceptMinimal Pro-cessing Technologies in the Food IndustryWoodhead Publishing2002

[12] S Bernardi B B Golineli and C J Contreras-CastilloldquoRevisao aspectos da aplicacao de culturas starter na producaode embutidos carneos fermentadosrdquo Brazilian Journal of FoodTechnology vol 13 no 2 pp 133ndash140 2010

[13] F Irlinger ldquoSafety assessment of dairy microorganismscoagulase-negative staphylococcirdquo International Journal of FoodMicrobiology vol 126 no 3 pp 302ndash310 2008

[14] B Ghebremedhin F Layer W Konig and B Konig ldquoGeneticclassification and distinguishing of Staphylococcus species basedon different partial gap 16S rRNA hsp60 rpoB sodA and tufgene sequencesrdquo Journal of Clinical Microbiology vol 46 no 3pp 1019ndash1025 2008

[15] I Babic K Markov D Kovacevic et al ldquoIdentification andcharacterization of potential autochthonous starter culturesfrom aCroatian lsquobrandrsquo product lsquoslavonski kulenrsquordquoMeat Sciencevol 88 no 3 pp 517ndash524 2011

[16] MMehrotra GWang andWM Johnson ldquoMultiplex PCR forthe detection of genes for Staphylococcus aureus enterotoxinsexfoliative toxins toxic shock syndrome toxin and methicillinresistancerdquo Journal of Clinical Microbiology vol 38 no 3 pp1032ndash1041 2000

[17] WM Johnson SD Tyler E P Ewan F EAshtonDR Pollardand K R Rozee ldquoDetection of genes for enterotoxins exfolia-tive toxins and toxic shock syndrome toxin 1 in Staphylococcusaureus by the polymerase chain reactionrdquo Journal of ClinicalMicrobiology vol 29 no 3 pp 426ndash430 1991

[18] J Mclauchlin G L Narayanan VMithani andG OrsquoNeill ldquoThedetection of enterotoxins and toxic shock syndrome toxin genesin Staphylococcus aureus by polymerase chain reactionrdquo Journalof Food Protection vol 63 no 4 pp 479ndash488 2000

[19] J P Rosec and O Gigaud ldquoStaphylococcal enterotoxin genes ofclassical and new types detected by PCR in Francerdquo Interna-tional Journal of Food Microbiology vol 77 no 1-2 pp 61ndash702002

[20] K Omoe D-L Hu H Takahashi-Omoe A Nakane andK Shinagawa ldquoIdentification and characterization of a newstaphylococcal enterotoxin-related putative toxin encoded bytwo kinds of plasmidsrdquo Infection and Immunity vol 71 no 10pp 6088ndash6094 2003

[21] N Sergeev D Volokhov V Chizhikov and A Rasooly ldquoSimul-taneous analysis of multiple staphylococcal enterotoxin genesby an oligonucleotide microarray assayrdquo Journal of ClinicalMicrobiology vol 42 no 5 pp 2134ndash2143 2004

[22] S Holtfreter D Grumann M Schmudde et al ldquoClonal distri-bution of superantigen genes in clinical Staphylococcus aureusisolatesrdquo Journal of Clinical Microbiology vol 45 no 8 pp2669ndash2680 2007

[23] W J Mason J S Blevins K Beenken NWibowo N Ojha andM S Smeltzer ldquoMultiplex PCR protocol for the diagnosis ofstaphylococcal infectionrdquo Journal of Clinical Microbiology vol39 no 9 pp 3332ndash3338 2001

[24] E Rahimi and H G Safai ldquoDetection of classical enterotoxinsof Staphylococcus aureus strains isolated frombovine subclinicalmastitis in Isfahan Iranrdquo Veterinary Microbiology vol 141 no3-4 pp 393ndash394 2010

[25] M A Larkin G Blackshields N P Brown et al ldquoClustalW andClustal X version 20rdquo Bioinformatics vol 23 no 21 pp 2947ndash2948 2007

[26] P H A Sneath and R R Sokal Unweighted Pair Group Methodwith Arithmetic Mean Numerical Taxonomy W H FreemanSan Francisco Calif USA 1973

[27] NCCLS ldquoPerformance standards for antimicrobial susceptibil-ity testing 14th informational supplementrdquo Tech Rep M 100-S14 NCCLS 2004

[28] S Corbiere Morot-Bizot S Leroy and R Talon ldquoMonitoringof staphylococcal starters in two French processing plantsmanufacturing dry fermented sausagesrdquo Journal of AppliedMicrobiology vol 102 no 1 pp 238ndash244 2007

[29] R Talon S Leroy I Lebert et al ldquoSafety improvement andpreservation of typical sensory qualities of traditional dryfermented sausages using autochthonous starter culturesrdquo Inter-national Journal of Food Microbiology vol 126 no 1-2 pp 227ndash234 2008

[30] G Mauriello A Casaburi G Blaiotta and F Villani ldquoIsolationand technological properties of coagulase negative staphylo-cocci from fermented sausages of Southern ItalyrdquoMeat Sciencevol 67 no 1 pp 149ndash158 2004

[31] M Janssens N Myter L De Vuyst and F Leroy ldquoSpeciesdiversity and metabolic impact of the microbiota are low inspontaneously acidified Belgian sausages with an added starterculture of Staphylococcus carnosusrdquo Food Microbiology vol 29no 2 pp 167ndash177 2012

[32] G Blaiotta D Ercolini G Mauriello G Salzano and F VillanildquoRapid and reliable identification of Staphylococcus equorum bya species-specific PCR assay targeting the sodA generdquo Systematicand Applied Microbiology vol 27 no 6 pp 696ndash702 2004

[33] L Kozacinski E Drosinos F Caklovica L Cocolin J Gasparik-Reichardt and S Veskovic ldquoInvestigation of microbial associa-tion of traditionally fermented sausagesrdquo Food Technology andBiotechnology vol 46 no 1 pp 93ndash106 2008

[34] S Leroy P Giammarinaro J-P Chacornac I Lebert and RTalon ldquoBiodiversity of indigenous staphylococci of naturallyfermented dry sausages and manufacturing environments ofsmall-scale processing unitsrdquo Food Microbiology vol 27 no 2pp 294ndash301 2010

[35] E Coton M-H Desmonts S Leroy et al ldquoBiodiversity ofCoagulase-Negative Staphylococci in French cheeses dry fer-mented sausages processing environments and clinical sam-plesrdquo International Journal of Food Microbiology vol 137 no 2-3 pp 221ndash229 2010

[36] S Hosseinzadeh and H D Saei ldquoStaphylococcal species associ-ated with bovinemastitis in the NorthWest of Iran emerging ofcoagulase-negative staphylococcirdquo International Journal of Vet-erinary Science and Medicine vol 2 no 1 pp 27ndash34 2014

[37] A Piette and G Verschraegen ldquoRole of coagulase-negativestaphylococci in human diseaserdquo Veterinary Microbiology vol134 no 1-2 pp 45ndash54 2009

[38] T M Moura F S Campos P A DrsquoAzevedo et al ldquoPrevalenceof enterotoxin-encoding genes and antimicrobial resistancein coagulase-negative and coagulase-positive Staphylococcusisolates from black pudding in Southern Brazilrdquo Revista daSociedade Brasileira deMedicina Tropical vol 45 no 5 pp 579ndash585 2012

[39] H I Suzuki T Lefebure P P Bitar and M J StanhopeldquoComparative genomic analysis of the genus Staphylococcusincluding Staphylococcus aureus and its newly described sister

BioMed Research International 17

species Staphylococcus simiaerdquo BMC Genomics vol 13 article38 2012

[40] R P Lamers G Muthukrishnan T A Castoe S Tafur A MCole and C L Parkinson ldquoPhylogenetic relationships amongStaphylococcus species and refinement of cluster groups basedon multilocus datardquo BMC Evolutionary Biology vol 12 article171 2012

[41] I V Pinchuk E J Beswick and V E Reyes ldquoStaphylococcalenterotoxinsrdquo Toxins vol 2 no 8 pp 2177ndash2197 2010

[42] S R Monday and G A Bohach ldquoGenes encoding staphylo-coccal enterotoxins G and I are linked and separated by DNArelated to other staphylococcal enterotoxinsrdquo Journal of NaturalToxins vol 10 no 1 pp 1ndash8 2001

[43] D Y Thomas S Jarraud B Lemercier et al ldquoStaphylococcalenterotoxin-like toxins U2 and V two new staphylococcalsuperantigens arising from recombination within the entero-toxin gene clusterrdquo Infection and Immunity vol 74 pp 4724ndash4734 2006

[44] N Indrawattana O Sungkhachat N Sookrung et al ldquoStaphylo-coccus aureus clinical isolates antibiotic susceptibility molecu-lar characteristics and ability to form biofilmrdquo BioMed ResearchInternational vol 2013 Article ID 314654 11 pages 2013

[45] V Pereira C Lopes A Castro J Silva P Gibbs and P TeixeiraldquoCharacterization for enterotoxin production virulence factorsand antibiotic susceptibility of Staphylococcus aureus isolatesfrom various foods in Portugalrdquo Food Microbiology vol 26 no3 pp 278ndash282 2009

[46] I Anacarso C Condo C Sabia and et al ldquoAntimicrobialresistance and other related virulence factors in staphylococcusspp isolated from food environmental and humans in ItalyrdquoUniversal Journal Microbiology Research vol 1 no 1 pp 1ndash92013

[47] B Martın M Garriga M Hugas S Bover-Cid M T Veciana-Nogues and T Aymerich ldquoMolecular technological and safetycharacterization of Gram-positive catalase-positive cocci fromslightly fermented sausagesrdquo International Journal of FoodMicrobiology vol 107 no 2 pp 148ndash158 2006

[48] NM Broekema T Van Tam T AMonson S AMarshall andD M Warshauer ldquoComparison of cefoxitin and oxacillin diskdiffusionmethods for detection ofmecA-mediated resistance inStaphylococcus aureus in a large-scale studyrdquo Journal of ClinicalMicrobiology vol 47 no 1 pp 217ndash219 2009

[49] E Cercenado F Garcıa-Garrote and E Bouza ldquoIn vitro activityof linezolid against multiply resistant Gram-positive clinicalisolatesrdquo Journal of Antimicrobial Chemotherapy vol 47 no 1pp 77ndash81 2001

[50] A Aydin K Muratoglu M Sudagidan K Bostan B Okukluand SHarsa ldquoPrevalence and antibiotic resistance of foodborneStaphylococcus aureus isolates in Turkeyrdquo Foodborne Pathogensand Disease vol 8 no 1 pp 63ndash69 2011

[51] D Sergelidis A Abrahim T Papadopoulos et al ldquoIsolation ofmethicillin-resistant Staphylococcus spp from ready-to-eat fishproductsrdquo Letters in Applied Microbiology vol 59 no 5 pp500ndash506 2014

[52] I PhillipsM Casewell T Cox et al ldquoDoes the use of antibioticsin food animals pose a risk to human health A critical reviewof published datardquo Journal of Antimicrobial Chemotherapy vol53 no 1 pp 28ndash52 2004

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Page 6: Research Article Safety Evaluation of the Coagulase ...downloads.hindawi.com/journals/bmri/2015/483548.pdf · Research Article Safety Evaluation of the Coagulase-Negative Staphylococci

6 BioMed Research International

Table2Genotypicandph

enotypiccharacteriz

ationof

CNSstrainsfrom

salami

Strainsidentificatio

nandcharacteriz

ation

Salamiorig

inStaphylococcus

species

Genotypic

Phenotypic

GenBa

nkaccessionnu

mbera

ndsim

ilarity(

)Presence

ofenterotoxingenes

mRN

Adetection

Enterotoxinprod

uctio

n(ngm

Lminus1)

Com

mercial

Staphylococcus

sppKF

1354451(96)

seaseb

mdashmdash

Scarnosus

KJ8620021(96)

seasebsedsehseiand

selj

mdash12plusmn01

Scarnosus

NR1164341(98)

sebsecsedandsee

see

03

Ssaprophyticus

AB6

977171

(98)

sehseiandselm

seiselm

03

Ssaprophyticus

EU4309921(99)

seasebselm

seln

selo

and

tstH1

sebselm

and

selo

13plusmn01

Ssaprophyticus

HQ6995101(97)

mdashmdash

mdashSsaprophyticus

JX4901221(99)

seaseh

seh

05plusmn01

Ssaprophyticus

KJ00

46231(96)

sec

mdash04

Ssaprophyticus

KJ9496061(98)

seasebseca

ndseh

seasebandseh

mdashSsciuriJX9664361(98)

sedsei

sei

10plusmn01

SxylosusA

M8827001(97)

mdashmdash

mdashSxylosusCP

0072081(99)

mdashmdash

mdashSxylosusC

P0087241(98)

seatstH1

sea

14SxylosusK

C4565901(98)

mdashmdash

mdash

Artisa

nal

SepidermidisKF

6005891(97)

secsedseesegsehandsei

seesei

07plusmn01

ShominisJX5199881(97)

seasebsecsedseljselm

seln

andselo

selnselo

09plusmn01

Ssaprophyticus

HF9

372521(97)

seb

seb

05

Ssaprophyticus

subspbovisK

J6991511

(98)

secselm

seln

andtstH1

seln

09plusmn01

Ssuccinus

KC3298241(99)

seasebsecseljandseln

sebsec

13SxylosusK

F1980801(97)

secselm

andtstH1

mdash05plusmn01

Thep

resenceo

fenterotoxin

genesseasebsecsedseesegsehsei

seljselkselm

seln

selo

selq

selrselu

and

tstH1w

asteste

dby

PCRusingthes

pecific

seto

fprim

ers

sea-seee

nterotoxin

prod

uctio

nwas

evaluatedby

immun

e-sorbentassays(EL

ISA)u

singthed

etectio

nkitR

IDASC

REEN

SETAB

CD

EV

aluesa

redisplay

edas

them

eansplusmnSD

ofassays

perfo

rmed

indu

plicate

mRN

Atranscrip

tsfora

llenterotoxingenesw

eree

valuated

byrealtim

eRT-PC

Rtests

CT

values

ared

isplayedas

meansplusmnSD

ofRT

-PCR

tests

perfo

rmed

indu

plicateseamdash

SxylosusC

P00872413

0plusmn04sebmdash

SxylosusC

P008724133plusmn20Ssaprophyticu

sJX4901221302plusmn04Ssaprophyticus

subspbovisK

J6991511

32plusmn01SxylosusK

F19808013

6plusmn10

and

ShominisJX519988134plusmn10secmdashSsaprophyticus

subspbovisK

J6991511

31plusmn11seemdashSsaprophyticus

subspbovisK

J6991511306plusmn05

Ssuccinus

KC3298241285plusmn01andSsaprophyticus

HF9

3725213

2plusmn04sehmdash

SxylosusC

P0087241 31plusmn10Ssaprophyticu

sJX9

6643613

32plusmn06seimdash

Ssaprophyticus

AB6

977171

30plusmn10

Ssaprophyticu

sJX9664361306plusmn10

seln

mdashShominisJX5199881303plusmn10

Ssaprophyticu

sEU430992131plusmn03andselomdashShominisJX519988132plusmn10

BioMed Research International 7

O S succinus KC3298241Δ S xylosus AM8827001

O S saprophyticus KJ6991511Δ S saprophyticus AB6977171

Δ S xylosus CP0087241Δ S xylosus KC4565901Δ S saprophyticus KJ9496061Δ S saprophyticus JX4901221Δ S saprophyticus KJ0046231

Δ S xylosus CP0072081Δ S carnosus NR1164341Δ S saprophyticus HQ6995101

O S epidermidis KF0882111O S hominis JX5199881Δ S saprophyticus EU4309921

O S xylosus KF1980801Δ S carnosus KJ8620021Δ S sciuri JX9664361O S saprophyticus HF9372521

04 03 02 01 00

Figure 1 Phylogenetic tree generated from the multiple alignments of the 16S rDNA sequences of CNS strains found in salami usingthe ClustalX 20 software The phylogenetic tree was constructed by using the Mega 60 software and the unweighted pair group method(UPGMA) Bootstrap values ranged from 00 to 04 Strains found in commercial (Δ) or artisanal salami (O)

between these CNS strains Suzuki et al 2012 demonstrateda bootstrap value higher than 90 for interspecies similaritiesbetween S saprophyticus S epidermidis S hominis and Scarnosus

33 Genotypic and Phenotypic Characterization of CNSStrains Twelve distinct combinations of staphylococcalenterotoxins geneswere found in the 15CNS strains compris-ing SEs AndashE GndashJ and also the enterotoxin-like toxins (SElL)KndashR and U (Table 2)

Fifteen strains (79) carried at least one gene encodingenterotoxins in their genomes (Figure 2) The seb and secgenes were the most predominant harbored by 57 ofthe strains followed by sea carried by 50 whereas thesedsehselm genes showed intermediate incidence harboredby 33 of the strains while seiseln and tsH1 were found in27 of strains Finally see selj and selo showed low incidence(13) and seg was carried by only 1 of strains (Table 2)

The relationships between superantigenic toxin geno-types and toxin gene-encoding mobile genetic elementsin CNS strains were evaluated Distinct combinations ofSaPI and plasmids or plasmids and genes of egc operonenterotoxins were found in the CNS strains obtained fromsalami

Four strains presented only the classical enterotoxingenes namely S saprophyticus KJ0046231 and HF9372521S xylosus CP0087241 and S carnosus NR1164341 Anotherstrain S saprophyticus AB6977171 presented only the newlydescribed enterotoxins seh sei and selm and finally 10 strainspresented a combination of classical and newly described

enterotoxin genes in their genomes with at least one of eachenterotoxin type (Table 2)

Previous studies have shown that sea is the most com-mon toxin associated with Staphylococcus food poisoningfollowed by sed and see with SEHand SEI being considered asplaying only a minor role [39] Among the 15 enterotoxigenicstrains found in salami 73 of them harbored at least the seagene or combinations of these 05 genes Strains S carnosusKJ8620021 found in commercial salami and S epidermidisKF6005891 found in artisanal salami carry 04 of these genesThe sea and seb enterotoxin genes are known to occupy thesame locus on the chromosome which may explain whythese enterotoxins are commonly found together in foodpoisoning outbreaks [36] The combination of sea and sebgenes was found in 05 strains S saprophyticus EU4309921 Scarnosus KJ8620021 S hominis JX5199881 S saprophyticusKJ9496061 and S succinus KC3298241 A single strain Scarnosus KJ8620021 showed the combination of sea and seigenes

Additionally some CNS are able to produce TSST-1 aloneor in combination with other enterotoxins Herein 04 of the19 strains (21) S saprophyticus EU4309921 S saprophyti-cus subsp bovis KJ6991511 and S xylosus CP0087241 andKF1980801 were shown to harbor the tstH1 combined withse andor sel enterotoxin genes (Table 2)

The staphylococci enterotoxins genes seg and sei [40] arepart of a chromosomal operon gene cluster (egc) comprisingfive genes designated as selo selm sei seln and seg Twoof the CNS strains determined in the present study Ssaprophyticus EU4309921 and S hominis JX5199881 were

8 BioMed Research International

sed

seb

sec

see

sea

M 2 3 4 51

500bp

(a)

sea

sebsebseb

M 1 2 3 4 5

(b)

sei

sehseg

1M

(c)

seh sehseh

M 1 2 3

(d)

1 2 4M 3 5

(e)

selm

seljseln

selo

21M

(f)

seln

selm

M 1 2

(g)

Figure 2 Uniplex duplex and multiplex PCR screening for the detection of enterotoxin genes in CNS strains from salami (a) Lane M100 bp DNA ladder plus (Fermentas Foster City CA USA) lane 1 S aureus ATCC 29231 harboring sea gene lane 2 S aureus NCTC 10654harboring seb gene lane 3 S aureus ATCC19095 harboring the sec gene lane 4 S aureus ATCC 13563 harboring the sed gene and lane 5 Saureus ATCC 27664 harboring the see gene (b) Lane M 100 bp DNA ladder plus lane 1 Staphylococcus spp lane 2 S carnosus NR116434lane 3 S carnosus KJ862002 lane 4 S carnosus KJ862002 and lane 5 S carnosus KJ8620021 (c) Lane M 100 bp DNA ladder plus lane1 S aureus ATCC 19095 harboring seg seh and sei genes (d) Lane M 100 bp DNA ladder plus lane 1 S saprophyticus AB6977171 lane 2S epidermidis KF 6005891 and lane 3 S sciuri JX9664361 (e) Lane M 100 bp DNA ladder plus lane 1 S xylosus KF1980801 lane 2 SsaprophyticusKJ6991511 (f) LaneM 100 bp DNA ladder plus lane 1 S aureusATCC 27154 harboring selj slem seln and selo genes (g) LaneM 100 bp DNA ladder plus lane 1 S xylosus KF1980801 and lane 2 S saprophyticus subsp bovis KJ6991511

BioMed Research International 9

shown to carry the selm seln and selo genes and the selmand seln sequences were detected in the genome of a singlestrain S saprophyticus subsp bovisKJ6991511 S epidermidisKF6005891 showed a combination of seg and sei genesand 03 strains harbored a single gene sei or selm or selnperhaps due to the high degree of genetic polymorphism inthe chromosomal assembly [41] Indrawattana et al 2013detected the seg-sei-selm-seln-selo of the highly prevalent egclocus was 261 in contrast to this study where 13 CNSpresents the combination of selm seln and selo genes [42]

The CNS strains clustered into groups near the bottom ofthe phylogenetic tree carried the classical enterotoxin geneswhile the species at the top of the tree showed high diversityamong the enterotoxin genes combining the classical and thenewly described genes in their genomes (Figure 1)

To assess the risk of staphylococcal food poisoning theability of the identified strains in harboring the se andsel genes and in expressing and producing enterotoxins invitro was evaluated The mRNA for each enterotoxin genewas evaluated by real time RT-PCR assays and enterotoxincontent was estimated by a sandwich enzyme immunoassayfor the combined detection of Staphylococcus enterotoxins(SET) A B C D and E

The fifteen enterotoxigenic CNS strains were able toexpress the classical enterotoxins SEA seb sec sed and seein concentrations ranging from 03 ngmLminus1 to 14 ngmLminus1as assessed by the in vitro assays (Table 2) The S sapro-phyticus AB6977171 KJ6991511 JX4901221 KJ0046231 andHF9372521 strains S xylosus KF1980801 and S carnosusNR1164341 produced low amount of enterotoxins lower than05 ngmLminus1 S epidermidisKF6005891 S saprophyticus bovisKJ6991511 and S hominis JX5199881 produced intermediateamount of enterotoxins ranging from 07 to 090 ngmLminus1while S sciuri JX9664361 S saprophyticus EU4309921 Scarnosus KJ8620021 S xylosus CP0087241 and S succi-nus KC3298241 produced enterotoxins in concentrationsge10 ngmLminus1

Although the sandwich enzyme immunoassay is consid-ered themost sensitivemethod to detect seandashsee enterotoxinsable to detect 0125 ngmLminus1 differences in the specificity andsensitivity of the assays for the detection of staphylococcalenterotoxins from foods are expected [43] A single strainStaphylococcus spp KF1354451 which harbors both the seaand seb genes was unable to produce seandashsee enterotoxins

The mRNA levels evaluated by the real time RT-PCRassays for enterotoxins were detected in 12 of 15 strains(86) (Table 2) Transcripts for the classical enterotoxinsgenes were detected when S saprophyticus HF9372521 Scarnosus NR1164341 S xylosus CP0087241 and S succinusKC3298241 were assayed Transcripts for newly describedenterotoxins were observed in S saprophyticus JX4901221and AB6977171 S sciuri JX9664361 S saprophyticus subspbovis KJ6991511 and S hominis JX5199881 Transcripts fromboth the classical and newly described enterotoxins weredetected in S saprophyticus EU4309921 and KJ9496061 andS epidermidis KF6005891

No mRNA transcripts were obtained for S sapro-phyticus KJ00462321 and S carnosus KJ8620021 although

enterotoxin production was detected by the enzyme-linkedimmunosorbent tests (Table 2)

The 04 strains that carry tstH1 in their genome Ssaprophyticus EU4309921 and KJ6991511 and S xylosusCP0087241 and KF980801 were not able to produce mRNAfor TSST-1 in the assay conditions

The production of classical enterotoxins in vitro(immunologic test) matched the results shown by real timeRT-PCR assays for the following strains S saprophyticusJX4901221 S sciuri JX9664361 S saprophyticusEU4309921S saprophyticus HF9372521 S saprophyticus subsp bovisKJ6991511 S hominis JX5199881 S epidermidis KF6005891and S succinus KC3298241

Ten strains namely S saprophyticusAB6977171 S sapro-phyticus EU4309921 S saprophyticus HF9372521 S sapro-phyticus KJ6991511 S saprophyticus KJ9496061 S succinusKC3298241 S hominis JX5199881 S xylosus KF1980801and S xylosus CP0087241 and S epidermidis KF6005891expressed mRNA for multiple se andor sel genes There isa differential transcription among these genes where themost frequent among the classical ones were seb and seeseatranscribed by 04 and 02 strains respectively and the mostfrequent among the newly described genes were sei andsehselnselo transcribed by 03 and 02 strains respectively

No mRNA for the sec gene was detected although SsaprophyticusAB6977171 was able to produce the enterotoxinin vitro as detected by the ELISA assays

34 Enterotoxin Gene Homologies between Salami CNSand CPS Strains The nucleotide sequencing of six entero-toxin genesmdashtwo of them sec and see encoding classi-cal enterotoxinsmdashand four of them encoding the newlydescribed genes seg seh selm and seln fromCNS strains wascompared to the enterotoxin genes from S aureus or S pas-teuri two coagulase-positive staphylococci strains (Figure 3)The homology between the CNS and CPS enterotoxin genesvaried from 65 to 98 The homology of sec seg sehselm and seln between CNS from salami and S aureus was98 60 98 65 and 70 respectively The homologybetween see from CNS found in salami and in S pasteuri was98

Although further studies should be performed it seemsthat the sequences encoding enterotoxins can be conservedamong coagulase-negative and coagulase-positive staphy-lococci as shown in the phylogenetic analysis of the 06enterotoxin genes (Figure 4)

35 Antimicrobial Multiresistance of CNS Strains (MRCNS)Another safety hazard associated with CNS strains besidesthe ability of producing enterotoxins in food matrices is theantimicrobial resistance to antimicrobial agents commonlyused to treat staphylococci infections The antimicrobialresistance carried by CNS strains from food matrices can bespread to the population by the consumption of an apparentlysafe food

Among the 19 CNS strains identified in salami 14 showedmultiresistance to antimicrobial agentsThree strains showedthe highest MAR indices 093 and 080 07 strains presented

10 BioMed Research International

lowast lowastlowastlowastlowastlowast lowast lowast lowastlowast lowast lowast lowastlowast lowastlowastlowastlowast lowastlowastlowast lowast

lowastlowast lowast lowast lowastlowastlowastlowast lowast lowast lowastlowast lowast

lowastlowastlowastlowast lowast lowast lowast

lowastlowast lowast lowast lowastlowast lowastlowast lowast lowastlowast lowastlowastlowastlowastlowast lowastlowast lowast lowastlowast lowastlowast

lowastlowast lowastlowastlowast lowast lowast lowast lowast lowast lowastlowastlowastlowastlowast

lowastlowast lowastlowast lowastlowast lowastlowast lowast lowast lowastlowast lowast lowast lowastlowastlowast lowast lowastlowast lowastlowastlowast

lowast lowast lowast lowast lowast lowast lowastlowast lowast lowastlowastlowast lowast lowastlowastlowast lowast lowast lowastlowastlowast lowast lowastlowastlowast lowast

lowast lowast lowast lowastlowastlowastlowast lowast lowastlowastlowast lowast lowast lowastlowastlowast lowast lowast lowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast lowast lowast lowast lowast lowastlowast lowast lowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

Enterotoxin C (sec) GATAAAAATGAATAAGAGTCGATTTATTTCATGCGTAATTTTGATATTCGCACTTATACT------------------------------------------------------------

AGTTCTTTTTACACCCAACGTATTAGCAGAGAGCCAACCAGACCCTACGCCAGATGAGTT------------------------------------------------------------

GCACAAAGCGAGTAAATTCACTGGTTTGATGGAAAATATGAAAGTTTTATATGATGATCA------------------------------------------------------------

TTATGTATCAGCAACTAAAGTTAAGTCTGTAGATAAATTTTTGGCACATGATTTAATTTA------------------------------------------------------------

TAACATTAGTGATAAAAAACTGAAAAATTATGACAAAGTGAAAACAGAGTTATTAAATGA------------------------------------------------------------

AGGTTTAGCAAAGAAGTACAAAGATGAAGTAGTTGATGTGTATGGATCAAATTACTATGT------------------------------------------------------------

AAACTGCTATTTTTCATCCAAAGATAATGTAGGTAAAGTTACAGGTGGCAAAACTTGTAT------------------------------------------------------------

GTATGGAGGAATAACAAAACATGAAGGAAACCACTTTGATAATGGGAACTTACAAAATGT------------------------------------------------------------

ACTTATAAGAGTTTATGAAAATAAAAGAAACACAATTTCTTTTGAAGTGCAAACTGATAA------------------------------------------------------------

GAAAAGTGTAACAGCTCAAGAACTAGACATAAAAGCTAGGAATTTTTTAATTAATAAAAA------------------------------------------------------------

AAATTTGTATGAGTTTAACAGTTCACCATATGAAACAGGATATATAAAATTTATTGAAAA----TTGTATGAGTTTAACAGTTCACCATATGAAACAGGATATATAAAATTTATTGAAAA

TAACGGCAATACTTTTTGGTATGATATGATGCCTGCACCAGGCGATAAGTTTGACCAATCTAACGGCAATACTTTTTGGTATGATATGATGCCTGCACCAGGCGATAAGTTTGACCAATC

TAAATATTTAATGATGTACAACGACAATAAAACGGTTGATTCTAAAAGTGTGAAGATAGATAAATATTTAATGATGTACAACGACAATAAAACAGTTGATTCTAAAAGTGTGAAGATAGA

AGTCCACCTTACAACAAAGAATGGATAATGTTAATCCGATTTTGATATAAAAAGTGAAAGAGTCCACCTTACAACAAAGAATGGATAATGTAATCCGATTATGGACAAGTGTGCTTTTTT

Enterotoxin C TATTAGATATATTTGAAAGGTAAGTACTTCGGTGCTTGCCTTTTTAGGATGCATATATATEnterotoxin C CNS

Enterotoxin CEnterotoxin C CNS

Enterotoxin CEnterotoxin C CNSEnterotoxin CEnterotoxin C CNSEnterotoxin CEnterotoxin C CNSEnterotoxin CEnterotoxin C CNSEnterotoxin CEnterotoxin C CNSEnterotoxin CEnterotoxin C CNSEnterotoxin CEnterotoxin C CNSEnterotoxin CEnterotoxin C CNS

Enterotoxin CEnterotoxin C CNSEnterotoxin CEnterotoxin C CNS

Enterotoxin CEnterotoxin C CNS

Enterotoxin CEnterotoxin C CNS

Enterotoxin CEnterotoxin C CNS

TTTGTGATCGTCGGCCA-----ACCGATACCGTCGTATTCTTGTACCGCGCAAGGCGAAGlowast lowast lowastlowastlowast lowast lowast lowast lowast lowastlowast lowast lowastlowastlowast lowast lowast lowast lowast

Enterotoxin E (see) Enterotoxin E TGCTTTAGGCGGGGTCTTACAGCATATTATCGTAGAAGATGCACAAGCTGCACGTAAAGCS pasteuri TGCTTTAGGCGCATCGTTACAACATGTCATCGTAGAAGATGAAAAAGATGGTCGTCAGGC

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast lowastlowastlowastlowast lowastlowastlowast lowast lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast lowast lowastlowastlowast lowastlowast lowastlowastlowast lowast lowastlowastlowast

Enterotoxin E ATTAGAATTCTTGAAATCAAAACA-AGCAGGACGTGCTACATTTCTACCATTAAATGTCAS pasteuri

Enterotoxin ES pasteuri

GATACAATT-TTTAAAACAACGTAGTTTAGGACGTGCCACATTCCTACCTTTAAATGTTA lowastlowast lowastlowastlowastlowast lowastlowast lowastlowastlowast lowastlowastlowast lowast lowastlowastlowastlowastlowastlowastlowastlowastlowast lowastlowastlowastlowastlowast lowastlowastlowastlowastlowast lowastlowastlowastlowastlowastlowastlowastlowast lowast

TCAAACCTAGAAATATTTCAACCAAGATATATlowast lowastlowastlowastlowast lowastlowastlowast lowastlowastlowastlowastlowast

Enterotoxin G (seg) Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

---------------------------attaagaaatactt--ccattttaatactcttaGGGGATATTTCCGGGAAACCGGGGCTAATACTGGATAACTTTTCTCTCCGAACGAAGAGA

gaatcaacaactttattatctccg--taaat-----atttaaaaacttatatggaacaaaGATTCAACGATGGTCTTGGTTTTCAATTAAGATGAACCCGCGGCGCTTAGCTGGTGGGAC

aggtactagtt-----------------------------------------cttttttaAGGTCACGGCTCACCCAAGCCCCCATGCGTATACGACCTGGAAGGGTGATCGGCCACATT

ggaaataagtcaaaccaaaaacttgtattgttcttttcagtaaat---------------GGGACTGAGACACAGCACAAACTCCTACGGGAGGCAGCAATAGGGAATCTTCCGCAATGG

------------------------------------------------------------CCGAAAGTCTGACGGAGCAACGCCGCGAGACTGAAGAATGTCTTCGGATCGTAAAACTCT

------------------------tttatatatccagattcaaa-----tgcagaaccatGTTGTTAGGGAAGAACAAGTATCGTTCGAATAGGGCGGTACCTTGACGGTACCTAACCAG

caaactcgtatagctttttttct---------ttagtgagccagtgtcttgctttg----AAAGCTTTGCTAACTACCTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCG

-taatctagttcctgaatagtaaccatattcttatttgtagttattgta--aatccaagtGAATTATTGGGCGTAAAGCGCGCGCATGCGGTCTTTTAAATCTGATGTCAAAGCCCACGG

gattgtct----------attgtcgattgttacctgtacagt------------aattaaGCTCAACCGTGGAGGGTCATTGGAAACTGTAAGACTTGGAGTGCAGAAGAGGATAGTGGA

Figure 3 Continued

BioMed Research International 11

lowast lowast lowast lowastlowast lowastlowast lowastlowastlowast lowast lowastlowast lowast lowast lowast lowast

lowast lowast lowastlowastlowast lowastlowast lowast lowastlowastlowast lowast lowastlowast lowastlowastlowast lowast lowastlowast lowastlowast lowast lowast lowastlowast lowast

lowastlowast lowast lowast lowastlowastlowastlowastlowast lowast lowast lowastlowast lowast lowast lowast lowast lowast lowastlowastlowast

lowastlowast lowast lowastlowast lowastlowastlowast lowastlowast lowastlowast lowast lowast lowast lowast lowastlowast lowastlowast lowast lowast lowast lowastlowast

lowastlowast lowastlowastlowastlowast

lowast lowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowast lowast lowastlowastlowastlowast lowast lowastlowast lowastlowast lowast lowastlowastlowastlowast lowast lowastlowast lowast lowast lowastlowastlowastlowastlowastlowastlowast

lowastlowast lowast lowast lowast lowast lowastlowastlowast lowastlowast lowastlowast lowast lowastlowastlowastlowastlowastlowastlowastlowast lowastlowastlowast

lowast lowast lowast lowast lowast lowast lowastlowast lowastlowast lowast lowast lowastlowastlowast lowast lowast lowastlowastlowast lowast lowast

lowastlowast lowastlowastlowastlowastlowastlowast lowastlowast lowastlowastlowast lowastlowast lowast lowastlowast lowastlowast lowastlowastlowast lowast lowast

lowastlowast lowast lowastlowastlowast lowast lowastlowastlowastlowastlowast lowast lowast lowast lowastlowastlowast lowast lowastlowast lowast

lowastlowast lowast lowast lowast lowast lowastlowast lowast lowastlowast lowast lowastlowast lowast lowastlowast lowastlowastlowastlowast lowast lowast lowastlowast

lowast lowastlowastlowast lowast lowastlowast lowast lowast lowastlowast lowast lowastlowast lowast lowastlowastlowastlowastlowastlowast lowast

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

tttatctctttcattttctgaactattaaa----tgtaagaccacc---atacatacaacATTCCACGTGTAACGGTGAAAATGAGTAGAGATGTGGAGGAACACCAGTGGAGAAACGCG

aacctccaaaattttggtttatatccggttc-----------agatttaggtattatacaACTCTCTGGTCTGTTACTGACGCTGAGGCGCGAAAGCGTGGGAGCAAACAGGATTATATA

tgtataaaaatatggaacgccaaaaatgtctacttttttatctttataattgttagctaaCCCCTGGTAGTCCAAGCCGTAAACGATGAGTGCTAGTGTTACAGGGT------------T

ttctgtattttctaattcagttttaacctcattataactcttatactcaattggaaaaatTCCGCCCTTTACTGCTGCAGCTAACG---CATTAG-GCACTCCGCCTGGGGAGTACAACT

taaatcatgagataaaaactgtctagaattaataactcctcttccttcaacaggtggagaGCAA------GGTTGAAACTCAAAGCA----TTGACCGGGGGCC---CGATAAGCTGAGG

cgtataaagattcattacat----tacccatagttcccttattgtttttataatcacttaTACATGGTGTTTAATTCCAAACCACGCCAAGAACCCTCACGAGGTCTTGACATCCTCTGG

ctttattt----------------------------------------------------ACCTCCCTAGAGAATAAGGGCTTCCCCGTCGGGGATGAAATGAACAGGTGGTGCATGGAT

-------agttcgtc---AGTCTTCAGCTCGTGCAT

Enterotoxin H (seh)

------------------------------------------------------------ATCTATGAATATAAATCTTTTAATATTGGTTTAATATAAATATTCCATTTCTAGAAACCT

------------------------------------------------------------ATATTTTATGTTTGTATAAATTCATAAATTGTTCGAAATTTATATATTAAAAGTTGTTTT

------------------------------------------------------------TGTTGTTTTATAATTAAGTTAATGAAATATATTGAGGAGTTTTAAAATGATTAATAAAAT

------------------------------------------------------------TAAAATATTATTTTCGTTTTTAGCATTATTACTTTCATTCACATCATATGCGAAAGCAGA

------------ -----------TTACGATTTAGCTTTAGCTAATGCATATGGTCAATAAGATTTACACGATAAAAGTGAGTTAACAGATTTAGCTTTAGCTAATGCATATGGTCAATA

TAATCACCCATTCATTAAAGAAAATATTAAGAGTGATGAAATAAGTGGAGAAAAAGATTTTAATCACCCATTCATTAAAGAAAATATTAAGAGTGATGAAATAAGTGGAGAAAAAGATTT

AATATTTAGAAATCAAGGTGATAGTGGCAATGATTTGAGAGTAAAGTTTGCAACTGCTGAAATATTTAGAAATCAAGGTGATAGTGGCAATGATTTGAGAGTAAAGTTTGCAACTGCTGA

TTTAGCTCAGAAGTTTAAAAATAAAAATGTAGATATATATGGGGCATCTTTTTATTATAATTTAGCTCAGAAGTTTAAAAATAAAAATGTAGATATATATGGGGCATCTTTTTATTATAA

GTGTGAAAAAATAAATGAAAATATTTCTGAATGTCTATATGGAGGTACAACACTAAATAAGTGTGAAAAAATAAGTGAAAATATTTCTGAATGTCTATATGGAGGTACAACACTAAATAG

TGAAAAATTGGCACAGGAAAGGGTGATTGGTGCTAATGTTTGGGTAGATGGTATTCAAAATGAAAAATTGGCACAGGAAAGGGTGATTGGTGCTAATGTTTGGGTAGATGGTATTCAAAA

AGAAACAGAATTAATACGAACAAATAAGAAAAATGTGACATTGCAAGAATTAGATATAAAAGAAACAGAATTAATACGAACAAATAAGAAAAATGTGACATTGCAAGAATTAGATATAAA

Enterotoxin H CNS GATCAGAAAAATATTGTCCGATAAATATAAAATTTATTATAAAGACAGCGAAATAAGGG-Enterotoxin H

Enterotoxin H CNSEnterotoxin H

Enterotoxin H CNSEnterotoxin HEnterotoxin H CNSEnterotoxin HEnterotoxin H CNSEnterotoxin HEnterotoxin H CNSEnterotoxin HEnterotoxin H CNSEnterotoxin H

Enterotoxin H CNSEnterotoxin H

Enterotoxin H CNSEnterotoxin H

Enterotoxin H CNSEnterotoxin H

Enterotoxin H CNSEnterotoxin H

Enterotoxin H CNSEnterotoxin H

GATCAGAAAAATATTGTCCGATAAATATAAAATTTATTATAAAGACAGCGAAATAAGTAA

Enterotoxin N (seln) TGATTTAGCTTTAGCTAATGCATATGGTCAATAT-AATCACCCATTCATTAAAGAAAA-----TAGATATTTATGGACTGTATTATGGAAATAAATGTGTAGGCTTAACTGAAGAAAAAA

TATTAAGAGTGATGAAATAAGTGGAGAAAAAGATTTAATATTTAGAAATCAAGGTGATAGCATCATGCTTATACGGAGGAGTTAC------GA--TATATGATGGAAATCAATTAGATGA

TGGCAATGATTTGAGAGTAAAG--------------TTTGCAA-CTGCTGATTTAGCTCAAGAGAGAGTTATAGGCGTTAATGTATTTAAAGATGGTATCCAACAAGAAGGTTTTGTTAT

GAAGTTTAAAAATAAAAATGTAGATATATATGGGGCATCTTTTTATTATAAGTGTGAAAAAAAAACTAAAAAGGCTAAAGTAACAGTACAA------GAATTAGATACTAAAGTTCGATT

AATAAGTGAAAATATTTCTGAATGTCTATATGGAGGTACAACACTAAATAATGAAAAATT------------TAAATTAGAAAATTTATATAAAATATACAATAAAGATACCGGTAACAT

Enterotoxin N CNSEnterotoxin N

Enterotoxin N CNSEnterotoxin N

Enterotoxin N CNSEnterotoxin N

Enterotoxin N CNSEnterotoxin N

Enterotoxin N CNSEnterotoxin N

Figure 3 Continued

12 BioMed Research International

lowastlowastlowast lowastlowast lowastlowast lowastlowast lowast lowastlowast lowastlowastlowast lowastlowast lowast lowastlowastlowastlowast

lowastlowast lowast lowastlowast lowast lowast lowastlowast lowastlowastlowastlowastlowast lowast lowast lowast lowastlowast lowast lowast lowastlowast lowast lowast

lowast lowast

lowast lowastlowast lowast lowastlowast lowast lowast lowast lowast lowast lowast lowast lowastlowast lowast lowastlowastlowastlowast lowast

lowast lowastlowast lowast lowast lowastlowast lowast lowastlowastlowast lowast lowastlowast lowast lowastlowast lowastlowast lowast lowast

lowastlowastlowast lowast lowast lowastlowast lowastlowast lowast lowast lowast

lowast lowast lowast lowastlowastlowast lowastlowastlowast lowastlowast lowast lowastlowastlowastlowastlowast lowastlowastlowast lowast

lowastlowast lowast lowast lowastlowast lowastlowastlowast lowastlowast lowast lowast lowastlowastlowastlowast lowastlowast

lowast lowast lowast lowastlowast lowast lowastlowast lowastlowastlowastlowastlowastlowast lowastlowast lowast lowastlowast lowastlowastlowast

lowast lowast lowast lowast lowastlowast lowastlowastlowastlowast lowast lowast lowastlowastlowast lowast lowast lowast lowast lowast

lowast lowast lowast lowast lowastlowastlowast lowast lowast lowastlowast lowastlowast lowast lowastlowastlowast lowast lowastlowastlowast lowast

lowast lowast lowastlowastlowast lowast lowastlowast lowastlowastlowast lowastlowastlowastlowastlowast lowastlowast lowast lowastlowastlowast lowast lowast lowastlowastlowast

lowastlowastlowast lowast lowastlowast lowastlowast lowastlowast lowast lowast lowast lowast lowastlowast lowastlowast lowastlowast lowast lowast

lowast lowast lowast lowast lowastlowast lowast lowast lowastlowast lowastlowast lowast lowastlowastlowast lowastlowastlowastlowast lowast lowastlowastlowastlowastlowast lowast

lowastlowast lowast lowast lowastlowastlowast lowastlowast lowast lowastlowast lowast lowastlowast lowastlowast lowastlowast lowast lowast lowast

lowastlowastlowastlowastlowast lowastlowast lowastlowast lowast lowastlowast lowast lowast lowast lowast lowastlowast lowastlowast

lowast lowast lowastlowast lowastlowast lowastlowastlowastlowastlowastlowast lowastlowast lowast lowast lowast lowast lowast lowast lowast lowast lowast lowast lowast

lowastlowast lowastlowast lowast lowastlowastlowastlowast lowast lowast lowastlowastlowast lowastlowast lowast lowastlowastlowast lowast

lowastlowastlowastlowast lowastlowast lowast lowast lowast lowast lowastlowast lowast lowast lowast lowastlowastlowastlowastlowast lowastlowast lowastlowastlowast lowast

lowast lowastlowastlowast lowastlowastlowastlowastlowast lowast lowast lowast lowastlowast lowast lowast lowastlowast lowast lowastlowastlowast lowastlowast lowast

lowastlowast lowast lowast lowast lowastlowast lowast lowastlowast lowastlowast lowastlowast lowast lowastlowastlowastlowast

ATTAATACGAACAAATAAGAAAAATGTGACATTGCAAGAATTAGATATAAAGATCAGAAA-TTAT----------TATGATTTATATAAC--------------ATAAAAGGTTCAG---

AATATTGTCCGATAAATATAAAATTTATTATAAAGACAGCGAAATAAGGTAAAAGG------TAGGAGCAGAGTTTTTTCAATTTTATAGTGATAATAGAACAGTTAGCTCATCTAATTA

Enterotoxin N CNS -TAAA---Enterotoxin N

lowast lowastlowastlowastlowastlowast lowastlowast lowast lowast lowast lowastlowast lowast lowastlowastlowastlowastlowastlowast lowastlowast lowastlowast

GGCACAGGAAAGGGTGATTGGTGCTAATGTTTGGGTAGATGGTATTCAAAAAGAAACAGAACAA----AAAGGATGCATTTTCTTT-----CATTCTAATAATCATCAAAATCAATCATT

Enterotoxin N CNSEnterotoxin N

Enterotoxin N CNSEnterotoxin N

Enterotoxin N CNSEnterotoxin N

TCATATCG

Enterotoxin M (selm)

ttgctattcgcaaaatcatatcgcaaccgctgatgtcggagtt--ttgaatcttaggaacEnterotoxin M CNSEnterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

--------------AGTATGTTTAAATCCCCGCTCACAGTTATGTTTAACTCTTCCTTTC

tattatggta-gctatccaattgaagaccaccaaagtattaatcctgaaaataatcatctTTTTCACACCGCCCCGTCGGGTGTTGCGCACGGGGTTGTTTAACCCCCCCGGAAAAAATT

ttcgcatcaattagttttttctatggataattcgacagtaacagctgaatttaagaacgtEnterotoxin M CNS

Enterotoxin M CNS

TTCCCTCCCTCCTCCTTACTC---------------CCT----------------CCCCT

tgatgatgtaaaggaattcaaaaatcatgctgtagatgtatatggtctaagttatagtggEnterotoxin M CNS TTTTCCTACAAATCCTTTCTCCCCTCCCGGAGTAGAAGTAAA------------------

atattgtttgaaaaacaaatatatatacggtggagttacattagcaggtgat-tatttagEnterotoxin M CNS -------------------TAAAACAATACCCCTACCACCTTAACAATTTTTTTATTGAG

agaaatctagacgtattcct-attaatctttgggttaatggagaacatcaaactatatctEnterotoxin M CNS CCCCTCCCCCCCCTTCTCATTTTTTCTCTTTGAC-----------GTTCCCCCCATTTCT

actgacaaagtatcaactaataaaa-agttagtaacagctcaagaaattgatactaaattEnterotoxin M CNS GCCAATGCTTAACCCACCCTTAAAGGCCATCGGCACATATTAGACACTCC-----CCACC

aagaagatatctacaagaagaatataatatttatggctttaatgatac---aaataaaggEnterotoxin M CNS CGCCCCCTCTTTTTATTAAGGACACAACTCTTTTTATTTTCCCCCAAGAACTAATTCTGA

a-agaaattatggtaataagtcaaaatttagttctgga---------tttaatgcaggaaEnterotoxin M CNS AAAAAAACGTTCCGCTTAGTTCAGAATTTCCTTTGAGCCTTTTATCCTTTTGTACGCGAA

aaatattatttca--------tttgaatgatggttcatcattttcttatgacttatttga

Enterotoxin M CNS AAACCATCAGCCATTGAATCAGTTATCCCATTGCCCTAACTAACCCAATATCTAATCTAA

tactggaacaggacaagctgaaagtttctt-aaaaatatataatgacaacaaaactgtcgEnterotoxin M CNS TTTTCGCCTACGAACGAATAATAGCTTGTGGCGAAAAATATCCAGGATTATCTACTGTAG

aaact--gaaaaattccatttagatgtagaaatatcttataaggacgaaagttgaagtatEnterotoxin M CNS CGCCTCGCCAGACGTCCTTTGAATTGA--ATATGACTCCTACTCCCCCCCGCACCGTCAA

ttcaatcataacttagtaaaggaaatgccatgaaaaaatttaaatatagtttt---atatEnterotoxin M CNS TTCAAACA---------------GGTGGAAGTTAATACTGAATAGTAGGTCTTCTTCCCG

tagtttttatattactttttaacattaaagatcttacgtatgct---caaggtgatattgEnterotoxin M CNS TGCTATTCTTAGAACTTTTAAATTATGTAAACCCAATTTTTAGTGCACTCTTTCTCCCTC

gtg-taggtaacttaagaaatttctatacaaaacatgattatatagatttaaaaggcgtcEnterotoxin M CNS GTTACTCTTAGATGGAGAAGCGTTAAGACAGAAGCTTTGTAT-------------TCCCA

acagataaaaa-cctacctattgcaaatcaacttgaattttcaac-----aggt----acEnterotoxin M CNS GCAGAGTGCAACCTTTCACAATGAAGTTTTTATCTAATTTACACGCCTTTAGGCTCCAAA

caatgatttgatctcagaatctaataattgggacgaaataagtaaatttaaaggaaagaaEnterotoxin M CNS TACGTATTAAATCTCCTCCTGT--TTATCTGACCCTAAG-ATAAAACACGAAAAATTATG

actggatatttttggcattgattataatggtccttgtaaatctaaatacatgtatggaggEnterotoxin M CNS CGTGCCCACTCATCCTAT-------TACTTTCATTATATCTATAAAA-------------

Figure 3 Alignment of the enterotoxin gene sequences found in coagulase-negative staphylococci (CNS) and in coagulase-positivestaphylococci (CPS)

BioMed Research International 13

Enterotoxin (seg) S aureus AY1587031

Enterotoxin (sec) CNS

Enterotoxin (see) S pasteuri NC0227371

Enterotoxin (see) CNS

Enterotoxin (seh) CNS

Enterotoxin (selm) S aureus AF2857601

Enterotoxin (seln) CNS

Enterotoxin (seh) S aureus AJ9375481

Enterotoxin (seln) S aureus AY1587031

Enterotoxin (sec) S aureus X516611

Enterotoxin (selm) CNS

Enterotoxin (seg) CNS

150 100

100

100

100

100

99

98

96

97

72

50 0

Figure 4 Phylogenetic tree generated from the multiple alignments of the enterotoxin sequences of CNS and CPS strains using the ClustalX20 software package The phylogenetic tree was constructed using the Mega 60 software and the unweighted pair group method (UPGMA)

MAR indices varying from 066 to 046 and the remaining04 strains showed MAR indices le026 (Table 3)

The 14 MRCNS strains were resistant to 120573-lactams(oxacillin penicillin andor cefoxitin) and to vancomycincorresponding to 73 of the total CNS strains identifiedwhile 09 strains (64) showed resistance to tetracycline andgentamicin 08 strains (57) were resistant to neomycinerythromycin and chloramphenicol 07 strains (50) wereresistant to sulfamethoprim 05 strains (36) were resistantto linezolid 03 strains (21) were resistant to rifampicin 02strains (14) were resistant to ciprofloxacin and cefepimeand 01 strain (7) was resistant to clindamycin (Table 3)

The multiresistance of CNS strains demonstrated hereinis consistent with previous studies on coagulase-negativeand coagulase-positive staphylococci that have found severalresistant and multiresistant Staphylococcus aureus strains inraw milk meat and fermented meat products [43]

Surprisingly the resistance to chloramphenicol is verysimilar to that estimated for MRCNS strains isolated fromhuman clinical samples Indeed there is no direct correla-tion between the researched features and the origin of thestaphylococci strains since different virulence factors arewidespread such as antibiotic resistance [44] reinforcing thefact that the ability of foodmatrices strains to produce SE andSEl and their multiresistance character must be consideredwhen evaluating the safety hazards of food poisoning

S epidermidis KF6005891 and S hominis JX5199881showed the highestMAR indexes (093 and 080 resp)Thesestrains found in artisanal salami are homologous to strainsisolated from human skin microbiota and should be carefullyconsidered among the potential pathogenic staphylococcifound in food from animal origins which can be caused bycontamination by poor hygienic conditions during salamimanufacturing

Depending on the conditions some species of coagulase-negative staphylococci can present health risks since theyhave shown resistance to antibiotics of therapeutic impor-tance such as beta lactams [15] However multiresistantstrains like S carnosusKJ18620021 and S xylosusCP0072081and CP0087241 were intentionally introduced in the foodmatrix since they are part of the culture starter used inItalian-salami manufacturing As previously discussed foodcan be a reservoir of multiresistant microorganisms that canspread by the consumption of an apparently safe food Dueto the intensive and indiscriminate use of antibiotics forhuman and veterinarian therapeutic purposes multiresistantstaphylococci strains are being selected and reproduced infood matrices [45]

The cefoxitin and oxacillin disk diffusion tests are rec-ommended for determining the resistance and susceptibilitybreakpoint for MRSA surveillance cultures [46] In thisstudy there was a good correlation between the disc testzone diameters for oxacillin and cefoxitin (Table 3) TheMICs for ampicillin andmethicillinoxacillincefoxitin for allsusceptible CNS strainswere determined by the diffusion disctest using 003 to 2mgmLminus1 of each antimicrobial agent

S saprophyticus HF9372521 showed an MIC of003mgmLminus1 formethicillin strain S carnosusKJ8620021 Sxylosus strains CP0087241 and CP0072081 S saprophyticusJX4901221 S hominis JX5199881 and S succinusKC3298241 were resistant to 006mgmLminus1 and two strainsS saprophyticus KJ6991511 and S saprophyticus KJ0046231showed an MIC of 05mgmLminus1 (Table 4) Two of themethicillin-resistant strains S saprophyticus KJ0046231 andS xylosus CP0072081 harbor no enterotoxin gene

Five strains S saprophyticus JX4901221 S carnosusKJ8620021 S xylosusCP0087241 S xylosusKF1980801 andS saprophyticus KJ0046231 showed an MIC for ampicillin

14 BioMed Research International

Table 3 Multiple resistance to antimicrobial as found in CNS strains from salami

Salami origin CNS strains Antimicrobial agent resistance Multiple antimicrobialresistance (MAR) indexlowast

Commercial Staphylococcus spp KF1354451 CIP CLO CPM GEN NEO OXA PENSXT TET and VAN 066

Commercial S carnosus KJ8620021 CIP GEN LZD NEO OXA SXT andTET 046

Commercial S saprophyticus AB6977171 CFO CLO ERI PEN OXA TET andVAN 046

Commercial S saprophyticus EU4309921 CFO CLO ERI GEN LZD NEO OXAPEN SXT TET and VAN 080

Commercial S saprophyticus JX4901221 CFO CLO ERI GEN NEO PEN OXASXT TET and VAN 066

Commercial S saprophyticus KJ0046231 CFO OXA and PEN 020

Commercial S sciuri JX9664361 CFO CLO GEN NEO OXA PEN andTET 046

Commercial S xylosus CP0072081 OXA LZD PEN and VAN 026

Commercial S xylosus CP0087241 CFO CLO ERI GEN NEO OXA PENTET and VAN 060

Artisanal S epidermidis KF6005891 CFO CLI CLO CPM ERI GEN LZDNEO OXA PEN RIF SXT and TET 093

Artisanal S hominis JX5199881 CIP CPM CPO ERI GEN LZD NEOOXA PEN RIF SXT and VAN 080

Artisanal S saprophyticusHF9372521 CFO OXA PEN and SXT 026Artisanal S saprophyticus KJ6991511 OXA PEN and VAN 020

Artisanal S succinus KC3298241 CFO CLO ERI GEN OXA PEN RIFSXT TET and VAN 066

Artisanal S xylosus KF1980801 CFO CLO ERI GEN NEO OXA PENTET and VAN 060

lowastTheMAR index of an isolate is defined as 119886119887 where 119886 represents the number of antimicrobials to which the isolate was resistant and 119887 represents the numberof antimicrobials to which the isolate was subjectedS aureus strains ATCC WB81 (sea) ATCC 13563 (sed) and ATCC 27664 (see) showing a MAR index of 05 and S aureus strains ATCC14458 (seb) andATCCWB72 (sec) and S xylosus ATCC 29971 showing a MAR index of 03 were used as reference strainsCPM cefepime CFO cefoxitin CLO chloramphenicol CIP ciprofloxacin CLI clindamycin ERI erythromycin GEN gentamycin NEO neomycin LZDlinezolid RIF rifampicin TET tetracycline OXA oxacillin PEN penicillin SXT sulfamethoprim and VAN vancomycin

Table 4 Minimal inhibitory concentration (MIC) of compounds used in antimicrobial therapy against staphylococci infections

Salami origin GenBank accession number and similarity () MIC mgmLminus1

Methicillin Ampicillin Vancomycin Linezolid

Commercial

S carnosus KJ8620021 006 003 mdash mdashS xylosus CP0087241 006 003 006 mdash

S saprophyticus JX4901221 006 003 05 mdashS saprophyticus AB6977171 mdash mdash 05 mdashS saprophyticus EU4309921 mdash mdash 025 0125

S succinus KC3298241 006 025 003 mdash

Artisanal

S hominis JX5199881 006 05 mdash mdashS saprophyticus HF9372521 003 mdash mdash mdash

S xylosus KF1980801 003 05 mdashS saprophyticus KJ6991511 05 025 003 mdashS saprophyticus KJ0046231 05 003 003 mdash

S xylosus CP0072081 006 mdash mdash 025Strains S epidermidis KF6005891 and Staphylococcus spp KF1354451 were not susceptible to the antimicrobial concentrations tested in the present study

BioMed Research International 15

of 003mgmLminus1 two strains S succinus KC3298241 and Ssaprophyticus KJ6991511 showed an MIC of 025mgmLminus1and S hominis JX5199881 showed an MIC of 05mgmLminus1(Table 4) S saprophyticus KJ0046231 also showed an MICof 003mgmLminus1 but does not harbor enterotoxin genes It isimportant to note that most of the CNS strains that showedresistance to ampicillin were also resistant to methicillin ashighlighted by the MIC tests

Five strains S saprophyticus HF9372521 S saprophyti-cus KJ6991511 S saprophyticus JX4901221 S saprophyticusKJ0046231 and S succinus KC3298241 showed an MICfor vancomycin of 003mgmLminus1 one strain S xylosusCP0087241 showed an MIC of 006mgmLminus1 one strain Ssaprophyticus EU4309921 showed an MIC of 025mgmLminus1and two strains S xylosus KF1980801 and S saprophyticusAB6977171 showed an MIC of 05mgmLminus1 (Table 4)

Additionally five CNS strains S xylosus CP0087241S saprophyticus JX 4901221 S succinus KC3298241 Ssaprophyticus KJ6991511 and S saprophyticus KJ0046231showed resistance tomethicillin as demonstrated by theMICdeterminations (Table 4)

Previous studies have demonstrated that linezolid isactive against Gram-positive bacteria including methicillin-resistant staphylococci [47] In the present study 05 strainsshowed linezolid resistance in the disc diffusion test but itwas impossible to establish MIC values for S epidermidisKF6005891 Staphylococcus hominis JX5199881 and Staphy-lococcus carnosus KJ8620021The remaining strains S sapro-phyticus EU4309921 and S xylosus CP0072081 presentedan MIC for linezolid of 0125mgmLminus1 and 025mgmLminus1respectively

The MIC of 003mgmLminus1 for penicillin is in accordancewith the previous values estimated for resistant S aureusstrains found in several food matrices such as meat dairyproducts and ready-to-eat food [48]

There is still a lack of information on the antimicro-bial resistance of staphylococci strains from food matricesalthough they are the most worrisome vehicles of dissemi-nation of antibiotic-resistant pathogens [49]

The high resistance found for salami staphylococci strainscan be ascribed to their inappropriate use as growth promot-ers of antimicrobial agents like oxacillin vancomycin chlo-ramphenicol neomycin and erythromycin which are com-monly used in veterinary medicine to treat infections [50]Antimicrobial therapy of infections staphylococci is basedon results of susceptibility tests in vitro [51] Methicillin-resistant coagulase-negative Staphylococcus spp found inready-to-eat products such as meats fish and dairy productsalso offer risks and although they are not classical foodpoisoning bacteria their presence in food offers significantrisks to public health due to the possible spread of antibioticresistance [52]

The standardization of salami quality should includediagnostic methods to screen and quantify the presence ofclassical and newly described enterotoxins directly in thefood matrices as a routine procedure to be conducted by themeat product industry in Brazil

The safety of salami consumption could also be enhancedby the inclusion of amicrobial barrier such as the inclusion ofprobiotic strains producing natural antibiotics or competitiveflora or even the addition of natural bioagents against spoilageor pathogenic microorganisms

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors acknowledge the financial support fromFundacao Carlos Chagas Filho de Amparo a Pesquisa doEstado do Rio de Janeiro (FAPERJ Rio de Janeiro Brazil)Conselho Nacional de Desenvolvimento Cientıfico eTecnologico (CNPq Brasılia Brazil) and Coordenacao deAperfeicoamento de Pessoal de Nıvel Superior (CAPESBrasılia Brazil)

References

[1] ICMSF International Comission on Microbiological Specifica-tions for FoodsMicroorganisms in FoodsTheir Significance andMethods of Enumerations University of Toronto Press TorontoCanada 3rd edition 1983

[2] J F Veras L S Carmo L C Tong et al ldquoA study of theenterotoxigenicity of coagulase-negative and coagulase-positivestaphylococcal isolates from food poisoning outbreaks inMinasGerais Brazilrdquo International Journal of Infectious Diseases vol12 no 4 pp 410ndash415 2008

[3] K Omoe D-L Hu H Takahashi-Omoe A Nakane and KShinagawa ldquoComprehensive analysis of classical and newlydescribed staphylococcal superantigenic toxin genes in Staphy-lococcus aureus isolatesrdquo FEMS Microbiology Letters vol 246no 2 pp 191ndash198 2005

[4] J Kadariya T C Smith and D Thapaliya ldquoStaphylococcusaureus and staphylococcal food-borne disease an ongoing chal-lenge in public healthrdquoBioMedResearch International vol 2014Article ID 827965 9 pages 2014

[5] KOmoeD-LHuH KOno et al ldquoEmetic potentials of newlyidentified staphylococcal enterotoxin-like toxinsrdquo Infection andImmunity vol 81 no 10 pp 3627ndash3631 2013

[6] J K McCormick J M Yarwood and P M SchlievertldquoToxic shock syndrome and bacterial superantigens an updaterdquoAnnual Review of Microbiology vol 55 pp 77ndash104 2001

[7] T Baba F Takeuchi M Kuroda et al ldquoGenome and virulencedeterminants of high virulence community-acquired MRSArdquoThe Lancet vol 359 no 9320 pp 1819ndash1827 2002

[8] Associacao Brasileira de Industria Produtora e Exportadorada Carne Suınas (ABIPECS) ldquoStatistics slaughter ranking2000ndash2014rdquo 2014 httpabpa-brcombrsetoressuinoculturapublicacoesrelatorios-anuais

[9] N Zdolec M Hadziosmanovic L Kozacinski et al ldquoMicrobialand physicochemical succession in fermented sausages pro-duced with bacteriocinogenic culture of Lactobacillus sakei andsemi-purified bacteriocin mesenterocin Yrdquo Meat Science vol80 no 2 pp 480ndash487 2008

16 BioMed Research International

[10] F Ravyts L D Vuyst and F Leroy ldquoBacterial diversity andfunctionalities in food fermentationsrdquo Engineering in Life Sci-ences vol 12 no 4 pp 356ndash367 2012

[11] TOhlsson andN BengtssonTheHurdle ConceptMinimal Pro-cessing Technologies in the Food IndustryWoodhead Publishing2002

[12] S Bernardi B B Golineli and C J Contreras-CastilloldquoRevisao aspectos da aplicacao de culturas starter na producaode embutidos carneos fermentadosrdquo Brazilian Journal of FoodTechnology vol 13 no 2 pp 133ndash140 2010

[13] F Irlinger ldquoSafety assessment of dairy microorganismscoagulase-negative staphylococcirdquo International Journal of FoodMicrobiology vol 126 no 3 pp 302ndash310 2008

[14] B Ghebremedhin F Layer W Konig and B Konig ldquoGeneticclassification and distinguishing of Staphylococcus species basedon different partial gap 16S rRNA hsp60 rpoB sodA and tufgene sequencesrdquo Journal of Clinical Microbiology vol 46 no 3pp 1019ndash1025 2008

[15] I Babic K Markov D Kovacevic et al ldquoIdentification andcharacterization of potential autochthonous starter culturesfrom aCroatian lsquobrandrsquo product lsquoslavonski kulenrsquordquoMeat Sciencevol 88 no 3 pp 517ndash524 2011

[16] MMehrotra GWang andWM Johnson ldquoMultiplex PCR forthe detection of genes for Staphylococcus aureus enterotoxinsexfoliative toxins toxic shock syndrome toxin and methicillinresistancerdquo Journal of Clinical Microbiology vol 38 no 3 pp1032ndash1041 2000

[17] WM Johnson SD Tyler E P Ewan F EAshtonDR Pollardand K R Rozee ldquoDetection of genes for enterotoxins exfolia-tive toxins and toxic shock syndrome toxin 1 in Staphylococcusaureus by the polymerase chain reactionrdquo Journal of ClinicalMicrobiology vol 29 no 3 pp 426ndash430 1991

[18] J Mclauchlin G L Narayanan VMithani andG OrsquoNeill ldquoThedetection of enterotoxins and toxic shock syndrome toxin genesin Staphylococcus aureus by polymerase chain reactionrdquo Journalof Food Protection vol 63 no 4 pp 479ndash488 2000

[19] J P Rosec and O Gigaud ldquoStaphylococcal enterotoxin genes ofclassical and new types detected by PCR in Francerdquo Interna-tional Journal of Food Microbiology vol 77 no 1-2 pp 61ndash702002

[20] K Omoe D-L Hu H Takahashi-Omoe A Nakane andK Shinagawa ldquoIdentification and characterization of a newstaphylococcal enterotoxin-related putative toxin encoded bytwo kinds of plasmidsrdquo Infection and Immunity vol 71 no 10pp 6088ndash6094 2003

[21] N Sergeev D Volokhov V Chizhikov and A Rasooly ldquoSimul-taneous analysis of multiple staphylococcal enterotoxin genesby an oligonucleotide microarray assayrdquo Journal of ClinicalMicrobiology vol 42 no 5 pp 2134ndash2143 2004

[22] S Holtfreter D Grumann M Schmudde et al ldquoClonal distri-bution of superantigen genes in clinical Staphylococcus aureusisolatesrdquo Journal of Clinical Microbiology vol 45 no 8 pp2669ndash2680 2007

[23] W J Mason J S Blevins K Beenken NWibowo N Ojha andM S Smeltzer ldquoMultiplex PCR protocol for the diagnosis ofstaphylococcal infectionrdquo Journal of Clinical Microbiology vol39 no 9 pp 3332ndash3338 2001

[24] E Rahimi and H G Safai ldquoDetection of classical enterotoxinsof Staphylococcus aureus strains isolated frombovine subclinicalmastitis in Isfahan Iranrdquo Veterinary Microbiology vol 141 no3-4 pp 393ndash394 2010

[25] M A Larkin G Blackshields N P Brown et al ldquoClustalW andClustal X version 20rdquo Bioinformatics vol 23 no 21 pp 2947ndash2948 2007

[26] P H A Sneath and R R Sokal Unweighted Pair Group Methodwith Arithmetic Mean Numerical Taxonomy W H FreemanSan Francisco Calif USA 1973

[27] NCCLS ldquoPerformance standards for antimicrobial susceptibil-ity testing 14th informational supplementrdquo Tech Rep M 100-S14 NCCLS 2004

[28] S Corbiere Morot-Bizot S Leroy and R Talon ldquoMonitoringof staphylococcal starters in two French processing plantsmanufacturing dry fermented sausagesrdquo Journal of AppliedMicrobiology vol 102 no 1 pp 238ndash244 2007

[29] R Talon S Leroy I Lebert et al ldquoSafety improvement andpreservation of typical sensory qualities of traditional dryfermented sausages using autochthonous starter culturesrdquo Inter-national Journal of Food Microbiology vol 126 no 1-2 pp 227ndash234 2008

[30] G Mauriello A Casaburi G Blaiotta and F Villani ldquoIsolationand technological properties of coagulase negative staphylo-cocci from fermented sausages of Southern ItalyrdquoMeat Sciencevol 67 no 1 pp 149ndash158 2004

[31] M Janssens N Myter L De Vuyst and F Leroy ldquoSpeciesdiversity and metabolic impact of the microbiota are low inspontaneously acidified Belgian sausages with an added starterculture of Staphylococcus carnosusrdquo Food Microbiology vol 29no 2 pp 167ndash177 2012

[32] G Blaiotta D Ercolini G Mauriello G Salzano and F VillanildquoRapid and reliable identification of Staphylococcus equorum bya species-specific PCR assay targeting the sodA generdquo Systematicand Applied Microbiology vol 27 no 6 pp 696ndash702 2004

[33] L Kozacinski E Drosinos F Caklovica L Cocolin J Gasparik-Reichardt and S Veskovic ldquoInvestigation of microbial associa-tion of traditionally fermented sausagesrdquo Food Technology andBiotechnology vol 46 no 1 pp 93ndash106 2008

[34] S Leroy P Giammarinaro J-P Chacornac I Lebert and RTalon ldquoBiodiversity of indigenous staphylococci of naturallyfermented dry sausages and manufacturing environments ofsmall-scale processing unitsrdquo Food Microbiology vol 27 no 2pp 294ndash301 2010

[35] E Coton M-H Desmonts S Leroy et al ldquoBiodiversity ofCoagulase-Negative Staphylococci in French cheeses dry fer-mented sausages processing environments and clinical sam-plesrdquo International Journal of Food Microbiology vol 137 no 2-3 pp 221ndash229 2010

[36] S Hosseinzadeh and H D Saei ldquoStaphylococcal species associ-ated with bovinemastitis in the NorthWest of Iran emerging ofcoagulase-negative staphylococcirdquo International Journal of Vet-erinary Science and Medicine vol 2 no 1 pp 27ndash34 2014

[37] A Piette and G Verschraegen ldquoRole of coagulase-negativestaphylococci in human diseaserdquo Veterinary Microbiology vol134 no 1-2 pp 45ndash54 2009

[38] T M Moura F S Campos P A DrsquoAzevedo et al ldquoPrevalenceof enterotoxin-encoding genes and antimicrobial resistancein coagulase-negative and coagulase-positive Staphylococcusisolates from black pudding in Southern Brazilrdquo Revista daSociedade Brasileira deMedicina Tropical vol 45 no 5 pp 579ndash585 2012

[39] H I Suzuki T Lefebure P P Bitar and M J StanhopeldquoComparative genomic analysis of the genus Staphylococcusincluding Staphylococcus aureus and its newly described sister

BioMed Research International 17

species Staphylococcus simiaerdquo BMC Genomics vol 13 article38 2012

[40] R P Lamers G Muthukrishnan T A Castoe S Tafur A MCole and C L Parkinson ldquoPhylogenetic relationships amongStaphylococcus species and refinement of cluster groups basedon multilocus datardquo BMC Evolutionary Biology vol 12 article171 2012

[41] I V Pinchuk E J Beswick and V E Reyes ldquoStaphylococcalenterotoxinsrdquo Toxins vol 2 no 8 pp 2177ndash2197 2010

[42] S R Monday and G A Bohach ldquoGenes encoding staphylo-coccal enterotoxins G and I are linked and separated by DNArelated to other staphylococcal enterotoxinsrdquo Journal of NaturalToxins vol 10 no 1 pp 1ndash8 2001

[43] D Y Thomas S Jarraud B Lemercier et al ldquoStaphylococcalenterotoxin-like toxins U2 and V two new staphylococcalsuperantigens arising from recombination within the entero-toxin gene clusterrdquo Infection and Immunity vol 74 pp 4724ndash4734 2006

[44] N Indrawattana O Sungkhachat N Sookrung et al ldquoStaphylo-coccus aureus clinical isolates antibiotic susceptibility molecu-lar characteristics and ability to form biofilmrdquo BioMed ResearchInternational vol 2013 Article ID 314654 11 pages 2013

[45] V Pereira C Lopes A Castro J Silva P Gibbs and P TeixeiraldquoCharacterization for enterotoxin production virulence factorsand antibiotic susceptibility of Staphylococcus aureus isolatesfrom various foods in Portugalrdquo Food Microbiology vol 26 no3 pp 278ndash282 2009

[46] I Anacarso C Condo C Sabia and et al ldquoAntimicrobialresistance and other related virulence factors in staphylococcusspp isolated from food environmental and humans in ItalyrdquoUniversal Journal Microbiology Research vol 1 no 1 pp 1ndash92013

[47] B Martın M Garriga M Hugas S Bover-Cid M T Veciana-Nogues and T Aymerich ldquoMolecular technological and safetycharacterization of Gram-positive catalase-positive cocci fromslightly fermented sausagesrdquo International Journal of FoodMicrobiology vol 107 no 2 pp 148ndash158 2006

[48] NM Broekema T Van Tam T AMonson S AMarshall andD M Warshauer ldquoComparison of cefoxitin and oxacillin diskdiffusionmethods for detection ofmecA-mediated resistance inStaphylococcus aureus in a large-scale studyrdquo Journal of ClinicalMicrobiology vol 47 no 1 pp 217ndash219 2009

[49] E Cercenado F Garcıa-Garrote and E Bouza ldquoIn vitro activityof linezolid against multiply resistant Gram-positive clinicalisolatesrdquo Journal of Antimicrobial Chemotherapy vol 47 no 1pp 77ndash81 2001

[50] A Aydin K Muratoglu M Sudagidan K Bostan B Okukluand SHarsa ldquoPrevalence and antibiotic resistance of foodborneStaphylococcus aureus isolates in Turkeyrdquo Foodborne Pathogensand Disease vol 8 no 1 pp 63ndash69 2011

[51] D Sergelidis A Abrahim T Papadopoulos et al ldquoIsolation ofmethicillin-resistant Staphylococcus spp from ready-to-eat fishproductsrdquo Letters in Applied Microbiology vol 59 no 5 pp500ndash506 2014

[52] I PhillipsM Casewell T Cox et al ldquoDoes the use of antibioticsin food animals pose a risk to human health A critical reviewof published datardquo Journal of Antimicrobial Chemotherapy vol53 no 1 pp 28ndash52 2004

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Page 7: Research Article Safety Evaluation of the Coagulase ...downloads.hindawi.com/journals/bmri/2015/483548.pdf · Research Article Safety Evaluation of the Coagulase-Negative Staphylococci

BioMed Research International 7

O S succinus KC3298241Δ S xylosus AM8827001

O S saprophyticus KJ6991511Δ S saprophyticus AB6977171

Δ S xylosus CP0087241Δ S xylosus KC4565901Δ S saprophyticus KJ9496061Δ S saprophyticus JX4901221Δ S saprophyticus KJ0046231

Δ S xylosus CP0072081Δ S carnosus NR1164341Δ S saprophyticus HQ6995101

O S epidermidis KF0882111O S hominis JX5199881Δ S saprophyticus EU4309921

O S xylosus KF1980801Δ S carnosus KJ8620021Δ S sciuri JX9664361O S saprophyticus HF9372521

04 03 02 01 00

Figure 1 Phylogenetic tree generated from the multiple alignments of the 16S rDNA sequences of CNS strains found in salami usingthe ClustalX 20 software The phylogenetic tree was constructed by using the Mega 60 software and the unweighted pair group method(UPGMA) Bootstrap values ranged from 00 to 04 Strains found in commercial (Δ) or artisanal salami (O)

between these CNS strains Suzuki et al 2012 demonstrateda bootstrap value higher than 90 for interspecies similaritiesbetween S saprophyticus S epidermidis S hominis and Scarnosus

33 Genotypic and Phenotypic Characterization of CNSStrains Twelve distinct combinations of staphylococcalenterotoxins geneswere found in the 15CNS strains compris-ing SEs AndashE GndashJ and also the enterotoxin-like toxins (SElL)KndashR and U (Table 2)

Fifteen strains (79) carried at least one gene encodingenterotoxins in their genomes (Figure 2) The seb and secgenes were the most predominant harbored by 57 ofthe strains followed by sea carried by 50 whereas thesedsehselm genes showed intermediate incidence harboredby 33 of the strains while seiseln and tsH1 were found in27 of strains Finally see selj and selo showed low incidence(13) and seg was carried by only 1 of strains (Table 2)

The relationships between superantigenic toxin geno-types and toxin gene-encoding mobile genetic elementsin CNS strains were evaluated Distinct combinations ofSaPI and plasmids or plasmids and genes of egc operonenterotoxins were found in the CNS strains obtained fromsalami

Four strains presented only the classical enterotoxingenes namely S saprophyticus KJ0046231 and HF9372521S xylosus CP0087241 and S carnosus NR1164341 Anotherstrain S saprophyticus AB6977171 presented only the newlydescribed enterotoxins seh sei and selm and finally 10 strainspresented a combination of classical and newly described

enterotoxin genes in their genomes with at least one of eachenterotoxin type (Table 2)

Previous studies have shown that sea is the most com-mon toxin associated with Staphylococcus food poisoningfollowed by sed and see with SEHand SEI being considered asplaying only a minor role [39] Among the 15 enterotoxigenicstrains found in salami 73 of them harbored at least the seagene or combinations of these 05 genes Strains S carnosusKJ8620021 found in commercial salami and S epidermidisKF6005891 found in artisanal salami carry 04 of these genesThe sea and seb enterotoxin genes are known to occupy thesame locus on the chromosome which may explain whythese enterotoxins are commonly found together in foodpoisoning outbreaks [36] The combination of sea and sebgenes was found in 05 strains S saprophyticus EU4309921 Scarnosus KJ8620021 S hominis JX5199881 S saprophyticusKJ9496061 and S succinus KC3298241 A single strain Scarnosus KJ8620021 showed the combination of sea and seigenes

Additionally some CNS are able to produce TSST-1 aloneor in combination with other enterotoxins Herein 04 of the19 strains (21) S saprophyticus EU4309921 S saprophyti-cus subsp bovis KJ6991511 and S xylosus CP0087241 andKF1980801 were shown to harbor the tstH1 combined withse andor sel enterotoxin genes (Table 2)

The staphylococci enterotoxins genes seg and sei [40] arepart of a chromosomal operon gene cluster (egc) comprisingfive genes designated as selo selm sei seln and seg Twoof the CNS strains determined in the present study Ssaprophyticus EU4309921 and S hominis JX5199881 were

8 BioMed Research International

sed

seb

sec

see

sea

M 2 3 4 51

500bp

(a)

sea

sebsebseb

M 1 2 3 4 5

(b)

sei

sehseg

1M

(c)

seh sehseh

M 1 2 3

(d)

1 2 4M 3 5

(e)

selm

seljseln

selo

21M

(f)

seln

selm

M 1 2

(g)

Figure 2 Uniplex duplex and multiplex PCR screening for the detection of enterotoxin genes in CNS strains from salami (a) Lane M100 bp DNA ladder plus (Fermentas Foster City CA USA) lane 1 S aureus ATCC 29231 harboring sea gene lane 2 S aureus NCTC 10654harboring seb gene lane 3 S aureus ATCC19095 harboring the sec gene lane 4 S aureus ATCC 13563 harboring the sed gene and lane 5 Saureus ATCC 27664 harboring the see gene (b) Lane M 100 bp DNA ladder plus lane 1 Staphylococcus spp lane 2 S carnosus NR116434lane 3 S carnosus KJ862002 lane 4 S carnosus KJ862002 and lane 5 S carnosus KJ8620021 (c) Lane M 100 bp DNA ladder plus lane1 S aureus ATCC 19095 harboring seg seh and sei genes (d) Lane M 100 bp DNA ladder plus lane 1 S saprophyticus AB6977171 lane 2S epidermidis KF 6005891 and lane 3 S sciuri JX9664361 (e) Lane M 100 bp DNA ladder plus lane 1 S xylosus KF1980801 lane 2 SsaprophyticusKJ6991511 (f) LaneM 100 bp DNA ladder plus lane 1 S aureusATCC 27154 harboring selj slem seln and selo genes (g) LaneM 100 bp DNA ladder plus lane 1 S xylosus KF1980801 and lane 2 S saprophyticus subsp bovis KJ6991511

BioMed Research International 9

shown to carry the selm seln and selo genes and the selmand seln sequences were detected in the genome of a singlestrain S saprophyticus subsp bovisKJ6991511 S epidermidisKF6005891 showed a combination of seg and sei genesand 03 strains harbored a single gene sei or selm or selnperhaps due to the high degree of genetic polymorphism inthe chromosomal assembly [41] Indrawattana et al 2013detected the seg-sei-selm-seln-selo of the highly prevalent egclocus was 261 in contrast to this study where 13 CNSpresents the combination of selm seln and selo genes [42]

The CNS strains clustered into groups near the bottom ofthe phylogenetic tree carried the classical enterotoxin geneswhile the species at the top of the tree showed high diversityamong the enterotoxin genes combining the classical and thenewly described genes in their genomes (Figure 1)

To assess the risk of staphylococcal food poisoning theability of the identified strains in harboring the se andsel genes and in expressing and producing enterotoxins invitro was evaluated The mRNA for each enterotoxin genewas evaluated by real time RT-PCR assays and enterotoxincontent was estimated by a sandwich enzyme immunoassayfor the combined detection of Staphylococcus enterotoxins(SET) A B C D and E

The fifteen enterotoxigenic CNS strains were able toexpress the classical enterotoxins SEA seb sec sed and seein concentrations ranging from 03 ngmLminus1 to 14 ngmLminus1as assessed by the in vitro assays (Table 2) The S sapro-phyticus AB6977171 KJ6991511 JX4901221 KJ0046231 andHF9372521 strains S xylosus KF1980801 and S carnosusNR1164341 produced low amount of enterotoxins lower than05 ngmLminus1 S epidermidisKF6005891 S saprophyticus bovisKJ6991511 and S hominis JX5199881 produced intermediateamount of enterotoxins ranging from 07 to 090 ngmLminus1while S sciuri JX9664361 S saprophyticus EU4309921 Scarnosus KJ8620021 S xylosus CP0087241 and S succi-nus KC3298241 produced enterotoxins in concentrationsge10 ngmLminus1

Although the sandwich enzyme immunoassay is consid-ered themost sensitivemethod to detect seandashsee enterotoxinsable to detect 0125 ngmLminus1 differences in the specificity andsensitivity of the assays for the detection of staphylococcalenterotoxins from foods are expected [43] A single strainStaphylococcus spp KF1354451 which harbors both the seaand seb genes was unable to produce seandashsee enterotoxins

The mRNA levels evaluated by the real time RT-PCRassays for enterotoxins were detected in 12 of 15 strains(86) (Table 2) Transcripts for the classical enterotoxinsgenes were detected when S saprophyticus HF9372521 Scarnosus NR1164341 S xylosus CP0087241 and S succinusKC3298241 were assayed Transcripts for newly describedenterotoxins were observed in S saprophyticus JX4901221and AB6977171 S sciuri JX9664361 S saprophyticus subspbovis KJ6991511 and S hominis JX5199881 Transcripts fromboth the classical and newly described enterotoxins weredetected in S saprophyticus EU4309921 and KJ9496061 andS epidermidis KF6005891

No mRNA transcripts were obtained for S sapro-phyticus KJ00462321 and S carnosus KJ8620021 although

enterotoxin production was detected by the enzyme-linkedimmunosorbent tests (Table 2)

The 04 strains that carry tstH1 in their genome Ssaprophyticus EU4309921 and KJ6991511 and S xylosusCP0087241 and KF980801 were not able to produce mRNAfor TSST-1 in the assay conditions

The production of classical enterotoxins in vitro(immunologic test) matched the results shown by real timeRT-PCR assays for the following strains S saprophyticusJX4901221 S sciuri JX9664361 S saprophyticusEU4309921S saprophyticus HF9372521 S saprophyticus subsp bovisKJ6991511 S hominis JX5199881 S epidermidis KF6005891and S succinus KC3298241

Ten strains namely S saprophyticusAB6977171 S sapro-phyticus EU4309921 S saprophyticus HF9372521 S sapro-phyticus KJ6991511 S saprophyticus KJ9496061 S succinusKC3298241 S hominis JX5199881 S xylosus KF1980801and S xylosus CP0087241 and S epidermidis KF6005891expressed mRNA for multiple se andor sel genes There isa differential transcription among these genes where themost frequent among the classical ones were seb and seeseatranscribed by 04 and 02 strains respectively and the mostfrequent among the newly described genes were sei andsehselnselo transcribed by 03 and 02 strains respectively

No mRNA for the sec gene was detected although SsaprophyticusAB6977171 was able to produce the enterotoxinin vitro as detected by the ELISA assays

34 Enterotoxin Gene Homologies between Salami CNSand CPS Strains The nucleotide sequencing of six entero-toxin genesmdashtwo of them sec and see encoding classi-cal enterotoxinsmdashand four of them encoding the newlydescribed genes seg seh selm and seln fromCNS strains wascompared to the enterotoxin genes from S aureus or S pas-teuri two coagulase-positive staphylococci strains (Figure 3)The homology between the CNS and CPS enterotoxin genesvaried from 65 to 98 The homology of sec seg sehselm and seln between CNS from salami and S aureus was98 60 98 65 and 70 respectively The homologybetween see from CNS found in salami and in S pasteuri was98

Although further studies should be performed it seemsthat the sequences encoding enterotoxins can be conservedamong coagulase-negative and coagulase-positive staphy-lococci as shown in the phylogenetic analysis of the 06enterotoxin genes (Figure 4)

35 Antimicrobial Multiresistance of CNS Strains (MRCNS)Another safety hazard associated with CNS strains besidesthe ability of producing enterotoxins in food matrices is theantimicrobial resistance to antimicrobial agents commonlyused to treat staphylococci infections The antimicrobialresistance carried by CNS strains from food matrices can bespread to the population by the consumption of an apparentlysafe food

Among the 19 CNS strains identified in salami 14 showedmultiresistance to antimicrobial agentsThree strains showedthe highest MAR indices 093 and 080 07 strains presented

10 BioMed Research International

lowast lowastlowastlowastlowastlowast lowast lowast lowastlowast lowast lowast lowastlowast lowastlowastlowastlowast lowastlowastlowast lowast

lowastlowast lowast lowast lowastlowastlowastlowast lowast lowast lowastlowast lowast

lowastlowastlowastlowast lowast lowast lowast

lowastlowast lowast lowast lowastlowast lowastlowast lowast lowastlowast lowastlowastlowastlowastlowast lowastlowast lowast lowastlowast lowastlowast

lowastlowast lowastlowastlowast lowast lowast lowast lowast lowast lowastlowastlowastlowastlowast

lowastlowast lowastlowast lowastlowast lowastlowast lowast lowast lowastlowast lowast lowast lowastlowastlowast lowast lowastlowast lowastlowastlowast

lowast lowast lowast lowast lowast lowast lowastlowast lowast lowastlowastlowast lowast lowastlowastlowast lowast lowast lowastlowastlowast lowast lowastlowastlowast lowast

lowast lowast lowast lowastlowastlowastlowast lowast lowastlowastlowast lowast lowast lowastlowastlowast lowast lowast lowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast lowast lowast lowast lowast lowastlowast lowast lowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

Enterotoxin C (sec) GATAAAAATGAATAAGAGTCGATTTATTTCATGCGTAATTTTGATATTCGCACTTATACT------------------------------------------------------------

AGTTCTTTTTACACCCAACGTATTAGCAGAGAGCCAACCAGACCCTACGCCAGATGAGTT------------------------------------------------------------

GCACAAAGCGAGTAAATTCACTGGTTTGATGGAAAATATGAAAGTTTTATATGATGATCA------------------------------------------------------------

TTATGTATCAGCAACTAAAGTTAAGTCTGTAGATAAATTTTTGGCACATGATTTAATTTA------------------------------------------------------------

TAACATTAGTGATAAAAAACTGAAAAATTATGACAAAGTGAAAACAGAGTTATTAAATGA------------------------------------------------------------

AGGTTTAGCAAAGAAGTACAAAGATGAAGTAGTTGATGTGTATGGATCAAATTACTATGT------------------------------------------------------------

AAACTGCTATTTTTCATCCAAAGATAATGTAGGTAAAGTTACAGGTGGCAAAACTTGTAT------------------------------------------------------------

GTATGGAGGAATAACAAAACATGAAGGAAACCACTTTGATAATGGGAACTTACAAAATGT------------------------------------------------------------

ACTTATAAGAGTTTATGAAAATAAAAGAAACACAATTTCTTTTGAAGTGCAAACTGATAA------------------------------------------------------------

GAAAAGTGTAACAGCTCAAGAACTAGACATAAAAGCTAGGAATTTTTTAATTAATAAAAA------------------------------------------------------------

AAATTTGTATGAGTTTAACAGTTCACCATATGAAACAGGATATATAAAATTTATTGAAAA----TTGTATGAGTTTAACAGTTCACCATATGAAACAGGATATATAAAATTTATTGAAAA

TAACGGCAATACTTTTTGGTATGATATGATGCCTGCACCAGGCGATAAGTTTGACCAATCTAACGGCAATACTTTTTGGTATGATATGATGCCTGCACCAGGCGATAAGTTTGACCAATC

TAAATATTTAATGATGTACAACGACAATAAAACGGTTGATTCTAAAAGTGTGAAGATAGATAAATATTTAATGATGTACAACGACAATAAAACAGTTGATTCTAAAAGTGTGAAGATAGA

AGTCCACCTTACAACAAAGAATGGATAATGTTAATCCGATTTTGATATAAAAAGTGAAAGAGTCCACCTTACAACAAAGAATGGATAATGTAATCCGATTATGGACAAGTGTGCTTTTTT

Enterotoxin C TATTAGATATATTTGAAAGGTAAGTACTTCGGTGCTTGCCTTTTTAGGATGCATATATATEnterotoxin C CNS

Enterotoxin CEnterotoxin C CNS

Enterotoxin CEnterotoxin C CNSEnterotoxin CEnterotoxin C CNSEnterotoxin CEnterotoxin C CNSEnterotoxin CEnterotoxin C CNSEnterotoxin CEnterotoxin C CNSEnterotoxin CEnterotoxin C CNSEnterotoxin CEnterotoxin C CNSEnterotoxin CEnterotoxin C CNS

Enterotoxin CEnterotoxin C CNSEnterotoxin CEnterotoxin C CNS

Enterotoxin CEnterotoxin C CNS

Enterotoxin CEnterotoxin C CNS

Enterotoxin CEnterotoxin C CNS

TTTGTGATCGTCGGCCA-----ACCGATACCGTCGTATTCTTGTACCGCGCAAGGCGAAGlowast lowast lowastlowastlowast lowast lowast lowast lowast lowastlowast lowast lowastlowastlowast lowast lowast lowast lowast

Enterotoxin E (see) Enterotoxin E TGCTTTAGGCGGGGTCTTACAGCATATTATCGTAGAAGATGCACAAGCTGCACGTAAAGCS pasteuri TGCTTTAGGCGCATCGTTACAACATGTCATCGTAGAAGATGAAAAAGATGGTCGTCAGGC

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast lowastlowastlowastlowast lowastlowastlowast lowast lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast lowast lowastlowastlowast lowastlowast lowastlowastlowast lowast lowastlowastlowast

Enterotoxin E ATTAGAATTCTTGAAATCAAAACA-AGCAGGACGTGCTACATTTCTACCATTAAATGTCAS pasteuri

Enterotoxin ES pasteuri

GATACAATT-TTTAAAACAACGTAGTTTAGGACGTGCCACATTCCTACCTTTAAATGTTA lowastlowast lowastlowastlowastlowast lowastlowast lowastlowastlowast lowastlowastlowast lowast lowastlowastlowastlowastlowastlowastlowastlowastlowast lowastlowastlowastlowastlowast lowastlowastlowastlowastlowast lowastlowastlowastlowastlowastlowastlowastlowast lowast

TCAAACCTAGAAATATTTCAACCAAGATATATlowast lowastlowastlowastlowast lowastlowastlowast lowastlowastlowastlowastlowast

Enterotoxin G (seg) Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

---------------------------attaagaaatactt--ccattttaatactcttaGGGGATATTTCCGGGAAACCGGGGCTAATACTGGATAACTTTTCTCTCCGAACGAAGAGA

gaatcaacaactttattatctccg--taaat-----atttaaaaacttatatggaacaaaGATTCAACGATGGTCTTGGTTTTCAATTAAGATGAACCCGCGGCGCTTAGCTGGTGGGAC

aggtactagtt-----------------------------------------cttttttaAGGTCACGGCTCACCCAAGCCCCCATGCGTATACGACCTGGAAGGGTGATCGGCCACATT

ggaaataagtcaaaccaaaaacttgtattgttcttttcagtaaat---------------GGGACTGAGACACAGCACAAACTCCTACGGGAGGCAGCAATAGGGAATCTTCCGCAATGG

------------------------------------------------------------CCGAAAGTCTGACGGAGCAACGCCGCGAGACTGAAGAATGTCTTCGGATCGTAAAACTCT

------------------------tttatatatccagattcaaa-----tgcagaaccatGTTGTTAGGGAAGAACAAGTATCGTTCGAATAGGGCGGTACCTTGACGGTACCTAACCAG

caaactcgtatagctttttttct---------ttagtgagccagtgtcttgctttg----AAAGCTTTGCTAACTACCTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCG

-taatctagttcctgaatagtaaccatattcttatttgtagttattgta--aatccaagtGAATTATTGGGCGTAAAGCGCGCGCATGCGGTCTTTTAAATCTGATGTCAAAGCCCACGG

gattgtct----------attgtcgattgttacctgtacagt------------aattaaGCTCAACCGTGGAGGGTCATTGGAAACTGTAAGACTTGGAGTGCAGAAGAGGATAGTGGA

Figure 3 Continued

BioMed Research International 11

lowast lowast lowast lowastlowast lowastlowast lowastlowastlowast lowast lowastlowast lowast lowast lowast lowast

lowast lowast lowastlowastlowast lowastlowast lowast lowastlowastlowast lowast lowastlowast lowastlowastlowast lowast lowastlowast lowastlowast lowast lowast lowastlowast lowast

lowastlowast lowast lowast lowastlowastlowastlowastlowast lowast lowast lowastlowast lowast lowast lowast lowast lowast lowastlowastlowast

lowastlowast lowast lowastlowast lowastlowastlowast lowastlowast lowastlowast lowast lowast lowast lowast lowastlowast lowastlowast lowast lowast lowast lowastlowast

lowastlowast lowastlowastlowastlowast

lowast lowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowast lowast lowastlowastlowastlowast lowast lowastlowast lowastlowast lowast lowastlowastlowastlowast lowast lowastlowast lowast lowast lowastlowastlowastlowastlowastlowastlowast

lowastlowast lowast lowast lowast lowast lowastlowastlowast lowastlowast lowastlowast lowast lowastlowastlowastlowastlowastlowastlowastlowast lowastlowastlowast

lowast lowast lowast lowast lowast lowast lowastlowast lowastlowast lowast lowast lowastlowastlowast lowast lowast lowastlowastlowast lowast lowast

lowastlowast lowastlowastlowastlowastlowastlowast lowastlowast lowastlowastlowast lowastlowast lowast lowastlowast lowastlowast lowastlowastlowast lowast lowast

lowastlowast lowast lowastlowastlowast lowast lowastlowastlowastlowastlowast lowast lowast lowast lowastlowastlowast lowast lowastlowast lowast

lowastlowast lowast lowast lowast lowast lowastlowast lowast lowastlowast lowast lowastlowast lowast lowastlowast lowastlowastlowastlowast lowast lowast lowastlowast

lowast lowastlowastlowast lowast lowastlowast lowast lowast lowastlowast lowast lowastlowast lowast lowastlowastlowastlowastlowastlowast lowast

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

tttatctctttcattttctgaactattaaa----tgtaagaccacc---atacatacaacATTCCACGTGTAACGGTGAAAATGAGTAGAGATGTGGAGGAACACCAGTGGAGAAACGCG

aacctccaaaattttggtttatatccggttc-----------agatttaggtattatacaACTCTCTGGTCTGTTACTGACGCTGAGGCGCGAAAGCGTGGGAGCAAACAGGATTATATA

tgtataaaaatatggaacgccaaaaatgtctacttttttatctttataattgttagctaaCCCCTGGTAGTCCAAGCCGTAAACGATGAGTGCTAGTGTTACAGGGT------------T

ttctgtattttctaattcagttttaacctcattataactcttatactcaattggaaaaatTCCGCCCTTTACTGCTGCAGCTAACG---CATTAG-GCACTCCGCCTGGGGAGTACAACT

taaatcatgagataaaaactgtctagaattaataactcctcttccttcaacaggtggagaGCAA------GGTTGAAACTCAAAGCA----TTGACCGGGGGCC---CGATAAGCTGAGG

cgtataaagattcattacat----tacccatagttcccttattgtttttataatcacttaTACATGGTGTTTAATTCCAAACCACGCCAAGAACCCTCACGAGGTCTTGACATCCTCTGG

ctttattt----------------------------------------------------ACCTCCCTAGAGAATAAGGGCTTCCCCGTCGGGGATGAAATGAACAGGTGGTGCATGGAT

-------agttcgtc---AGTCTTCAGCTCGTGCAT

Enterotoxin H (seh)

------------------------------------------------------------ATCTATGAATATAAATCTTTTAATATTGGTTTAATATAAATATTCCATTTCTAGAAACCT

------------------------------------------------------------ATATTTTATGTTTGTATAAATTCATAAATTGTTCGAAATTTATATATTAAAAGTTGTTTT

------------------------------------------------------------TGTTGTTTTATAATTAAGTTAATGAAATATATTGAGGAGTTTTAAAATGATTAATAAAAT

------------------------------------------------------------TAAAATATTATTTTCGTTTTTAGCATTATTACTTTCATTCACATCATATGCGAAAGCAGA

------------ -----------TTACGATTTAGCTTTAGCTAATGCATATGGTCAATAAGATTTACACGATAAAAGTGAGTTAACAGATTTAGCTTTAGCTAATGCATATGGTCAATA

TAATCACCCATTCATTAAAGAAAATATTAAGAGTGATGAAATAAGTGGAGAAAAAGATTTTAATCACCCATTCATTAAAGAAAATATTAAGAGTGATGAAATAAGTGGAGAAAAAGATTT

AATATTTAGAAATCAAGGTGATAGTGGCAATGATTTGAGAGTAAAGTTTGCAACTGCTGAAATATTTAGAAATCAAGGTGATAGTGGCAATGATTTGAGAGTAAAGTTTGCAACTGCTGA

TTTAGCTCAGAAGTTTAAAAATAAAAATGTAGATATATATGGGGCATCTTTTTATTATAATTTAGCTCAGAAGTTTAAAAATAAAAATGTAGATATATATGGGGCATCTTTTTATTATAA

GTGTGAAAAAATAAATGAAAATATTTCTGAATGTCTATATGGAGGTACAACACTAAATAAGTGTGAAAAAATAAGTGAAAATATTTCTGAATGTCTATATGGAGGTACAACACTAAATAG

TGAAAAATTGGCACAGGAAAGGGTGATTGGTGCTAATGTTTGGGTAGATGGTATTCAAAATGAAAAATTGGCACAGGAAAGGGTGATTGGTGCTAATGTTTGGGTAGATGGTATTCAAAA

AGAAACAGAATTAATACGAACAAATAAGAAAAATGTGACATTGCAAGAATTAGATATAAAAGAAACAGAATTAATACGAACAAATAAGAAAAATGTGACATTGCAAGAATTAGATATAAA

Enterotoxin H CNS GATCAGAAAAATATTGTCCGATAAATATAAAATTTATTATAAAGACAGCGAAATAAGGG-Enterotoxin H

Enterotoxin H CNSEnterotoxin H

Enterotoxin H CNSEnterotoxin HEnterotoxin H CNSEnterotoxin HEnterotoxin H CNSEnterotoxin HEnterotoxin H CNSEnterotoxin HEnterotoxin H CNSEnterotoxin H

Enterotoxin H CNSEnterotoxin H

Enterotoxin H CNSEnterotoxin H

Enterotoxin H CNSEnterotoxin H

Enterotoxin H CNSEnterotoxin H

Enterotoxin H CNSEnterotoxin H

GATCAGAAAAATATTGTCCGATAAATATAAAATTTATTATAAAGACAGCGAAATAAGTAA

Enterotoxin N (seln) TGATTTAGCTTTAGCTAATGCATATGGTCAATAT-AATCACCCATTCATTAAAGAAAA-----TAGATATTTATGGACTGTATTATGGAAATAAATGTGTAGGCTTAACTGAAGAAAAAA

TATTAAGAGTGATGAAATAAGTGGAGAAAAAGATTTAATATTTAGAAATCAAGGTGATAGCATCATGCTTATACGGAGGAGTTAC------GA--TATATGATGGAAATCAATTAGATGA

TGGCAATGATTTGAGAGTAAAG--------------TTTGCAA-CTGCTGATTTAGCTCAAGAGAGAGTTATAGGCGTTAATGTATTTAAAGATGGTATCCAACAAGAAGGTTTTGTTAT

GAAGTTTAAAAATAAAAATGTAGATATATATGGGGCATCTTTTTATTATAAGTGTGAAAAAAAAACTAAAAAGGCTAAAGTAACAGTACAA------GAATTAGATACTAAAGTTCGATT

AATAAGTGAAAATATTTCTGAATGTCTATATGGAGGTACAACACTAAATAATGAAAAATT------------TAAATTAGAAAATTTATATAAAATATACAATAAAGATACCGGTAACAT

Enterotoxin N CNSEnterotoxin N

Enterotoxin N CNSEnterotoxin N

Enterotoxin N CNSEnterotoxin N

Enterotoxin N CNSEnterotoxin N

Enterotoxin N CNSEnterotoxin N

Figure 3 Continued

12 BioMed Research International

lowastlowastlowast lowastlowast lowastlowast lowastlowast lowast lowastlowast lowastlowastlowast lowastlowast lowast lowastlowastlowastlowast

lowastlowast lowast lowastlowast lowast lowast lowastlowast lowastlowastlowastlowastlowast lowast lowast lowast lowastlowast lowast lowast lowastlowast lowast lowast

lowast lowast

lowast lowastlowast lowast lowastlowast lowast lowast lowast lowast lowast lowast lowast lowastlowast lowast lowastlowastlowastlowast lowast

lowast lowastlowast lowast lowast lowastlowast lowast lowastlowastlowast lowast lowastlowast lowast lowastlowast lowastlowast lowast lowast

lowastlowastlowast lowast lowast lowastlowast lowastlowast lowast lowast lowast

lowast lowast lowast lowastlowastlowast lowastlowastlowast lowastlowast lowast lowastlowastlowastlowastlowast lowastlowastlowast lowast

lowastlowast lowast lowast lowastlowast lowastlowastlowast lowastlowast lowast lowast lowastlowastlowastlowast lowastlowast

lowast lowast lowast lowastlowast lowast lowastlowast lowastlowastlowastlowastlowastlowast lowastlowast lowast lowastlowast lowastlowastlowast

lowast lowast lowast lowast lowastlowast lowastlowastlowastlowast lowast lowast lowastlowastlowast lowast lowast lowast lowast lowast

lowast lowast lowast lowast lowastlowastlowast lowast lowast lowastlowast lowastlowast lowast lowastlowastlowast lowast lowastlowastlowast lowast

lowast lowast lowastlowastlowast lowast lowastlowast lowastlowastlowast lowastlowastlowastlowastlowast lowastlowast lowast lowastlowastlowast lowast lowast lowastlowastlowast

lowastlowastlowast lowast lowastlowast lowastlowast lowastlowast lowast lowast lowast lowast lowastlowast lowastlowast lowastlowast lowast lowast

lowast lowast lowast lowast lowastlowast lowast lowast lowastlowast lowastlowast lowast lowastlowastlowast lowastlowastlowastlowast lowast lowastlowastlowastlowastlowast lowast

lowastlowast lowast lowast lowastlowastlowast lowastlowast lowast lowastlowast lowast lowastlowast lowastlowast lowastlowast lowast lowast lowast

lowastlowastlowastlowastlowast lowastlowast lowastlowast lowast lowastlowast lowast lowast lowast lowast lowastlowast lowastlowast

lowast lowast lowastlowast lowastlowast lowastlowastlowastlowastlowastlowast lowastlowast lowast lowast lowast lowast lowast lowast lowast lowast lowast lowast lowast

lowastlowast lowastlowast lowast lowastlowastlowastlowast lowast lowast lowastlowastlowast lowastlowast lowast lowastlowastlowast lowast

lowastlowastlowastlowast lowastlowast lowast lowast lowast lowast lowastlowast lowast lowast lowast lowastlowastlowastlowastlowast lowastlowast lowastlowastlowast lowast

lowast lowastlowastlowast lowastlowastlowastlowastlowast lowast lowast lowast lowastlowast lowast lowast lowastlowast lowast lowastlowastlowast lowastlowast lowast

lowastlowast lowast lowast lowast lowastlowast lowast lowastlowast lowastlowast lowastlowast lowast lowastlowastlowastlowast

ATTAATACGAACAAATAAGAAAAATGTGACATTGCAAGAATTAGATATAAAGATCAGAAA-TTAT----------TATGATTTATATAAC--------------ATAAAAGGTTCAG---

AATATTGTCCGATAAATATAAAATTTATTATAAAGACAGCGAAATAAGGTAAAAGG------TAGGAGCAGAGTTTTTTCAATTTTATAGTGATAATAGAACAGTTAGCTCATCTAATTA

Enterotoxin N CNS -TAAA---Enterotoxin N

lowast lowastlowastlowastlowastlowast lowastlowast lowast lowast lowast lowastlowast lowast lowastlowastlowastlowastlowastlowast lowastlowast lowastlowast

GGCACAGGAAAGGGTGATTGGTGCTAATGTTTGGGTAGATGGTATTCAAAAAGAAACAGAACAA----AAAGGATGCATTTTCTTT-----CATTCTAATAATCATCAAAATCAATCATT

Enterotoxin N CNSEnterotoxin N

Enterotoxin N CNSEnterotoxin N

Enterotoxin N CNSEnterotoxin N

TCATATCG

Enterotoxin M (selm)

ttgctattcgcaaaatcatatcgcaaccgctgatgtcggagtt--ttgaatcttaggaacEnterotoxin M CNSEnterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

--------------AGTATGTTTAAATCCCCGCTCACAGTTATGTTTAACTCTTCCTTTC

tattatggta-gctatccaattgaagaccaccaaagtattaatcctgaaaataatcatctTTTTCACACCGCCCCGTCGGGTGTTGCGCACGGGGTTGTTTAACCCCCCCGGAAAAAATT

ttcgcatcaattagttttttctatggataattcgacagtaacagctgaatttaagaacgtEnterotoxin M CNS

Enterotoxin M CNS

TTCCCTCCCTCCTCCTTACTC---------------CCT----------------CCCCT

tgatgatgtaaaggaattcaaaaatcatgctgtagatgtatatggtctaagttatagtggEnterotoxin M CNS TTTTCCTACAAATCCTTTCTCCCCTCCCGGAGTAGAAGTAAA------------------

atattgtttgaaaaacaaatatatatacggtggagttacattagcaggtgat-tatttagEnterotoxin M CNS -------------------TAAAACAATACCCCTACCACCTTAACAATTTTTTTATTGAG

agaaatctagacgtattcct-attaatctttgggttaatggagaacatcaaactatatctEnterotoxin M CNS CCCCTCCCCCCCCTTCTCATTTTTTCTCTTTGAC-----------GTTCCCCCCATTTCT

actgacaaagtatcaactaataaaa-agttagtaacagctcaagaaattgatactaaattEnterotoxin M CNS GCCAATGCTTAACCCACCCTTAAAGGCCATCGGCACATATTAGACACTCC-----CCACC

aagaagatatctacaagaagaatataatatttatggctttaatgatac---aaataaaggEnterotoxin M CNS CGCCCCCTCTTTTTATTAAGGACACAACTCTTTTTATTTTCCCCCAAGAACTAATTCTGA

a-agaaattatggtaataagtcaaaatttagttctgga---------tttaatgcaggaaEnterotoxin M CNS AAAAAAACGTTCCGCTTAGTTCAGAATTTCCTTTGAGCCTTTTATCCTTTTGTACGCGAA

aaatattatttca--------tttgaatgatggttcatcattttcttatgacttatttga

Enterotoxin M CNS AAACCATCAGCCATTGAATCAGTTATCCCATTGCCCTAACTAACCCAATATCTAATCTAA

tactggaacaggacaagctgaaagtttctt-aaaaatatataatgacaacaaaactgtcgEnterotoxin M CNS TTTTCGCCTACGAACGAATAATAGCTTGTGGCGAAAAATATCCAGGATTATCTACTGTAG

aaact--gaaaaattccatttagatgtagaaatatcttataaggacgaaagttgaagtatEnterotoxin M CNS CGCCTCGCCAGACGTCCTTTGAATTGA--ATATGACTCCTACTCCCCCCCGCACCGTCAA

ttcaatcataacttagtaaaggaaatgccatgaaaaaatttaaatatagtttt---atatEnterotoxin M CNS TTCAAACA---------------GGTGGAAGTTAATACTGAATAGTAGGTCTTCTTCCCG

tagtttttatattactttttaacattaaagatcttacgtatgct---caaggtgatattgEnterotoxin M CNS TGCTATTCTTAGAACTTTTAAATTATGTAAACCCAATTTTTAGTGCACTCTTTCTCCCTC

gtg-taggtaacttaagaaatttctatacaaaacatgattatatagatttaaaaggcgtcEnterotoxin M CNS GTTACTCTTAGATGGAGAAGCGTTAAGACAGAAGCTTTGTAT-------------TCCCA

acagataaaaa-cctacctattgcaaatcaacttgaattttcaac-----aggt----acEnterotoxin M CNS GCAGAGTGCAACCTTTCACAATGAAGTTTTTATCTAATTTACACGCCTTTAGGCTCCAAA

caatgatttgatctcagaatctaataattgggacgaaataagtaaatttaaaggaaagaaEnterotoxin M CNS TACGTATTAAATCTCCTCCTGT--TTATCTGACCCTAAG-ATAAAACACGAAAAATTATG

actggatatttttggcattgattataatggtccttgtaaatctaaatacatgtatggaggEnterotoxin M CNS CGTGCCCACTCATCCTAT-------TACTTTCATTATATCTATAAAA-------------

Figure 3 Alignment of the enterotoxin gene sequences found in coagulase-negative staphylococci (CNS) and in coagulase-positivestaphylococci (CPS)

BioMed Research International 13

Enterotoxin (seg) S aureus AY1587031

Enterotoxin (sec) CNS

Enterotoxin (see) S pasteuri NC0227371

Enterotoxin (see) CNS

Enterotoxin (seh) CNS

Enterotoxin (selm) S aureus AF2857601

Enterotoxin (seln) CNS

Enterotoxin (seh) S aureus AJ9375481

Enterotoxin (seln) S aureus AY1587031

Enterotoxin (sec) S aureus X516611

Enterotoxin (selm) CNS

Enterotoxin (seg) CNS

150 100

100

100

100

100

99

98

96

97

72

50 0

Figure 4 Phylogenetic tree generated from the multiple alignments of the enterotoxin sequences of CNS and CPS strains using the ClustalX20 software package The phylogenetic tree was constructed using the Mega 60 software and the unweighted pair group method (UPGMA)

MAR indices varying from 066 to 046 and the remaining04 strains showed MAR indices le026 (Table 3)

The 14 MRCNS strains were resistant to 120573-lactams(oxacillin penicillin andor cefoxitin) and to vancomycincorresponding to 73 of the total CNS strains identifiedwhile 09 strains (64) showed resistance to tetracycline andgentamicin 08 strains (57) were resistant to neomycinerythromycin and chloramphenicol 07 strains (50) wereresistant to sulfamethoprim 05 strains (36) were resistantto linezolid 03 strains (21) were resistant to rifampicin 02strains (14) were resistant to ciprofloxacin and cefepimeand 01 strain (7) was resistant to clindamycin (Table 3)

The multiresistance of CNS strains demonstrated hereinis consistent with previous studies on coagulase-negativeand coagulase-positive staphylococci that have found severalresistant and multiresistant Staphylococcus aureus strains inraw milk meat and fermented meat products [43]

Surprisingly the resistance to chloramphenicol is verysimilar to that estimated for MRCNS strains isolated fromhuman clinical samples Indeed there is no direct correla-tion between the researched features and the origin of thestaphylococci strains since different virulence factors arewidespread such as antibiotic resistance [44] reinforcing thefact that the ability of foodmatrices strains to produce SE andSEl and their multiresistance character must be consideredwhen evaluating the safety hazards of food poisoning

S epidermidis KF6005891 and S hominis JX5199881showed the highestMAR indexes (093 and 080 resp)Thesestrains found in artisanal salami are homologous to strainsisolated from human skin microbiota and should be carefullyconsidered among the potential pathogenic staphylococcifound in food from animal origins which can be caused bycontamination by poor hygienic conditions during salamimanufacturing

Depending on the conditions some species of coagulase-negative staphylococci can present health risks since theyhave shown resistance to antibiotics of therapeutic impor-tance such as beta lactams [15] However multiresistantstrains like S carnosusKJ18620021 and S xylosusCP0072081and CP0087241 were intentionally introduced in the foodmatrix since they are part of the culture starter used inItalian-salami manufacturing As previously discussed foodcan be a reservoir of multiresistant microorganisms that canspread by the consumption of an apparently safe food Dueto the intensive and indiscriminate use of antibiotics forhuman and veterinarian therapeutic purposes multiresistantstaphylococci strains are being selected and reproduced infood matrices [45]

The cefoxitin and oxacillin disk diffusion tests are rec-ommended for determining the resistance and susceptibilitybreakpoint for MRSA surveillance cultures [46] In thisstudy there was a good correlation between the disc testzone diameters for oxacillin and cefoxitin (Table 3) TheMICs for ampicillin andmethicillinoxacillincefoxitin for allsusceptible CNS strainswere determined by the diffusion disctest using 003 to 2mgmLminus1 of each antimicrobial agent

S saprophyticus HF9372521 showed an MIC of003mgmLminus1 formethicillin strain S carnosusKJ8620021 Sxylosus strains CP0087241 and CP0072081 S saprophyticusJX4901221 S hominis JX5199881 and S succinusKC3298241 were resistant to 006mgmLminus1 and two strainsS saprophyticus KJ6991511 and S saprophyticus KJ0046231showed an MIC of 05mgmLminus1 (Table 4) Two of themethicillin-resistant strains S saprophyticus KJ0046231 andS xylosus CP0072081 harbor no enterotoxin gene

Five strains S saprophyticus JX4901221 S carnosusKJ8620021 S xylosusCP0087241 S xylosusKF1980801 andS saprophyticus KJ0046231 showed an MIC for ampicillin

14 BioMed Research International

Table 3 Multiple resistance to antimicrobial as found in CNS strains from salami

Salami origin CNS strains Antimicrobial agent resistance Multiple antimicrobialresistance (MAR) indexlowast

Commercial Staphylococcus spp KF1354451 CIP CLO CPM GEN NEO OXA PENSXT TET and VAN 066

Commercial S carnosus KJ8620021 CIP GEN LZD NEO OXA SXT andTET 046

Commercial S saprophyticus AB6977171 CFO CLO ERI PEN OXA TET andVAN 046

Commercial S saprophyticus EU4309921 CFO CLO ERI GEN LZD NEO OXAPEN SXT TET and VAN 080

Commercial S saprophyticus JX4901221 CFO CLO ERI GEN NEO PEN OXASXT TET and VAN 066

Commercial S saprophyticus KJ0046231 CFO OXA and PEN 020

Commercial S sciuri JX9664361 CFO CLO GEN NEO OXA PEN andTET 046

Commercial S xylosus CP0072081 OXA LZD PEN and VAN 026

Commercial S xylosus CP0087241 CFO CLO ERI GEN NEO OXA PENTET and VAN 060

Artisanal S epidermidis KF6005891 CFO CLI CLO CPM ERI GEN LZDNEO OXA PEN RIF SXT and TET 093

Artisanal S hominis JX5199881 CIP CPM CPO ERI GEN LZD NEOOXA PEN RIF SXT and VAN 080

Artisanal S saprophyticusHF9372521 CFO OXA PEN and SXT 026Artisanal S saprophyticus KJ6991511 OXA PEN and VAN 020

Artisanal S succinus KC3298241 CFO CLO ERI GEN OXA PEN RIFSXT TET and VAN 066

Artisanal S xylosus KF1980801 CFO CLO ERI GEN NEO OXA PENTET and VAN 060

lowastTheMAR index of an isolate is defined as 119886119887 where 119886 represents the number of antimicrobials to which the isolate was resistant and 119887 represents the numberof antimicrobials to which the isolate was subjectedS aureus strains ATCC WB81 (sea) ATCC 13563 (sed) and ATCC 27664 (see) showing a MAR index of 05 and S aureus strains ATCC14458 (seb) andATCCWB72 (sec) and S xylosus ATCC 29971 showing a MAR index of 03 were used as reference strainsCPM cefepime CFO cefoxitin CLO chloramphenicol CIP ciprofloxacin CLI clindamycin ERI erythromycin GEN gentamycin NEO neomycin LZDlinezolid RIF rifampicin TET tetracycline OXA oxacillin PEN penicillin SXT sulfamethoprim and VAN vancomycin

Table 4 Minimal inhibitory concentration (MIC) of compounds used in antimicrobial therapy against staphylococci infections

Salami origin GenBank accession number and similarity () MIC mgmLminus1

Methicillin Ampicillin Vancomycin Linezolid

Commercial

S carnosus KJ8620021 006 003 mdash mdashS xylosus CP0087241 006 003 006 mdash

S saprophyticus JX4901221 006 003 05 mdashS saprophyticus AB6977171 mdash mdash 05 mdashS saprophyticus EU4309921 mdash mdash 025 0125

S succinus KC3298241 006 025 003 mdash

Artisanal

S hominis JX5199881 006 05 mdash mdashS saprophyticus HF9372521 003 mdash mdash mdash

S xylosus KF1980801 003 05 mdashS saprophyticus KJ6991511 05 025 003 mdashS saprophyticus KJ0046231 05 003 003 mdash

S xylosus CP0072081 006 mdash mdash 025Strains S epidermidis KF6005891 and Staphylococcus spp KF1354451 were not susceptible to the antimicrobial concentrations tested in the present study

BioMed Research International 15

of 003mgmLminus1 two strains S succinus KC3298241 and Ssaprophyticus KJ6991511 showed an MIC of 025mgmLminus1and S hominis JX5199881 showed an MIC of 05mgmLminus1(Table 4) S saprophyticus KJ0046231 also showed an MICof 003mgmLminus1 but does not harbor enterotoxin genes It isimportant to note that most of the CNS strains that showedresistance to ampicillin were also resistant to methicillin ashighlighted by the MIC tests

Five strains S saprophyticus HF9372521 S saprophyti-cus KJ6991511 S saprophyticus JX4901221 S saprophyticusKJ0046231 and S succinus KC3298241 showed an MICfor vancomycin of 003mgmLminus1 one strain S xylosusCP0087241 showed an MIC of 006mgmLminus1 one strain Ssaprophyticus EU4309921 showed an MIC of 025mgmLminus1and two strains S xylosus KF1980801 and S saprophyticusAB6977171 showed an MIC of 05mgmLminus1 (Table 4)

Additionally five CNS strains S xylosus CP0087241S saprophyticus JX 4901221 S succinus KC3298241 Ssaprophyticus KJ6991511 and S saprophyticus KJ0046231showed resistance tomethicillin as demonstrated by theMICdeterminations (Table 4)

Previous studies have demonstrated that linezolid isactive against Gram-positive bacteria including methicillin-resistant staphylococci [47] In the present study 05 strainsshowed linezolid resistance in the disc diffusion test but itwas impossible to establish MIC values for S epidermidisKF6005891 Staphylococcus hominis JX5199881 and Staphy-lococcus carnosus KJ8620021The remaining strains S sapro-phyticus EU4309921 and S xylosus CP0072081 presentedan MIC for linezolid of 0125mgmLminus1 and 025mgmLminus1respectively

The MIC of 003mgmLminus1 for penicillin is in accordancewith the previous values estimated for resistant S aureusstrains found in several food matrices such as meat dairyproducts and ready-to-eat food [48]

There is still a lack of information on the antimicro-bial resistance of staphylococci strains from food matricesalthough they are the most worrisome vehicles of dissemi-nation of antibiotic-resistant pathogens [49]

The high resistance found for salami staphylococci strainscan be ascribed to their inappropriate use as growth promot-ers of antimicrobial agents like oxacillin vancomycin chlo-ramphenicol neomycin and erythromycin which are com-monly used in veterinary medicine to treat infections [50]Antimicrobial therapy of infections staphylococci is basedon results of susceptibility tests in vitro [51] Methicillin-resistant coagulase-negative Staphylococcus spp found inready-to-eat products such as meats fish and dairy productsalso offer risks and although they are not classical foodpoisoning bacteria their presence in food offers significantrisks to public health due to the possible spread of antibioticresistance [52]

The standardization of salami quality should includediagnostic methods to screen and quantify the presence ofclassical and newly described enterotoxins directly in thefood matrices as a routine procedure to be conducted by themeat product industry in Brazil

The safety of salami consumption could also be enhancedby the inclusion of amicrobial barrier such as the inclusion ofprobiotic strains producing natural antibiotics or competitiveflora or even the addition of natural bioagents against spoilageor pathogenic microorganisms

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors acknowledge the financial support fromFundacao Carlos Chagas Filho de Amparo a Pesquisa doEstado do Rio de Janeiro (FAPERJ Rio de Janeiro Brazil)Conselho Nacional de Desenvolvimento Cientıfico eTecnologico (CNPq Brasılia Brazil) and Coordenacao deAperfeicoamento de Pessoal de Nıvel Superior (CAPESBrasılia Brazil)

References

[1] ICMSF International Comission on Microbiological Specifica-tions for FoodsMicroorganisms in FoodsTheir Significance andMethods of Enumerations University of Toronto Press TorontoCanada 3rd edition 1983

[2] J F Veras L S Carmo L C Tong et al ldquoA study of theenterotoxigenicity of coagulase-negative and coagulase-positivestaphylococcal isolates from food poisoning outbreaks inMinasGerais Brazilrdquo International Journal of Infectious Diseases vol12 no 4 pp 410ndash415 2008

[3] K Omoe D-L Hu H Takahashi-Omoe A Nakane and KShinagawa ldquoComprehensive analysis of classical and newlydescribed staphylococcal superantigenic toxin genes in Staphy-lococcus aureus isolatesrdquo FEMS Microbiology Letters vol 246no 2 pp 191ndash198 2005

[4] J Kadariya T C Smith and D Thapaliya ldquoStaphylococcusaureus and staphylococcal food-borne disease an ongoing chal-lenge in public healthrdquoBioMedResearch International vol 2014Article ID 827965 9 pages 2014

[5] KOmoeD-LHuH KOno et al ldquoEmetic potentials of newlyidentified staphylococcal enterotoxin-like toxinsrdquo Infection andImmunity vol 81 no 10 pp 3627ndash3631 2013

[6] J K McCormick J M Yarwood and P M SchlievertldquoToxic shock syndrome and bacterial superantigens an updaterdquoAnnual Review of Microbiology vol 55 pp 77ndash104 2001

[7] T Baba F Takeuchi M Kuroda et al ldquoGenome and virulencedeterminants of high virulence community-acquired MRSArdquoThe Lancet vol 359 no 9320 pp 1819ndash1827 2002

[8] Associacao Brasileira de Industria Produtora e Exportadorada Carne Suınas (ABIPECS) ldquoStatistics slaughter ranking2000ndash2014rdquo 2014 httpabpa-brcombrsetoressuinoculturapublicacoesrelatorios-anuais

[9] N Zdolec M Hadziosmanovic L Kozacinski et al ldquoMicrobialand physicochemical succession in fermented sausages pro-duced with bacteriocinogenic culture of Lactobacillus sakei andsemi-purified bacteriocin mesenterocin Yrdquo Meat Science vol80 no 2 pp 480ndash487 2008

16 BioMed Research International

[10] F Ravyts L D Vuyst and F Leroy ldquoBacterial diversity andfunctionalities in food fermentationsrdquo Engineering in Life Sci-ences vol 12 no 4 pp 356ndash367 2012

[11] TOhlsson andN BengtssonTheHurdle ConceptMinimal Pro-cessing Technologies in the Food IndustryWoodhead Publishing2002

[12] S Bernardi B B Golineli and C J Contreras-CastilloldquoRevisao aspectos da aplicacao de culturas starter na producaode embutidos carneos fermentadosrdquo Brazilian Journal of FoodTechnology vol 13 no 2 pp 133ndash140 2010

[13] F Irlinger ldquoSafety assessment of dairy microorganismscoagulase-negative staphylococcirdquo International Journal of FoodMicrobiology vol 126 no 3 pp 302ndash310 2008

[14] B Ghebremedhin F Layer W Konig and B Konig ldquoGeneticclassification and distinguishing of Staphylococcus species basedon different partial gap 16S rRNA hsp60 rpoB sodA and tufgene sequencesrdquo Journal of Clinical Microbiology vol 46 no 3pp 1019ndash1025 2008

[15] I Babic K Markov D Kovacevic et al ldquoIdentification andcharacterization of potential autochthonous starter culturesfrom aCroatian lsquobrandrsquo product lsquoslavonski kulenrsquordquoMeat Sciencevol 88 no 3 pp 517ndash524 2011

[16] MMehrotra GWang andWM Johnson ldquoMultiplex PCR forthe detection of genes for Staphylococcus aureus enterotoxinsexfoliative toxins toxic shock syndrome toxin and methicillinresistancerdquo Journal of Clinical Microbiology vol 38 no 3 pp1032ndash1041 2000

[17] WM Johnson SD Tyler E P Ewan F EAshtonDR Pollardand K R Rozee ldquoDetection of genes for enterotoxins exfolia-tive toxins and toxic shock syndrome toxin 1 in Staphylococcusaureus by the polymerase chain reactionrdquo Journal of ClinicalMicrobiology vol 29 no 3 pp 426ndash430 1991

[18] J Mclauchlin G L Narayanan VMithani andG OrsquoNeill ldquoThedetection of enterotoxins and toxic shock syndrome toxin genesin Staphylococcus aureus by polymerase chain reactionrdquo Journalof Food Protection vol 63 no 4 pp 479ndash488 2000

[19] J P Rosec and O Gigaud ldquoStaphylococcal enterotoxin genes ofclassical and new types detected by PCR in Francerdquo Interna-tional Journal of Food Microbiology vol 77 no 1-2 pp 61ndash702002

[20] K Omoe D-L Hu H Takahashi-Omoe A Nakane andK Shinagawa ldquoIdentification and characterization of a newstaphylococcal enterotoxin-related putative toxin encoded bytwo kinds of plasmidsrdquo Infection and Immunity vol 71 no 10pp 6088ndash6094 2003

[21] N Sergeev D Volokhov V Chizhikov and A Rasooly ldquoSimul-taneous analysis of multiple staphylococcal enterotoxin genesby an oligonucleotide microarray assayrdquo Journal of ClinicalMicrobiology vol 42 no 5 pp 2134ndash2143 2004

[22] S Holtfreter D Grumann M Schmudde et al ldquoClonal distri-bution of superantigen genes in clinical Staphylococcus aureusisolatesrdquo Journal of Clinical Microbiology vol 45 no 8 pp2669ndash2680 2007

[23] W J Mason J S Blevins K Beenken NWibowo N Ojha andM S Smeltzer ldquoMultiplex PCR protocol for the diagnosis ofstaphylococcal infectionrdquo Journal of Clinical Microbiology vol39 no 9 pp 3332ndash3338 2001

[24] E Rahimi and H G Safai ldquoDetection of classical enterotoxinsof Staphylococcus aureus strains isolated frombovine subclinicalmastitis in Isfahan Iranrdquo Veterinary Microbiology vol 141 no3-4 pp 393ndash394 2010

[25] M A Larkin G Blackshields N P Brown et al ldquoClustalW andClustal X version 20rdquo Bioinformatics vol 23 no 21 pp 2947ndash2948 2007

[26] P H A Sneath and R R Sokal Unweighted Pair Group Methodwith Arithmetic Mean Numerical Taxonomy W H FreemanSan Francisco Calif USA 1973

[27] NCCLS ldquoPerformance standards for antimicrobial susceptibil-ity testing 14th informational supplementrdquo Tech Rep M 100-S14 NCCLS 2004

[28] S Corbiere Morot-Bizot S Leroy and R Talon ldquoMonitoringof staphylococcal starters in two French processing plantsmanufacturing dry fermented sausagesrdquo Journal of AppliedMicrobiology vol 102 no 1 pp 238ndash244 2007

[29] R Talon S Leroy I Lebert et al ldquoSafety improvement andpreservation of typical sensory qualities of traditional dryfermented sausages using autochthonous starter culturesrdquo Inter-national Journal of Food Microbiology vol 126 no 1-2 pp 227ndash234 2008

[30] G Mauriello A Casaburi G Blaiotta and F Villani ldquoIsolationand technological properties of coagulase negative staphylo-cocci from fermented sausages of Southern ItalyrdquoMeat Sciencevol 67 no 1 pp 149ndash158 2004

[31] M Janssens N Myter L De Vuyst and F Leroy ldquoSpeciesdiversity and metabolic impact of the microbiota are low inspontaneously acidified Belgian sausages with an added starterculture of Staphylococcus carnosusrdquo Food Microbiology vol 29no 2 pp 167ndash177 2012

[32] G Blaiotta D Ercolini G Mauriello G Salzano and F VillanildquoRapid and reliable identification of Staphylococcus equorum bya species-specific PCR assay targeting the sodA generdquo Systematicand Applied Microbiology vol 27 no 6 pp 696ndash702 2004

[33] L Kozacinski E Drosinos F Caklovica L Cocolin J Gasparik-Reichardt and S Veskovic ldquoInvestigation of microbial associa-tion of traditionally fermented sausagesrdquo Food Technology andBiotechnology vol 46 no 1 pp 93ndash106 2008

[34] S Leroy P Giammarinaro J-P Chacornac I Lebert and RTalon ldquoBiodiversity of indigenous staphylococci of naturallyfermented dry sausages and manufacturing environments ofsmall-scale processing unitsrdquo Food Microbiology vol 27 no 2pp 294ndash301 2010

[35] E Coton M-H Desmonts S Leroy et al ldquoBiodiversity ofCoagulase-Negative Staphylococci in French cheeses dry fer-mented sausages processing environments and clinical sam-plesrdquo International Journal of Food Microbiology vol 137 no 2-3 pp 221ndash229 2010

[36] S Hosseinzadeh and H D Saei ldquoStaphylococcal species associ-ated with bovinemastitis in the NorthWest of Iran emerging ofcoagulase-negative staphylococcirdquo International Journal of Vet-erinary Science and Medicine vol 2 no 1 pp 27ndash34 2014

[37] A Piette and G Verschraegen ldquoRole of coagulase-negativestaphylococci in human diseaserdquo Veterinary Microbiology vol134 no 1-2 pp 45ndash54 2009

[38] T M Moura F S Campos P A DrsquoAzevedo et al ldquoPrevalenceof enterotoxin-encoding genes and antimicrobial resistancein coagulase-negative and coagulase-positive Staphylococcusisolates from black pudding in Southern Brazilrdquo Revista daSociedade Brasileira deMedicina Tropical vol 45 no 5 pp 579ndash585 2012

[39] H I Suzuki T Lefebure P P Bitar and M J StanhopeldquoComparative genomic analysis of the genus Staphylococcusincluding Staphylococcus aureus and its newly described sister

BioMed Research International 17

species Staphylococcus simiaerdquo BMC Genomics vol 13 article38 2012

[40] R P Lamers G Muthukrishnan T A Castoe S Tafur A MCole and C L Parkinson ldquoPhylogenetic relationships amongStaphylococcus species and refinement of cluster groups basedon multilocus datardquo BMC Evolutionary Biology vol 12 article171 2012

[41] I V Pinchuk E J Beswick and V E Reyes ldquoStaphylococcalenterotoxinsrdquo Toxins vol 2 no 8 pp 2177ndash2197 2010

[42] S R Monday and G A Bohach ldquoGenes encoding staphylo-coccal enterotoxins G and I are linked and separated by DNArelated to other staphylococcal enterotoxinsrdquo Journal of NaturalToxins vol 10 no 1 pp 1ndash8 2001

[43] D Y Thomas S Jarraud B Lemercier et al ldquoStaphylococcalenterotoxin-like toxins U2 and V two new staphylococcalsuperantigens arising from recombination within the entero-toxin gene clusterrdquo Infection and Immunity vol 74 pp 4724ndash4734 2006

[44] N Indrawattana O Sungkhachat N Sookrung et al ldquoStaphylo-coccus aureus clinical isolates antibiotic susceptibility molecu-lar characteristics and ability to form biofilmrdquo BioMed ResearchInternational vol 2013 Article ID 314654 11 pages 2013

[45] V Pereira C Lopes A Castro J Silva P Gibbs and P TeixeiraldquoCharacterization for enterotoxin production virulence factorsand antibiotic susceptibility of Staphylococcus aureus isolatesfrom various foods in Portugalrdquo Food Microbiology vol 26 no3 pp 278ndash282 2009

[46] I Anacarso C Condo C Sabia and et al ldquoAntimicrobialresistance and other related virulence factors in staphylococcusspp isolated from food environmental and humans in ItalyrdquoUniversal Journal Microbiology Research vol 1 no 1 pp 1ndash92013

[47] B Martın M Garriga M Hugas S Bover-Cid M T Veciana-Nogues and T Aymerich ldquoMolecular technological and safetycharacterization of Gram-positive catalase-positive cocci fromslightly fermented sausagesrdquo International Journal of FoodMicrobiology vol 107 no 2 pp 148ndash158 2006

[48] NM Broekema T Van Tam T AMonson S AMarshall andD M Warshauer ldquoComparison of cefoxitin and oxacillin diskdiffusionmethods for detection ofmecA-mediated resistance inStaphylococcus aureus in a large-scale studyrdquo Journal of ClinicalMicrobiology vol 47 no 1 pp 217ndash219 2009

[49] E Cercenado F Garcıa-Garrote and E Bouza ldquoIn vitro activityof linezolid against multiply resistant Gram-positive clinicalisolatesrdquo Journal of Antimicrobial Chemotherapy vol 47 no 1pp 77ndash81 2001

[50] A Aydin K Muratoglu M Sudagidan K Bostan B Okukluand SHarsa ldquoPrevalence and antibiotic resistance of foodborneStaphylococcus aureus isolates in Turkeyrdquo Foodborne Pathogensand Disease vol 8 no 1 pp 63ndash69 2011

[51] D Sergelidis A Abrahim T Papadopoulos et al ldquoIsolation ofmethicillin-resistant Staphylococcus spp from ready-to-eat fishproductsrdquo Letters in Applied Microbiology vol 59 no 5 pp500ndash506 2014

[52] I PhillipsM Casewell T Cox et al ldquoDoes the use of antibioticsin food animals pose a risk to human health A critical reviewof published datardquo Journal of Antimicrobial Chemotherapy vol53 no 1 pp 28ndash52 2004

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Page 8: Research Article Safety Evaluation of the Coagulase ...downloads.hindawi.com/journals/bmri/2015/483548.pdf · Research Article Safety Evaluation of the Coagulase-Negative Staphylococci

8 BioMed Research International

sed

seb

sec

see

sea

M 2 3 4 51

500bp

(a)

sea

sebsebseb

M 1 2 3 4 5

(b)

sei

sehseg

1M

(c)

seh sehseh

M 1 2 3

(d)

1 2 4M 3 5

(e)

selm

seljseln

selo

21M

(f)

seln

selm

M 1 2

(g)

Figure 2 Uniplex duplex and multiplex PCR screening for the detection of enterotoxin genes in CNS strains from salami (a) Lane M100 bp DNA ladder plus (Fermentas Foster City CA USA) lane 1 S aureus ATCC 29231 harboring sea gene lane 2 S aureus NCTC 10654harboring seb gene lane 3 S aureus ATCC19095 harboring the sec gene lane 4 S aureus ATCC 13563 harboring the sed gene and lane 5 Saureus ATCC 27664 harboring the see gene (b) Lane M 100 bp DNA ladder plus lane 1 Staphylococcus spp lane 2 S carnosus NR116434lane 3 S carnosus KJ862002 lane 4 S carnosus KJ862002 and lane 5 S carnosus KJ8620021 (c) Lane M 100 bp DNA ladder plus lane1 S aureus ATCC 19095 harboring seg seh and sei genes (d) Lane M 100 bp DNA ladder plus lane 1 S saprophyticus AB6977171 lane 2S epidermidis KF 6005891 and lane 3 S sciuri JX9664361 (e) Lane M 100 bp DNA ladder plus lane 1 S xylosus KF1980801 lane 2 SsaprophyticusKJ6991511 (f) LaneM 100 bp DNA ladder plus lane 1 S aureusATCC 27154 harboring selj slem seln and selo genes (g) LaneM 100 bp DNA ladder plus lane 1 S xylosus KF1980801 and lane 2 S saprophyticus subsp bovis KJ6991511

BioMed Research International 9

shown to carry the selm seln and selo genes and the selmand seln sequences were detected in the genome of a singlestrain S saprophyticus subsp bovisKJ6991511 S epidermidisKF6005891 showed a combination of seg and sei genesand 03 strains harbored a single gene sei or selm or selnperhaps due to the high degree of genetic polymorphism inthe chromosomal assembly [41] Indrawattana et al 2013detected the seg-sei-selm-seln-selo of the highly prevalent egclocus was 261 in contrast to this study where 13 CNSpresents the combination of selm seln and selo genes [42]

The CNS strains clustered into groups near the bottom ofthe phylogenetic tree carried the classical enterotoxin geneswhile the species at the top of the tree showed high diversityamong the enterotoxin genes combining the classical and thenewly described genes in their genomes (Figure 1)

To assess the risk of staphylococcal food poisoning theability of the identified strains in harboring the se andsel genes and in expressing and producing enterotoxins invitro was evaluated The mRNA for each enterotoxin genewas evaluated by real time RT-PCR assays and enterotoxincontent was estimated by a sandwich enzyme immunoassayfor the combined detection of Staphylococcus enterotoxins(SET) A B C D and E

The fifteen enterotoxigenic CNS strains were able toexpress the classical enterotoxins SEA seb sec sed and seein concentrations ranging from 03 ngmLminus1 to 14 ngmLminus1as assessed by the in vitro assays (Table 2) The S sapro-phyticus AB6977171 KJ6991511 JX4901221 KJ0046231 andHF9372521 strains S xylosus KF1980801 and S carnosusNR1164341 produced low amount of enterotoxins lower than05 ngmLminus1 S epidermidisKF6005891 S saprophyticus bovisKJ6991511 and S hominis JX5199881 produced intermediateamount of enterotoxins ranging from 07 to 090 ngmLminus1while S sciuri JX9664361 S saprophyticus EU4309921 Scarnosus KJ8620021 S xylosus CP0087241 and S succi-nus KC3298241 produced enterotoxins in concentrationsge10 ngmLminus1

Although the sandwich enzyme immunoassay is consid-ered themost sensitivemethod to detect seandashsee enterotoxinsable to detect 0125 ngmLminus1 differences in the specificity andsensitivity of the assays for the detection of staphylococcalenterotoxins from foods are expected [43] A single strainStaphylococcus spp KF1354451 which harbors both the seaand seb genes was unable to produce seandashsee enterotoxins

The mRNA levels evaluated by the real time RT-PCRassays for enterotoxins were detected in 12 of 15 strains(86) (Table 2) Transcripts for the classical enterotoxinsgenes were detected when S saprophyticus HF9372521 Scarnosus NR1164341 S xylosus CP0087241 and S succinusKC3298241 were assayed Transcripts for newly describedenterotoxins were observed in S saprophyticus JX4901221and AB6977171 S sciuri JX9664361 S saprophyticus subspbovis KJ6991511 and S hominis JX5199881 Transcripts fromboth the classical and newly described enterotoxins weredetected in S saprophyticus EU4309921 and KJ9496061 andS epidermidis KF6005891

No mRNA transcripts were obtained for S sapro-phyticus KJ00462321 and S carnosus KJ8620021 although

enterotoxin production was detected by the enzyme-linkedimmunosorbent tests (Table 2)

The 04 strains that carry tstH1 in their genome Ssaprophyticus EU4309921 and KJ6991511 and S xylosusCP0087241 and KF980801 were not able to produce mRNAfor TSST-1 in the assay conditions

The production of classical enterotoxins in vitro(immunologic test) matched the results shown by real timeRT-PCR assays for the following strains S saprophyticusJX4901221 S sciuri JX9664361 S saprophyticusEU4309921S saprophyticus HF9372521 S saprophyticus subsp bovisKJ6991511 S hominis JX5199881 S epidermidis KF6005891and S succinus KC3298241

Ten strains namely S saprophyticusAB6977171 S sapro-phyticus EU4309921 S saprophyticus HF9372521 S sapro-phyticus KJ6991511 S saprophyticus KJ9496061 S succinusKC3298241 S hominis JX5199881 S xylosus KF1980801and S xylosus CP0087241 and S epidermidis KF6005891expressed mRNA for multiple se andor sel genes There isa differential transcription among these genes where themost frequent among the classical ones were seb and seeseatranscribed by 04 and 02 strains respectively and the mostfrequent among the newly described genes were sei andsehselnselo transcribed by 03 and 02 strains respectively

No mRNA for the sec gene was detected although SsaprophyticusAB6977171 was able to produce the enterotoxinin vitro as detected by the ELISA assays

34 Enterotoxin Gene Homologies between Salami CNSand CPS Strains The nucleotide sequencing of six entero-toxin genesmdashtwo of them sec and see encoding classi-cal enterotoxinsmdashand four of them encoding the newlydescribed genes seg seh selm and seln fromCNS strains wascompared to the enterotoxin genes from S aureus or S pas-teuri two coagulase-positive staphylococci strains (Figure 3)The homology between the CNS and CPS enterotoxin genesvaried from 65 to 98 The homology of sec seg sehselm and seln between CNS from salami and S aureus was98 60 98 65 and 70 respectively The homologybetween see from CNS found in salami and in S pasteuri was98

Although further studies should be performed it seemsthat the sequences encoding enterotoxins can be conservedamong coagulase-negative and coagulase-positive staphy-lococci as shown in the phylogenetic analysis of the 06enterotoxin genes (Figure 4)

35 Antimicrobial Multiresistance of CNS Strains (MRCNS)Another safety hazard associated with CNS strains besidesthe ability of producing enterotoxins in food matrices is theantimicrobial resistance to antimicrobial agents commonlyused to treat staphylococci infections The antimicrobialresistance carried by CNS strains from food matrices can bespread to the population by the consumption of an apparentlysafe food

Among the 19 CNS strains identified in salami 14 showedmultiresistance to antimicrobial agentsThree strains showedthe highest MAR indices 093 and 080 07 strains presented

10 BioMed Research International

lowast lowastlowastlowastlowastlowast lowast lowast lowastlowast lowast lowast lowastlowast lowastlowastlowastlowast lowastlowastlowast lowast

lowastlowast lowast lowast lowastlowastlowastlowast lowast lowast lowastlowast lowast

lowastlowastlowastlowast lowast lowast lowast

lowastlowast lowast lowast lowastlowast lowastlowast lowast lowastlowast lowastlowastlowastlowastlowast lowastlowast lowast lowastlowast lowastlowast

lowastlowast lowastlowastlowast lowast lowast lowast lowast lowast lowastlowastlowastlowastlowast

lowastlowast lowastlowast lowastlowast lowastlowast lowast lowast lowastlowast lowast lowast lowastlowastlowast lowast lowastlowast lowastlowastlowast

lowast lowast lowast lowast lowast lowast lowastlowast lowast lowastlowastlowast lowast lowastlowastlowast lowast lowast lowastlowastlowast lowast lowastlowastlowast lowast

lowast lowast lowast lowastlowastlowastlowast lowast lowastlowastlowast lowast lowast lowastlowastlowast lowast lowast lowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast lowast lowast lowast lowast lowastlowast lowast lowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

Enterotoxin C (sec) GATAAAAATGAATAAGAGTCGATTTATTTCATGCGTAATTTTGATATTCGCACTTATACT------------------------------------------------------------

AGTTCTTTTTACACCCAACGTATTAGCAGAGAGCCAACCAGACCCTACGCCAGATGAGTT------------------------------------------------------------

GCACAAAGCGAGTAAATTCACTGGTTTGATGGAAAATATGAAAGTTTTATATGATGATCA------------------------------------------------------------

TTATGTATCAGCAACTAAAGTTAAGTCTGTAGATAAATTTTTGGCACATGATTTAATTTA------------------------------------------------------------

TAACATTAGTGATAAAAAACTGAAAAATTATGACAAAGTGAAAACAGAGTTATTAAATGA------------------------------------------------------------

AGGTTTAGCAAAGAAGTACAAAGATGAAGTAGTTGATGTGTATGGATCAAATTACTATGT------------------------------------------------------------

AAACTGCTATTTTTCATCCAAAGATAATGTAGGTAAAGTTACAGGTGGCAAAACTTGTAT------------------------------------------------------------

GTATGGAGGAATAACAAAACATGAAGGAAACCACTTTGATAATGGGAACTTACAAAATGT------------------------------------------------------------

ACTTATAAGAGTTTATGAAAATAAAAGAAACACAATTTCTTTTGAAGTGCAAACTGATAA------------------------------------------------------------

GAAAAGTGTAACAGCTCAAGAACTAGACATAAAAGCTAGGAATTTTTTAATTAATAAAAA------------------------------------------------------------

AAATTTGTATGAGTTTAACAGTTCACCATATGAAACAGGATATATAAAATTTATTGAAAA----TTGTATGAGTTTAACAGTTCACCATATGAAACAGGATATATAAAATTTATTGAAAA

TAACGGCAATACTTTTTGGTATGATATGATGCCTGCACCAGGCGATAAGTTTGACCAATCTAACGGCAATACTTTTTGGTATGATATGATGCCTGCACCAGGCGATAAGTTTGACCAATC

TAAATATTTAATGATGTACAACGACAATAAAACGGTTGATTCTAAAAGTGTGAAGATAGATAAATATTTAATGATGTACAACGACAATAAAACAGTTGATTCTAAAAGTGTGAAGATAGA

AGTCCACCTTACAACAAAGAATGGATAATGTTAATCCGATTTTGATATAAAAAGTGAAAGAGTCCACCTTACAACAAAGAATGGATAATGTAATCCGATTATGGACAAGTGTGCTTTTTT

Enterotoxin C TATTAGATATATTTGAAAGGTAAGTACTTCGGTGCTTGCCTTTTTAGGATGCATATATATEnterotoxin C CNS

Enterotoxin CEnterotoxin C CNS

Enterotoxin CEnterotoxin C CNSEnterotoxin CEnterotoxin C CNSEnterotoxin CEnterotoxin C CNSEnterotoxin CEnterotoxin C CNSEnterotoxin CEnterotoxin C CNSEnterotoxin CEnterotoxin C CNSEnterotoxin CEnterotoxin C CNSEnterotoxin CEnterotoxin C CNS

Enterotoxin CEnterotoxin C CNSEnterotoxin CEnterotoxin C CNS

Enterotoxin CEnterotoxin C CNS

Enterotoxin CEnterotoxin C CNS

Enterotoxin CEnterotoxin C CNS

TTTGTGATCGTCGGCCA-----ACCGATACCGTCGTATTCTTGTACCGCGCAAGGCGAAGlowast lowast lowastlowastlowast lowast lowast lowast lowast lowastlowast lowast lowastlowastlowast lowast lowast lowast lowast

Enterotoxin E (see) Enterotoxin E TGCTTTAGGCGGGGTCTTACAGCATATTATCGTAGAAGATGCACAAGCTGCACGTAAAGCS pasteuri TGCTTTAGGCGCATCGTTACAACATGTCATCGTAGAAGATGAAAAAGATGGTCGTCAGGC

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast lowastlowastlowastlowast lowastlowastlowast lowast lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast lowast lowastlowastlowast lowastlowast lowastlowastlowast lowast lowastlowastlowast

Enterotoxin E ATTAGAATTCTTGAAATCAAAACA-AGCAGGACGTGCTACATTTCTACCATTAAATGTCAS pasteuri

Enterotoxin ES pasteuri

GATACAATT-TTTAAAACAACGTAGTTTAGGACGTGCCACATTCCTACCTTTAAATGTTA lowastlowast lowastlowastlowastlowast lowastlowast lowastlowastlowast lowastlowastlowast lowast lowastlowastlowastlowastlowastlowastlowastlowastlowast lowastlowastlowastlowastlowast lowastlowastlowastlowastlowast lowastlowastlowastlowastlowastlowastlowastlowast lowast

TCAAACCTAGAAATATTTCAACCAAGATATATlowast lowastlowastlowastlowast lowastlowastlowast lowastlowastlowastlowastlowast

Enterotoxin G (seg) Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

---------------------------attaagaaatactt--ccattttaatactcttaGGGGATATTTCCGGGAAACCGGGGCTAATACTGGATAACTTTTCTCTCCGAACGAAGAGA

gaatcaacaactttattatctccg--taaat-----atttaaaaacttatatggaacaaaGATTCAACGATGGTCTTGGTTTTCAATTAAGATGAACCCGCGGCGCTTAGCTGGTGGGAC

aggtactagtt-----------------------------------------cttttttaAGGTCACGGCTCACCCAAGCCCCCATGCGTATACGACCTGGAAGGGTGATCGGCCACATT

ggaaataagtcaaaccaaaaacttgtattgttcttttcagtaaat---------------GGGACTGAGACACAGCACAAACTCCTACGGGAGGCAGCAATAGGGAATCTTCCGCAATGG

------------------------------------------------------------CCGAAAGTCTGACGGAGCAACGCCGCGAGACTGAAGAATGTCTTCGGATCGTAAAACTCT

------------------------tttatatatccagattcaaa-----tgcagaaccatGTTGTTAGGGAAGAACAAGTATCGTTCGAATAGGGCGGTACCTTGACGGTACCTAACCAG

caaactcgtatagctttttttct---------ttagtgagccagtgtcttgctttg----AAAGCTTTGCTAACTACCTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCG

-taatctagttcctgaatagtaaccatattcttatttgtagttattgta--aatccaagtGAATTATTGGGCGTAAAGCGCGCGCATGCGGTCTTTTAAATCTGATGTCAAAGCCCACGG

gattgtct----------attgtcgattgttacctgtacagt------------aattaaGCTCAACCGTGGAGGGTCATTGGAAACTGTAAGACTTGGAGTGCAGAAGAGGATAGTGGA

Figure 3 Continued

BioMed Research International 11

lowast lowast lowast lowastlowast lowastlowast lowastlowastlowast lowast lowastlowast lowast lowast lowast lowast

lowast lowast lowastlowastlowast lowastlowast lowast lowastlowastlowast lowast lowastlowast lowastlowastlowast lowast lowastlowast lowastlowast lowast lowast lowastlowast lowast

lowastlowast lowast lowast lowastlowastlowastlowastlowast lowast lowast lowastlowast lowast lowast lowast lowast lowast lowastlowastlowast

lowastlowast lowast lowastlowast lowastlowastlowast lowastlowast lowastlowast lowast lowast lowast lowast lowastlowast lowastlowast lowast lowast lowast lowastlowast

lowastlowast lowastlowastlowastlowast

lowast lowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowast lowast lowastlowastlowastlowast lowast lowastlowast lowastlowast lowast lowastlowastlowastlowast lowast lowastlowast lowast lowast lowastlowastlowastlowastlowastlowastlowast

lowastlowast lowast lowast lowast lowast lowastlowastlowast lowastlowast lowastlowast lowast lowastlowastlowastlowastlowastlowastlowastlowast lowastlowastlowast

lowast lowast lowast lowast lowast lowast lowastlowast lowastlowast lowast lowast lowastlowastlowast lowast lowast lowastlowastlowast lowast lowast

lowastlowast lowastlowastlowastlowastlowastlowast lowastlowast lowastlowastlowast lowastlowast lowast lowastlowast lowastlowast lowastlowastlowast lowast lowast

lowastlowast lowast lowastlowastlowast lowast lowastlowastlowastlowastlowast lowast lowast lowast lowastlowastlowast lowast lowastlowast lowast

lowastlowast lowast lowast lowast lowast lowastlowast lowast lowastlowast lowast lowastlowast lowast lowastlowast lowastlowastlowastlowast lowast lowast lowastlowast

lowast lowastlowastlowast lowast lowastlowast lowast lowast lowastlowast lowast lowastlowast lowast lowastlowastlowastlowastlowastlowast lowast

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

tttatctctttcattttctgaactattaaa----tgtaagaccacc---atacatacaacATTCCACGTGTAACGGTGAAAATGAGTAGAGATGTGGAGGAACACCAGTGGAGAAACGCG

aacctccaaaattttggtttatatccggttc-----------agatttaggtattatacaACTCTCTGGTCTGTTACTGACGCTGAGGCGCGAAAGCGTGGGAGCAAACAGGATTATATA

tgtataaaaatatggaacgccaaaaatgtctacttttttatctttataattgttagctaaCCCCTGGTAGTCCAAGCCGTAAACGATGAGTGCTAGTGTTACAGGGT------------T

ttctgtattttctaattcagttttaacctcattataactcttatactcaattggaaaaatTCCGCCCTTTACTGCTGCAGCTAACG---CATTAG-GCACTCCGCCTGGGGAGTACAACT

taaatcatgagataaaaactgtctagaattaataactcctcttccttcaacaggtggagaGCAA------GGTTGAAACTCAAAGCA----TTGACCGGGGGCC---CGATAAGCTGAGG

cgtataaagattcattacat----tacccatagttcccttattgtttttataatcacttaTACATGGTGTTTAATTCCAAACCACGCCAAGAACCCTCACGAGGTCTTGACATCCTCTGG

ctttattt----------------------------------------------------ACCTCCCTAGAGAATAAGGGCTTCCCCGTCGGGGATGAAATGAACAGGTGGTGCATGGAT

-------agttcgtc---AGTCTTCAGCTCGTGCAT

Enterotoxin H (seh)

------------------------------------------------------------ATCTATGAATATAAATCTTTTAATATTGGTTTAATATAAATATTCCATTTCTAGAAACCT

------------------------------------------------------------ATATTTTATGTTTGTATAAATTCATAAATTGTTCGAAATTTATATATTAAAAGTTGTTTT

------------------------------------------------------------TGTTGTTTTATAATTAAGTTAATGAAATATATTGAGGAGTTTTAAAATGATTAATAAAAT

------------------------------------------------------------TAAAATATTATTTTCGTTTTTAGCATTATTACTTTCATTCACATCATATGCGAAAGCAGA

------------ -----------TTACGATTTAGCTTTAGCTAATGCATATGGTCAATAAGATTTACACGATAAAAGTGAGTTAACAGATTTAGCTTTAGCTAATGCATATGGTCAATA

TAATCACCCATTCATTAAAGAAAATATTAAGAGTGATGAAATAAGTGGAGAAAAAGATTTTAATCACCCATTCATTAAAGAAAATATTAAGAGTGATGAAATAAGTGGAGAAAAAGATTT

AATATTTAGAAATCAAGGTGATAGTGGCAATGATTTGAGAGTAAAGTTTGCAACTGCTGAAATATTTAGAAATCAAGGTGATAGTGGCAATGATTTGAGAGTAAAGTTTGCAACTGCTGA

TTTAGCTCAGAAGTTTAAAAATAAAAATGTAGATATATATGGGGCATCTTTTTATTATAATTTAGCTCAGAAGTTTAAAAATAAAAATGTAGATATATATGGGGCATCTTTTTATTATAA

GTGTGAAAAAATAAATGAAAATATTTCTGAATGTCTATATGGAGGTACAACACTAAATAAGTGTGAAAAAATAAGTGAAAATATTTCTGAATGTCTATATGGAGGTACAACACTAAATAG

TGAAAAATTGGCACAGGAAAGGGTGATTGGTGCTAATGTTTGGGTAGATGGTATTCAAAATGAAAAATTGGCACAGGAAAGGGTGATTGGTGCTAATGTTTGGGTAGATGGTATTCAAAA

AGAAACAGAATTAATACGAACAAATAAGAAAAATGTGACATTGCAAGAATTAGATATAAAAGAAACAGAATTAATACGAACAAATAAGAAAAATGTGACATTGCAAGAATTAGATATAAA

Enterotoxin H CNS GATCAGAAAAATATTGTCCGATAAATATAAAATTTATTATAAAGACAGCGAAATAAGGG-Enterotoxin H

Enterotoxin H CNSEnterotoxin H

Enterotoxin H CNSEnterotoxin HEnterotoxin H CNSEnterotoxin HEnterotoxin H CNSEnterotoxin HEnterotoxin H CNSEnterotoxin HEnterotoxin H CNSEnterotoxin H

Enterotoxin H CNSEnterotoxin H

Enterotoxin H CNSEnterotoxin H

Enterotoxin H CNSEnterotoxin H

Enterotoxin H CNSEnterotoxin H

Enterotoxin H CNSEnterotoxin H

GATCAGAAAAATATTGTCCGATAAATATAAAATTTATTATAAAGACAGCGAAATAAGTAA

Enterotoxin N (seln) TGATTTAGCTTTAGCTAATGCATATGGTCAATAT-AATCACCCATTCATTAAAGAAAA-----TAGATATTTATGGACTGTATTATGGAAATAAATGTGTAGGCTTAACTGAAGAAAAAA

TATTAAGAGTGATGAAATAAGTGGAGAAAAAGATTTAATATTTAGAAATCAAGGTGATAGCATCATGCTTATACGGAGGAGTTAC------GA--TATATGATGGAAATCAATTAGATGA

TGGCAATGATTTGAGAGTAAAG--------------TTTGCAA-CTGCTGATTTAGCTCAAGAGAGAGTTATAGGCGTTAATGTATTTAAAGATGGTATCCAACAAGAAGGTTTTGTTAT

GAAGTTTAAAAATAAAAATGTAGATATATATGGGGCATCTTTTTATTATAAGTGTGAAAAAAAAACTAAAAAGGCTAAAGTAACAGTACAA------GAATTAGATACTAAAGTTCGATT

AATAAGTGAAAATATTTCTGAATGTCTATATGGAGGTACAACACTAAATAATGAAAAATT------------TAAATTAGAAAATTTATATAAAATATACAATAAAGATACCGGTAACAT

Enterotoxin N CNSEnterotoxin N

Enterotoxin N CNSEnterotoxin N

Enterotoxin N CNSEnterotoxin N

Enterotoxin N CNSEnterotoxin N

Enterotoxin N CNSEnterotoxin N

Figure 3 Continued

12 BioMed Research International

lowastlowastlowast lowastlowast lowastlowast lowastlowast lowast lowastlowast lowastlowastlowast lowastlowast lowast lowastlowastlowastlowast

lowastlowast lowast lowastlowast lowast lowast lowastlowast lowastlowastlowastlowastlowast lowast lowast lowast lowastlowast lowast lowast lowastlowast lowast lowast

lowast lowast

lowast lowastlowast lowast lowastlowast lowast lowast lowast lowast lowast lowast lowast lowastlowast lowast lowastlowastlowastlowast lowast

lowast lowastlowast lowast lowast lowastlowast lowast lowastlowastlowast lowast lowastlowast lowast lowastlowast lowastlowast lowast lowast

lowastlowastlowast lowast lowast lowastlowast lowastlowast lowast lowast lowast

lowast lowast lowast lowastlowastlowast lowastlowastlowast lowastlowast lowast lowastlowastlowastlowastlowast lowastlowastlowast lowast

lowastlowast lowast lowast lowastlowast lowastlowastlowast lowastlowast lowast lowast lowastlowastlowastlowast lowastlowast

lowast lowast lowast lowastlowast lowast lowastlowast lowastlowastlowastlowastlowastlowast lowastlowast lowast lowastlowast lowastlowastlowast

lowast lowast lowast lowast lowastlowast lowastlowastlowastlowast lowast lowast lowastlowastlowast lowast lowast lowast lowast lowast

lowast lowast lowast lowast lowastlowastlowast lowast lowast lowastlowast lowastlowast lowast lowastlowastlowast lowast lowastlowastlowast lowast

lowast lowast lowastlowastlowast lowast lowastlowast lowastlowastlowast lowastlowastlowastlowastlowast lowastlowast lowast lowastlowastlowast lowast lowast lowastlowastlowast

lowastlowastlowast lowast lowastlowast lowastlowast lowastlowast lowast lowast lowast lowast lowastlowast lowastlowast lowastlowast lowast lowast

lowast lowast lowast lowast lowastlowast lowast lowast lowastlowast lowastlowast lowast lowastlowastlowast lowastlowastlowastlowast lowast lowastlowastlowastlowastlowast lowast

lowastlowast lowast lowast lowastlowastlowast lowastlowast lowast lowastlowast lowast lowastlowast lowastlowast lowastlowast lowast lowast lowast

lowastlowastlowastlowastlowast lowastlowast lowastlowast lowast lowastlowast lowast lowast lowast lowast lowastlowast lowastlowast

lowast lowast lowastlowast lowastlowast lowastlowastlowastlowastlowastlowast lowastlowast lowast lowast lowast lowast lowast lowast lowast lowast lowast lowast lowast

lowastlowast lowastlowast lowast lowastlowastlowastlowast lowast lowast lowastlowastlowast lowastlowast lowast lowastlowastlowast lowast

lowastlowastlowastlowast lowastlowast lowast lowast lowast lowast lowastlowast lowast lowast lowast lowastlowastlowastlowastlowast lowastlowast lowastlowastlowast lowast

lowast lowastlowastlowast lowastlowastlowastlowastlowast lowast lowast lowast lowastlowast lowast lowast lowastlowast lowast lowastlowastlowast lowastlowast lowast

lowastlowast lowast lowast lowast lowastlowast lowast lowastlowast lowastlowast lowastlowast lowast lowastlowastlowastlowast

ATTAATACGAACAAATAAGAAAAATGTGACATTGCAAGAATTAGATATAAAGATCAGAAA-TTAT----------TATGATTTATATAAC--------------ATAAAAGGTTCAG---

AATATTGTCCGATAAATATAAAATTTATTATAAAGACAGCGAAATAAGGTAAAAGG------TAGGAGCAGAGTTTTTTCAATTTTATAGTGATAATAGAACAGTTAGCTCATCTAATTA

Enterotoxin N CNS -TAAA---Enterotoxin N

lowast lowastlowastlowastlowastlowast lowastlowast lowast lowast lowast lowastlowast lowast lowastlowastlowastlowastlowastlowast lowastlowast lowastlowast

GGCACAGGAAAGGGTGATTGGTGCTAATGTTTGGGTAGATGGTATTCAAAAAGAAACAGAACAA----AAAGGATGCATTTTCTTT-----CATTCTAATAATCATCAAAATCAATCATT

Enterotoxin N CNSEnterotoxin N

Enterotoxin N CNSEnterotoxin N

Enterotoxin N CNSEnterotoxin N

TCATATCG

Enterotoxin M (selm)

ttgctattcgcaaaatcatatcgcaaccgctgatgtcggagtt--ttgaatcttaggaacEnterotoxin M CNSEnterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

--------------AGTATGTTTAAATCCCCGCTCACAGTTATGTTTAACTCTTCCTTTC

tattatggta-gctatccaattgaagaccaccaaagtattaatcctgaaaataatcatctTTTTCACACCGCCCCGTCGGGTGTTGCGCACGGGGTTGTTTAACCCCCCCGGAAAAAATT

ttcgcatcaattagttttttctatggataattcgacagtaacagctgaatttaagaacgtEnterotoxin M CNS

Enterotoxin M CNS

TTCCCTCCCTCCTCCTTACTC---------------CCT----------------CCCCT

tgatgatgtaaaggaattcaaaaatcatgctgtagatgtatatggtctaagttatagtggEnterotoxin M CNS TTTTCCTACAAATCCTTTCTCCCCTCCCGGAGTAGAAGTAAA------------------

atattgtttgaaaaacaaatatatatacggtggagttacattagcaggtgat-tatttagEnterotoxin M CNS -------------------TAAAACAATACCCCTACCACCTTAACAATTTTTTTATTGAG

agaaatctagacgtattcct-attaatctttgggttaatggagaacatcaaactatatctEnterotoxin M CNS CCCCTCCCCCCCCTTCTCATTTTTTCTCTTTGAC-----------GTTCCCCCCATTTCT

actgacaaagtatcaactaataaaa-agttagtaacagctcaagaaattgatactaaattEnterotoxin M CNS GCCAATGCTTAACCCACCCTTAAAGGCCATCGGCACATATTAGACACTCC-----CCACC

aagaagatatctacaagaagaatataatatttatggctttaatgatac---aaataaaggEnterotoxin M CNS CGCCCCCTCTTTTTATTAAGGACACAACTCTTTTTATTTTCCCCCAAGAACTAATTCTGA

a-agaaattatggtaataagtcaaaatttagttctgga---------tttaatgcaggaaEnterotoxin M CNS AAAAAAACGTTCCGCTTAGTTCAGAATTTCCTTTGAGCCTTTTATCCTTTTGTACGCGAA

aaatattatttca--------tttgaatgatggttcatcattttcttatgacttatttga

Enterotoxin M CNS AAACCATCAGCCATTGAATCAGTTATCCCATTGCCCTAACTAACCCAATATCTAATCTAA

tactggaacaggacaagctgaaagtttctt-aaaaatatataatgacaacaaaactgtcgEnterotoxin M CNS TTTTCGCCTACGAACGAATAATAGCTTGTGGCGAAAAATATCCAGGATTATCTACTGTAG

aaact--gaaaaattccatttagatgtagaaatatcttataaggacgaaagttgaagtatEnterotoxin M CNS CGCCTCGCCAGACGTCCTTTGAATTGA--ATATGACTCCTACTCCCCCCCGCACCGTCAA

ttcaatcataacttagtaaaggaaatgccatgaaaaaatttaaatatagtttt---atatEnterotoxin M CNS TTCAAACA---------------GGTGGAAGTTAATACTGAATAGTAGGTCTTCTTCCCG

tagtttttatattactttttaacattaaagatcttacgtatgct---caaggtgatattgEnterotoxin M CNS TGCTATTCTTAGAACTTTTAAATTATGTAAACCCAATTTTTAGTGCACTCTTTCTCCCTC

gtg-taggtaacttaagaaatttctatacaaaacatgattatatagatttaaaaggcgtcEnterotoxin M CNS GTTACTCTTAGATGGAGAAGCGTTAAGACAGAAGCTTTGTAT-------------TCCCA

acagataaaaa-cctacctattgcaaatcaacttgaattttcaac-----aggt----acEnterotoxin M CNS GCAGAGTGCAACCTTTCACAATGAAGTTTTTATCTAATTTACACGCCTTTAGGCTCCAAA

caatgatttgatctcagaatctaataattgggacgaaataagtaaatttaaaggaaagaaEnterotoxin M CNS TACGTATTAAATCTCCTCCTGT--TTATCTGACCCTAAG-ATAAAACACGAAAAATTATG

actggatatttttggcattgattataatggtccttgtaaatctaaatacatgtatggaggEnterotoxin M CNS CGTGCCCACTCATCCTAT-------TACTTTCATTATATCTATAAAA-------------

Figure 3 Alignment of the enterotoxin gene sequences found in coagulase-negative staphylococci (CNS) and in coagulase-positivestaphylococci (CPS)

BioMed Research International 13

Enterotoxin (seg) S aureus AY1587031

Enterotoxin (sec) CNS

Enterotoxin (see) S pasteuri NC0227371

Enterotoxin (see) CNS

Enterotoxin (seh) CNS

Enterotoxin (selm) S aureus AF2857601

Enterotoxin (seln) CNS

Enterotoxin (seh) S aureus AJ9375481

Enterotoxin (seln) S aureus AY1587031

Enterotoxin (sec) S aureus X516611

Enterotoxin (selm) CNS

Enterotoxin (seg) CNS

150 100

100

100

100

100

99

98

96

97

72

50 0

Figure 4 Phylogenetic tree generated from the multiple alignments of the enterotoxin sequences of CNS and CPS strains using the ClustalX20 software package The phylogenetic tree was constructed using the Mega 60 software and the unweighted pair group method (UPGMA)

MAR indices varying from 066 to 046 and the remaining04 strains showed MAR indices le026 (Table 3)

The 14 MRCNS strains were resistant to 120573-lactams(oxacillin penicillin andor cefoxitin) and to vancomycincorresponding to 73 of the total CNS strains identifiedwhile 09 strains (64) showed resistance to tetracycline andgentamicin 08 strains (57) were resistant to neomycinerythromycin and chloramphenicol 07 strains (50) wereresistant to sulfamethoprim 05 strains (36) were resistantto linezolid 03 strains (21) were resistant to rifampicin 02strains (14) were resistant to ciprofloxacin and cefepimeand 01 strain (7) was resistant to clindamycin (Table 3)

The multiresistance of CNS strains demonstrated hereinis consistent with previous studies on coagulase-negativeand coagulase-positive staphylococci that have found severalresistant and multiresistant Staphylococcus aureus strains inraw milk meat and fermented meat products [43]

Surprisingly the resistance to chloramphenicol is verysimilar to that estimated for MRCNS strains isolated fromhuman clinical samples Indeed there is no direct correla-tion between the researched features and the origin of thestaphylococci strains since different virulence factors arewidespread such as antibiotic resistance [44] reinforcing thefact that the ability of foodmatrices strains to produce SE andSEl and their multiresistance character must be consideredwhen evaluating the safety hazards of food poisoning

S epidermidis KF6005891 and S hominis JX5199881showed the highestMAR indexes (093 and 080 resp)Thesestrains found in artisanal salami are homologous to strainsisolated from human skin microbiota and should be carefullyconsidered among the potential pathogenic staphylococcifound in food from animal origins which can be caused bycontamination by poor hygienic conditions during salamimanufacturing

Depending on the conditions some species of coagulase-negative staphylococci can present health risks since theyhave shown resistance to antibiotics of therapeutic impor-tance such as beta lactams [15] However multiresistantstrains like S carnosusKJ18620021 and S xylosusCP0072081and CP0087241 were intentionally introduced in the foodmatrix since they are part of the culture starter used inItalian-salami manufacturing As previously discussed foodcan be a reservoir of multiresistant microorganisms that canspread by the consumption of an apparently safe food Dueto the intensive and indiscriminate use of antibiotics forhuman and veterinarian therapeutic purposes multiresistantstaphylococci strains are being selected and reproduced infood matrices [45]

The cefoxitin and oxacillin disk diffusion tests are rec-ommended for determining the resistance and susceptibilitybreakpoint for MRSA surveillance cultures [46] In thisstudy there was a good correlation between the disc testzone diameters for oxacillin and cefoxitin (Table 3) TheMICs for ampicillin andmethicillinoxacillincefoxitin for allsusceptible CNS strainswere determined by the diffusion disctest using 003 to 2mgmLminus1 of each antimicrobial agent

S saprophyticus HF9372521 showed an MIC of003mgmLminus1 formethicillin strain S carnosusKJ8620021 Sxylosus strains CP0087241 and CP0072081 S saprophyticusJX4901221 S hominis JX5199881 and S succinusKC3298241 were resistant to 006mgmLminus1 and two strainsS saprophyticus KJ6991511 and S saprophyticus KJ0046231showed an MIC of 05mgmLminus1 (Table 4) Two of themethicillin-resistant strains S saprophyticus KJ0046231 andS xylosus CP0072081 harbor no enterotoxin gene

Five strains S saprophyticus JX4901221 S carnosusKJ8620021 S xylosusCP0087241 S xylosusKF1980801 andS saprophyticus KJ0046231 showed an MIC for ampicillin

14 BioMed Research International

Table 3 Multiple resistance to antimicrobial as found in CNS strains from salami

Salami origin CNS strains Antimicrobial agent resistance Multiple antimicrobialresistance (MAR) indexlowast

Commercial Staphylococcus spp KF1354451 CIP CLO CPM GEN NEO OXA PENSXT TET and VAN 066

Commercial S carnosus KJ8620021 CIP GEN LZD NEO OXA SXT andTET 046

Commercial S saprophyticus AB6977171 CFO CLO ERI PEN OXA TET andVAN 046

Commercial S saprophyticus EU4309921 CFO CLO ERI GEN LZD NEO OXAPEN SXT TET and VAN 080

Commercial S saprophyticus JX4901221 CFO CLO ERI GEN NEO PEN OXASXT TET and VAN 066

Commercial S saprophyticus KJ0046231 CFO OXA and PEN 020

Commercial S sciuri JX9664361 CFO CLO GEN NEO OXA PEN andTET 046

Commercial S xylosus CP0072081 OXA LZD PEN and VAN 026

Commercial S xylosus CP0087241 CFO CLO ERI GEN NEO OXA PENTET and VAN 060

Artisanal S epidermidis KF6005891 CFO CLI CLO CPM ERI GEN LZDNEO OXA PEN RIF SXT and TET 093

Artisanal S hominis JX5199881 CIP CPM CPO ERI GEN LZD NEOOXA PEN RIF SXT and VAN 080

Artisanal S saprophyticusHF9372521 CFO OXA PEN and SXT 026Artisanal S saprophyticus KJ6991511 OXA PEN and VAN 020

Artisanal S succinus KC3298241 CFO CLO ERI GEN OXA PEN RIFSXT TET and VAN 066

Artisanal S xylosus KF1980801 CFO CLO ERI GEN NEO OXA PENTET and VAN 060

lowastTheMAR index of an isolate is defined as 119886119887 where 119886 represents the number of antimicrobials to which the isolate was resistant and 119887 represents the numberof antimicrobials to which the isolate was subjectedS aureus strains ATCC WB81 (sea) ATCC 13563 (sed) and ATCC 27664 (see) showing a MAR index of 05 and S aureus strains ATCC14458 (seb) andATCCWB72 (sec) and S xylosus ATCC 29971 showing a MAR index of 03 were used as reference strainsCPM cefepime CFO cefoxitin CLO chloramphenicol CIP ciprofloxacin CLI clindamycin ERI erythromycin GEN gentamycin NEO neomycin LZDlinezolid RIF rifampicin TET tetracycline OXA oxacillin PEN penicillin SXT sulfamethoprim and VAN vancomycin

Table 4 Minimal inhibitory concentration (MIC) of compounds used in antimicrobial therapy against staphylococci infections

Salami origin GenBank accession number and similarity () MIC mgmLminus1

Methicillin Ampicillin Vancomycin Linezolid

Commercial

S carnosus KJ8620021 006 003 mdash mdashS xylosus CP0087241 006 003 006 mdash

S saprophyticus JX4901221 006 003 05 mdashS saprophyticus AB6977171 mdash mdash 05 mdashS saprophyticus EU4309921 mdash mdash 025 0125

S succinus KC3298241 006 025 003 mdash

Artisanal

S hominis JX5199881 006 05 mdash mdashS saprophyticus HF9372521 003 mdash mdash mdash

S xylosus KF1980801 003 05 mdashS saprophyticus KJ6991511 05 025 003 mdashS saprophyticus KJ0046231 05 003 003 mdash

S xylosus CP0072081 006 mdash mdash 025Strains S epidermidis KF6005891 and Staphylococcus spp KF1354451 were not susceptible to the antimicrobial concentrations tested in the present study

BioMed Research International 15

of 003mgmLminus1 two strains S succinus KC3298241 and Ssaprophyticus KJ6991511 showed an MIC of 025mgmLminus1and S hominis JX5199881 showed an MIC of 05mgmLminus1(Table 4) S saprophyticus KJ0046231 also showed an MICof 003mgmLminus1 but does not harbor enterotoxin genes It isimportant to note that most of the CNS strains that showedresistance to ampicillin were also resistant to methicillin ashighlighted by the MIC tests

Five strains S saprophyticus HF9372521 S saprophyti-cus KJ6991511 S saprophyticus JX4901221 S saprophyticusKJ0046231 and S succinus KC3298241 showed an MICfor vancomycin of 003mgmLminus1 one strain S xylosusCP0087241 showed an MIC of 006mgmLminus1 one strain Ssaprophyticus EU4309921 showed an MIC of 025mgmLminus1and two strains S xylosus KF1980801 and S saprophyticusAB6977171 showed an MIC of 05mgmLminus1 (Table 4)

Additionally five CNS strains S xylosus CP0087241S saprophyticus JX 4901221 S succinus KC3298241 Ssaprophyticus KJ6991511 and S saprophyticus KJ0046231showed resistance tomethicillin as demonstrated by theMICdeterminations (Table 4)

Previous studies have demonstrated that linezolid isactive against Gram-positive bacteria including methicillin-resistant staphylococci [47] In the present study 05 strainsshowed linezolid resistance in the disc diffusion test but itwas impossible to establish MIC values for S epidermidisKF6005891 Staphylococcus hominis JX5199881 and Staphy-lococcus carnosus KJ8620021The remaining strains S sapro-phyticus EU4309921 and S xylosus CP0072081 presentedan MIC for linezolid of 0125mgmLminus1 and 025mgmLminus1respectively

The MIC of 003mgmLminus1 for penicillin is in accordancewith the previous values estimated for resistant S aureusstrains found in several food matrices such as meat dairyproducts and ready-to-eat food [48]

There is still a lack of information on the antimicro-bial resistance of staphylococci strains from food matricesalthough they are the most worrisome vehicles of dissemi-nation of antibiotic-resistant pathogens [49]

The high resistance found for salami staphylococci strainscan be ascribed to their inappropriate use as growth promot-ers of antimicrobial agents like oxacillin vancomycin chlo-ramphenicol neomycin and erythromycin which are com-monly used in veterinary medicine to treat infections [50]Antimicrobial therapy of infections staphylococci is basedon results of susceptibility tests in vitro [51] Methicillin-resistant coagulase-negative Staphylococcus spp found inready-to-eat products such as meats fish and dairy productsalso offer risks and although they are not classical foodpoisoning bacteria their presence in food offers significantrisks to public health due to the possible spread of antibioticresistance [52]

The standardization of salami quality should includediagnostic methods to screen and quantify the presence ofclassical and newly described enterotoxins directly in thefood matrices as a routine procedure to be conducted by themeat product industry in Brazil

The safety of salami consumption could also be enhancedby the inclusion of amicrobial barrier such as the inclusion ofprobiotic strains producing natural antibiotics or competitiveflora or even the addition of natural bioagents against spoilageor pathogenic microorganisms

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors acknowledge the financial support fromFundacao Carlos Chagas Filho de Amparo a Pesquisa doEstado do Rio de Janeiro (FAPERJ Rio de Janeiro Brazil)Conselho Nacional de Desenvolvimento Cientıfico eTecnologico (CNPq Brasılia Brazil) and Coordenacao deAperfeicoamento de Pessoal de Nıvel Superior (CAPESBrasılia Brazil)

References

[1] ICMSF International Comission on Microbiological Specifica-tions for FoodsMicroorganisms in FoodsTheir Significance andMethods of Enumerations University of Toronto Press TorontoCanada 3rd edition 1983

[2] J F Veras L S Carmo L C Tong et al ldquoA study of theenterotoxigenicity of coagulase-negative and coagulase-positivestaphylococcal isolates from food poisoning outbreaks inMinasGerais Brazilrdquo International Journal of Infectious Diseases vol12 no 4 pp 410ndash415 2008

[3] K Omoe D-L Hu H Takahashi-Omoe A Nakane and KShinagawa ldquoComprehensive analysis of classical and newlydescribed staphylococcal superantigenic toxin genes in Staphy-lococcus aureus isolatesrdquo FEMS Microbiology Letters vol 246no 2 pp 191ndash198 2005

[4] J Kadariya T C Smith and D Thapaliya ldquoStaphylococcusaureus and staphylococcal food-borne disease an ongoing chal-lenge in public healthrdquoBioMedResearch International vol 2014Article ID 827965 9 pages 2014

[5] KOmoeD-LHuH KOno et al ldquoEmetic potentials of newlyidentified staphylococcal enterotoxin-like toxinsrdquo Infection andImmunity vol 81 no 10 pp 3627ndash3631 2013

[6] J K McCormick J M Yarwood and P M SchlievertldquoToxic shock syndrome and bacterial superantigens an updaterdquoAnnual Review of Microbiology vol 55 pp 77ndash104 2001

[7] T Baba F Takeuchi M Kuroda et al ldquoGenome and virulencedeterminants of high virulence community-acquired MRSArdquoThe Lancet vol 359 no 9320 pp 1819ndash1827 2002

[8] Associacao Brasileira de Industria Produtora e Exportadorada Carne Suınas (ABIPECS) ldquoStatistics slaughter ranking2000ndash2014rdquo 2014 httpabpa-brcombrsetoressuinoculturapublicacoesrelatorios-anuais

[9] N Zdolec M Hadziosmanovic L Kozacinski et al ldquoMicrobialand physicochemical succession in fermented sausages pro-duced with bacteriocinogenic culture of Lactobacillus sakei andsemi-purified bacteriocin mesenterocin Yrdquo Meat Science vol80 no 2 pp 480ndash487 2008

16 BioMed Research International

[10] F Ravyts L D Vuyst and F Leroy ldquoBacterial diversity andfunctionalities in food fermentationsrdquo Engineering in Life Sci-ences vol 12 no 4 pp 356ndash367 2012

[11] TOhlsson andN BengtssonTheHurdle ConceptMinimal Pro-cessing Technologies in the Food IndustryWoodhead Publishing2002

[12] S Bernardi B B Golineli and C J Contreras-CastilloldquoRevisao aspectos da aplicacao de culturas starter na producaode embutidos carneos fermentadosrdquo Brazilian Journal of FoodTechnology vol 13 no 2 pp 133ndash140 2010

[13] F Irlinger ldquoSafety assessment of dairy microorganismscoagulase-negative staphylococcirdquo International Journal of FoodMicrobiology vol 126 no 3 pp 302ndash310 2008

[14] B Ghebremedhin F Layer W Konig and B Konig ldquoGeneticclassification and distinguishing of Staphylococcus species basedon different partial gap 16S rRNA hsp60 rpoB sodA and tufgene sequencesrdquo Journal of Clinical Microbiology vol 46 no 3pp 1019ndash1025 2008

[15] I Babic K Markov D Kovacevic et al ldquoIdentification andcharacterization of potential autochthonous starter culturesfrom aCroatian lsquobrandrsquo product lsquoslavonski kulenrsquordquoMeat Sciencevol 88 no 3 pp 517ndash524 2011

[16] MMehrotra GWang andWM Johnson ldquoMultiplex PCR forthe detection of genes for Staphylococcus aureus enterotoxinsexfoliative toxins toxic shock syndrome toxin and methicillinresistancerdquo Journal of Clinical Microbiology vol 38 no 3 pp1032ndash1041 2000

[17] WM Johnson SD Tyler E P Ewan F EAshtonDR Pollardand K R Rozee ldquoDetection of genes for enterotoxins exfolia-tive toxins and toxic shock syndrome toxin 1 in Staphylococcusaureus by the polymerase chain reactionrdquo Journal of ClinicalMicrobiology vol 29 no 3 pp 426ndash430 1991

[18] J Mclauchlin G L Narayanan VMithani andG OrsquoNeill ldquoThedetection of enterotoxins and toxic shock syndrome toxin genesin Staphylococcus aureus by polymerase chain reactionrdquo Journalof Food Protection vol 63 no 4 pp 479ndash488 2000

[19] J P Rosec and O Gigaud ldquoStaphylococcal enterotoxin genes ofclassical and new types detected by PCR in Francerdquo Interna-tional Journal of Food Microbiology vol 77 no 1-2 pp 61ndash702002

[20] K Omoe D-L Hu H Takahashi-Omoe A Nakane andK Shinagawa ldquoIdentification and characterization of a newstaphylococcal enterotoxin-related putative toxin encoded bytwo kinds of plasmidsrdquo Infection and Immunity vol 71 no 10pp 6088ndash6094 2003

[21] N Sergeev D Volokhov V Chizhikov and A Rasooly ldquoSimul-taneous analysis of multiple staphylococcal enterotoxin genesby an oligonucleotide microarray assayrdquo Journal of ClinicalMicrobiology vol 42 no 5 pp 2134ndash2143 2004

[22] S Holtfreter D Grumann M Schmudde et al ldquoClonal distri-bution of superantigen genes in clinical Staphylococcus aureusisolatesrdquo Journal of Clinical Microbiology vol 45 no 8 pp2669ndash2680 2007

[23] W J Mason J S Blevins K Beenken NWibowo N Ojha andM S Smeltzer ldquoMultiplex PCR protocol for the diagnosis ofstaphylococcal infectionrdquo Journal of Clinical Microbiology vol39 no 9 pp 3332ndash3338 2001

[24] E Rahimi and H G Safai ldquoDetection of classical enterotoxinsof Staphylococcus aureus strains isolated frombovine subclinicalmastitis in Isfahan Iranrdquo Veterinary Microbiology vol 141 no3-4 pp 393ndash394 2010

[25] M A Larkin G Blackshields N P Brown et al ldquoClustalW andClustal X version 20rdquo Bioinformatics vol 23 no 21 pp 2947ndash2948 2007

[26] P H A Sneath and R R Sokal Unweighted Pair Group Methodwith Arithmetic Mean Numerical Taxonomy W H FreemanSan Francisco Calif USA 1973

[27] NCCLS ldquoPerformance standards for antimicrobial susceptibil-ity testing 14th informational supplementrdquo Tech Rep M 100-S14 NCCLS 2004

[28] S Corbiere Morot-Bizot S Leroy and R Talon ldquoMonitoringof staphylococcal starters in two French processing plantsmanufacturing dry fermented sausagesrdquo Journal of AppliedMicrobiology vol 102 no 1 pp 238ndash244 2007

[29] R Talon S Leroy I Lebert et al ldquoSafety improvement andpreservation of typical sensory qualities of traditional dryfermented sausages using autochthonous starter culturesrdquo Inter-national Journal of Food Microbiology vol 126 no 1-2 pp 227ndash234 2008

[30] G Mauriello A Casaburi G Blaiotta and F Villani ldquoIsolationand technological properties of coagulase negative staphylo-cocci from fermented sausages of Southern ItalyrdquoMeat Sciencevol 67 no 1 pp 149ndash158 2004

[31] M Janssens N Myter L De Vuyst and F Leroy ldquoSpeciesdiversity and metabolic impact of the microbiota are low inspontaneously acidified Belgian sausages with an added starterculture of Staphylococcus carnosusrdquo Food Microbiology vol 29no 2 pp 167ndash177 2012

[32] G Blaiotta D Ercolini G Mauriello G Salzano and F VillanildquoRapid and reliable identification of Staphylococcus equorum bya species-specific PCR assay targeting the sodA generdquo Systematicand Applied Microbiology vol 27 no 6 pp 696ndash702 2004

[33] L Kozacinski E Drosinos F Caklovica L Cocolin J Gasparik-Reichardt and S Veskovic ldquoInvestigation of microbial associa-tion of traditionally fermented sausagesrdquo Food Technology andBiotechnology vol 46 no 1 pp 93ndash106 2008

[34] S Leroy P Giammarinaro J-P Chacornac I Lebert and RTalon ldquoBiodiversity of indigenous staphylococci of naturallyfermented dry sausages and manufacturing environments ofsmall-scale processing unitsrdquo Food Microbiology vol 27 no 2pp 294ndash301 2010

[35] E Coton M-H Desmonts S Leroy et al ldquoBiodiversity ofCoagulase-Negative Staphylococci in French cheeses dry fer-mented sausages processing environments and clinical sam-plesrdquo International Journal of Food Microbiology vol 137 no 2-3 pp 221ndash229 2010

[36] S Hosseinzadeh and H D Saei ldquoStaphylococcal species associ-ated with bovinemastitis in the NorthWest of Iran emerging ofcoagulase-negative staphylococcirdquo International Journal of Vet-erinary Science and Medicine vol 2 no 1 pp 27ndash34 2014

[37] A Piette and G Verschraegen ldquoRole of coagulase-negativestaphylococci in human diseaserdquo Veterinary Microbiology vol134 no 1-2 pp 45ndash54 2009

[38] T M Moura F S Campos P A DrsquoAzevedo et al ldquoPrevalenceof enterotoxin-encoding genes and antimicrobial resistancein coagulase-negative and coagulase-positive Staphylococcusisolates from black pudding in Southern Brazilrdquo Revista daSociedade Brasileira deMedicina Tropical vol 45 no 5 pp 579ndash585 2012

[39] H I Suzuki T Lefebure P P Bitar and M J StanhopeldquoComparative genomic analysis of the genus Staphylococcusincluding Staphylococcus aureus and its newly described sister

BioMed Research International 17

species Staphylococcus simiaerdquo BMC Genomics vol 13 article38 2012

[40] R P Lamers G Muthukrishnan T A Castoe S Tafur A MCole and C L Parkinson ldquoPhylogenetic relationships amongStaphylococcus species and refinement of cluster groups basedon multilocus datardquo BMC Evolutionary Biology vol 12 article171 2012

[41] I V Pinchuk E J Beswick and V E Reyes ldquoStaphylococcalenterotoxinsrdquo Toxins vol 2 no 8 pp 2177ndash2197 2010

[42] S R Monday and G A Bohach ldquoGenes encoding staphylo-coccal enterotoxins G and I are linked and separated by DNArelated to other staphylococcal enterotoxinsrdquo Journal of NaturalToxins vol 10 no 1 pp 1ndash8 2001

[43] D Y Thomas S Jarraud B Lemercier et al ldquoStaphylococcalenterotoxin-like toxins U2 and V two new staphylococcalsuperantigens arising from recombination within the entero-toxin gene clusterrdquo Infection and Immunity vol 74 pp 4724ndash4734 2006

[44] N Indrawattana O Sungkhachat N Sookrung et al ldquoStaphylo-coccus aureus clinical isolates antibiotic susceptibility molecu-lar characteristics and ability to form biofilmrdquo BioMed ResearchInternational vol 2013 Article ID 314654 11 pages 2013

[45] V Pereira C Lopes A Castro J Silva P Gibbs and P TeixeiraldquoCharacterization for enterotoxin production virulence factorsand antibiotic susceptibility of Staphylococcus aureus isolatesfrom various foods in Portugalrdquo Food Microbiology vol 26 no3 pp 278ndash282 2009

[46] I Anacarso C Condo C Sabia and et al ldquoAntimicrobialresistance and other related virulence factors in staphylococcusspp isolated from food environmental and humans in ItalyrdquoUniversal Journal Microbiology Research vol 1 no 1 pp 1ndash92013

[47] B Martın M Garriga M Hugas S Bover-Cid M T Veciana-Nogues and T Aymerich ldquoMolecular technological and safetycharacterization of Gram-positive catalase-positive cocci fromslightly fermented sausagesrdquo International Journal of FoodMicrobiology vol 107 no 2 pp 148ndash158 2006

[48] NM Broekema T Van Tam T AMonson S AMarshall andD M Warshauer ldquoComparison of cefoxitin and oxacillin diskdiffusionmethods for detection ofmecA-mediated resistance inStaphylococcus aureus in a large-scale studyrdquo Journal of ClinicalMicrobiology vol 47 no 1 pp 217ndash219 2009

[49] E Cercenado F Garcıa-Garrote and E Bouza ldquoIn vitro activityof linezolid against multiply resistant Gram-positive clinicalisolatesrdquo Journal of Antimicrobial Chemotherapy vol 47 no 1pp 77ndash81 2001

[50] A Aydin K Muratoglu M Sudagidan K Bostan B Okukluand SHarsa ldquoPrevalence and antibiotic resistance of foodborneStaphylococcus aureus isolates in Turkeyrdquo Foodborne Pathogensand Disease vol 8 no 1 pp 63ndash69 2011

[51] D Sergelidis A Abrahim T Papadopoulos et al ldquoIsolation ofmethicillin-resistant Staphylococcus spp from ready-to-eat fishproductsrdquo Letters in Applied Microbiology vol 59 no 5 pp500ndash506 2014

[52] I PhillipsM Casewell T Cox et al ldquoDoes the use of antibioticsin food animals pose a risk to human health A critical reviewof published datardquo Journal of Antimicrobial Chemotherapy vol53 no 1 pp 28ndash52 2004

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Page 9: Research Article Safety Evaluation of the Coagulase ...downloads.hindawi.com/journals/bmri/2015/483548.pdf · Research Article Safety Evaluation of the Coagulase-Negative Staphylococci

BioMed Research International 9

shown to carry the selm seln and selo genes and the selmand seln sequences were detected in the genome of a singlestrain S saprophyticus subsp bovisKJ6991511 S epidermidisKF6005891 showed a combination of seg and sei genesand 03 strains harbored a single gene sei or selm or selnperhaps due to the high degree of genetic polymorphism inthe chromosomal assembly [41] Indrawattana et al 2013detected the seg-sei-selm-seln-selo of the highly prevalent egclocus was 261 in contrast to this study where 13 CNSpresents the combination of selm seln and selo genes [42]

The CNS strains clustered into groups near the bottom ofthe phylogenetic tree carried the classical enterotoxin geneswhile the species at the top of the tree showed high diversityamong the enterotoxin genes combining the classical and thenewly described genes in their genomes (Figure 1)

To assess the risk of staphylococcal food poisoning theability of the identified strains in harboring the se andsel genes and in expressing and producing enterotoxins invitro was evaluated The mRNA for each enterotoxin genewas evaluated by real time RT-PCR assays and enterotoxincontent was estimated by a sandwich enzyme immunoassayfor the combined detection of Staphylococcus enterotoxins(SET) A B C D and E

The fifteen enterotoxigenic CNS strains were able toexpress the classical enterotoxins SEA seb sec sed and seein concentrations ranging from 03 ngmLminus1 to 14 ngmLminus1as assessed by the in vitro assays (Table 2) The S sapro-phyticus AB6977171 KJ6991511 JX4901221 KJ0046231 andHF9372521 strains S xylosus KF1980801 and S carnosusNR1164341 produced low amount of enterotoxins lower than05 ngmLminus1 S epidermidisKF6005891 S saprophyticus bovisKJ6991511 and S hominis JX5199881 produced intermediateamount of enterotoxins ranging from 07 to 090 ngmLminus1while S sciuri JX9664361 S saprophyticus EU4309921 Scarnosus KJ8620021 S xylosus CP0087241 and S succi-nus KC3298241 produced enterotoxins in concentrationsge10 ngmLminus1

Although the sandwich enzyme immunoassay is consid-ered themost sensitivemethod to detect seandashsee enterotoxinsable to detect 0125 ngmLminus1 differences in the specificity andsensitivity of the assays for the detection of staphylococcalenterotoxins from foods are expected [43] A single strainStaphylococcus spp KF1354451 which harbors both the seaand seb genes was unable to produce seandashsee enterotoxins

The mRNA levels evaluated by the real time RT-PCRassays for enterotoxins were detected in 12 of 15 strains(86) (Table 2) Transcripts for the classical enterotoxinsgenes were detected when S saprophyticus HF9372521 Scarnosus NR1164341 S xylosus CP0087241 and S succinusKC3298241 were assayed Transcripts for newly describedenterotoxins were observed in S saprophyticus JX4901221and AB6977171 S sciuri JX9664361 S saprophyticus subspbovis KJ6991511 and S hominis JX5199881 Transcripts fromboth the classical and newly described enterotoxins weredetected in S saprophyticus EU4309921 and KJ9496061 andS epidermidis KF6005891

No mRNA transcripts were obtained for S sapro-phyticus KJ00462321 and S carnosus KJ8620021 although

enterotoxin production was detected by the enzyme-linkedimmunosorbent tests (Table 2)

The 04 strains that carry tstH1 in their genome Ssaprophyticus EU4309921 and KJ6991511 and S xylosusCP0087241 and KF980801 were not able to produce mRNAfor TSST-1 in the assay conditions

The production of classical enterotoxins in vitro(immunologic test) matched the results shown by real timeRT-PCR assays for the following strains S saprophyticusJX4901221 S sciuri JX9664361 S saprophyticusEU4309921S saprophyticus HF9372521 S saprophyticus subsp bovisKJ6991511 S hominis JX5199881 S epidermidis KF6005891and S succinus KC3298241

Ten strains namely S saprophyticusAB6977171 S sapro-phyticus EU4309921 S saprophyticus HF9372521 S sapro-phyticus KJ6991511 S saprophyticus KJ9496061 S succinusKC3298241 S hominis JX5199881 S xylosus KF1980801and S xylosus CP0087241 and S epidermidis KF6005891expressed mRNA for multiple se andor sel genes There isa differential transcription among these genes where themost frequent among the classical ones were seb and seeseatranscribed by 04 and 02 strains respectively and the mostfrequent among the newly described genes were sei andsehselnselo transcribed by 03 and 02 strains respectively

No mRNA for the sec gene was detected although SsaprophyticusAB6977171 was able to produce the enterotoxinin vitro as detected by the ELISA assays

34 Enterotoxin Gene Homologies between Salami CNSand CPS Strains The nucleotide sequencing of six entero-toxin genesmdashtwo of them sec and see encoding classi-cal enterotoxinsmdashand four of them encoding the newlydescribed genes seg seh selm and seln fromCNS strains wascompared to the enterotoxin genes from S aureus or S pas-teuri two coagulase-positive staphylococci strains (Figure 3)The homology between the CNS and CPS enterotoxin genesvaried from 65 to 98 The homology of sec seg sehselm and seln between CNS from salami and S aureus was98 60 98 65 and 70 respectively The homologybetween see from CNS found in salami and in S pasteuri was98

Although further studies should be performed it seemsthat the sequences encoding enterotoxins can be conservedamong coagulase-negative and coagulase-positive staphy-lococci as shown in the phylogenetic analysis of the 06enterotoxin genes (Figure 4)

35 Antimicrobial Multiresistance of CNS Strains (MRCNS)Another safety hazard associated with CNS strains besidesthe ability of producing enterotoxins in food matrices is theantimicrobial resistance to antimicrobial agents commonlyused to treat staphylococci infections The antimicrobialresistance carried by CNS strains from food matrices can bespread to the population by the consumption of an apparentlysafe food

Among the 19 CNS strains identified in salami 14 showedmultiresistance to antimicrobial agentsThree strains showedthe highest MAR indices 093 and 080 07 strains presented

10 BioMed Research International

lowast lowastlowastlowastlowastlowast lowast lowast lowastlowast lowast lowast lowastlowast lowastlowastlowastlowast lowastlowastlowast lowast

lowastlowast lowast lowast lowastlowastlowastlowast lowast lowast lowastlowast lowast

lowastlowastlowastlowast lowast lowast lowast

lowastlowast lowast lowast lowastlowast lowastlowast lowast lowastlowast lowastlowastlowastlowastlowast lowastlowast lowast lowastlowast lowastlowast

lowastlowast lowastlowastlowast lowast lowast lowast lowast lowast lowastlowastlowastlowastlowast

lowastlowast lowastlowast lowastlowast lowastlowast lowast lowast lowastlowast lowast lowast lowastlowastlowast lowast lowastlowast lowastlowastlowast

lowast lowast lowast lowast lowast lowast lowastlowast lowast lowastlowastlowast lowast lowastlowastlowast lowast lowast lowastlowastlowast lowast lowastlowastlowast lowast

lowast lowast lowast lowastlowastlowastlowast lowast lowastlowastlowast lowast lowast lowastlowastlowast lowast lowast lowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast lowast lowast lowast lowast lowastlowast lowast lowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

Enterotoxin C (sec) GATAAAAATGAATAAGAGTCGATTTATTTCATGCGTAATTTTGATATTCGCACTTATACT------------------------------------------------------------

AGTTCTTTTTACACCCAACGTATTAGCAGAGAGCCAACCAGACCCTACGCCAGATGAGTT------------------------------------------------------------

GCACAAAGCGAGTAAATTCACTGGTTTGATGGAAAATATGAAAGTTTTATATGATGATCA------------------------------------------------------------

TTATGTATCAGCAACTAAAGTTAAGTCTGTAGATAAATTTTTGGCACATGATTTAATTTA------------------------------------------------------------

TAACATTAGTGATAAAAAACTGAAAAATTATGACAAAGTGAAAACAGAGTTATTAAATGA------------------------------------------------------------

AGGTTTAGCAAAGAAGTACAAAGATGAAGTAGTTGATGTGTATGGATCAAATTACTATGT------------------------------------------------------------

AAACTGCTATTTTTCATCCAAAGATAATGTAGGTAAAGTTACAGGTGGCAAAACTTGTAT------------------------------------------------------------

GTATGGAGGAATAACAAAACATGAAGGAAACCACTTTGATAATGGGAACTTACAAAATGT------------------------------------------------------------

ACTTATAAGAGTTTATGAAAATAAAAGAAACACAATTTCTTTTGAAGTGCAAACTGATAA------------------------------------------------------------

GAAAAGTGTAACAGCTCAAGAACTAGACATAAAAGCTAGGAATTTTTTAATTAATAAAAA------------------------------------------------------------

AAATTTGTATGAGTTTAACAGTTCACCATATGAAACAGGATATATAAAATTTATTGAAAA----TTGTATGAGTTTAACAGTTCACCATATGAAACAGGATATATAAAATTTATTGAAAA

TAACGGCAATACTTTTTGGTATGATATGATGCCTGCACCAGGCGATAAGTTTGACCAATCTAACGGCAATACTTTTTGGTATGATATGATGCCTGCACCAGGCGATAAGTTTGACCAATC

TAAATATTTAATGATGTACAACGACAATAAAACGGTTGATTCTAAAAGTGTGAAGATAGATAAATATTTAATGATGTACAACGACAATAAAACAGTTGATTCTAAAAGTGTGAAGATAGA

AGTCCACCTTACAACAAAGAATGGATAATGTTAATCCGATTTTGATATAAAAAGTGAAAGAGTCCACCTTACAACAAAGAATGGATAATGTAATCCGATTATGGACAAGTGTGCTTTTTT

Enterotoxin C TATTAGATATATTTGAAAGGTAAGTACTTCGGTGCTTGCCTTTTTAGGATGCATATATATEnterotoxin C CNS

Enterotoxin CEnterotoxin C CNS

Enterotoxin CEnterotoxin C CNSEnterotoxin CEnterotoxin C CNSEnterotoxin CEnterotoxin C CNSEnterotoxin CEnterotoxin C CNSEnterotoxin CEnterotoxin C CNSEnterotoxin CEnterotoxin C CNSEnterotoxin CEnterotoxin C CNSEnterotoxin CEnterotoxin C CNS

Enterotoxin CEnterotoxin C CNSEnterotoxin CEnterotoxin C CNS

Enterotoxin CEnterotoxin C CNS

Enterotoxin CEnterotoxin C CNS

Enterotoxin CEnterotoxin C CNS

TTTGTGATCGTCGGCCA-----ACCGATACCGTCGTATTCTTGTACCGCGCAAGGCGAAGlowast lowast lowastlowastlowast lowast lowast lowast lowast lowastlowast lowast lowastlowastlowast lowast lowast lowast lowast

Enterotoxin E (see) Enterotoxin E TGCTTTAGGCGGGGTCTTACAGCATATTATCGTAGAAGATGCACAAGCTGCACGTAAAGCS pasteuri TGCTTTAGGCGCATCGTTACAACATGTCATCGTAGAAGATGAAAAAGATGGTCGTCAGGC

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast lowastlowastlowastlowast lowastlowastlowast lowast lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast lowast lowastlowastlowast lowastlowast lowastlowastlowast lowast lowastlowastlowast

Enterotoxin E ATTAGAATTCTTGAAATCAAAACA-AGCAGGACGTGCTACATTTCTACCATTAAATGTCAS pasteuri

Enterotoxin ES pasteuri

GATACAATT-TTTAAAACAACGTAGTTTAGGACGTGCCACATTCCTACCTTTAAATGTTA lowastlowast lowastlowastlowastlowast lowastlowast lowastlowastlowast lowastlowastlowast lowast lowastlowastlowastlowastlowastlowastlowastlowastlowast lowastlowastlowastlowastlowast lowastlowastlowastlowastlowast lowastlowastlowastlowastlowastlowastlowastlowast lowast

TCAAACCTAGAAATATTTCAACCAAGATATATlowast lowastlowastlowastlowast lowastlowastlowast lowastlowastlowastlowastlowast

Enterotoxin G (seg) Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

---------------------------attaagaaatactt--ccattttaatactcttaGGGGATATTTCCGGGAAACCGGGGCTAATACTGGATAACTTTTCTCTCCGAACGAAGAGA

gaatcaacaactttattatctccg--taaat-----atttaaaaacttatatggaacaaaGATTCAACGATGGTCTTGGTTTTCAATTAAGATGAACCCGCGGCGCTTAGCTGGTGGGAC

aggtactagtt-----------------------------------------cttttttaAGGTCACGGCTCACCCAAGCCCCCATGCGTATACGACCTGGAAGGGTGATCGGCCACATT

ggaaataagtcaaaccaaaaacttgtattgttcttttcagtaaat---------------GGGACTGAGACACAGCACAAACTCCTACGGGAGGCAGCAATAGGGAATCTTCCGCAATGG

------------------------------------------------------------CCGAAAGTCTGACGGAGCAACGCCGCGAGACTGAAGAATGTCTTCGGATCGTAAAACTCT

------------------------tttatatatccagattcaaa-----tgcagaaccatGTTGTTAGGGAAGAACAAGTATCGTTCGAATAGGGCGGTACCTTGACGGTACCTAACCAG

caaactcgtatagctttttttct---------ttagtgagccagtgtcttgctttg----AAAGCTTTGCTAACTACCTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCG

-taatctagttcctgaatagtaaccatattcttatttgtagttattgta--aatccaagtGAATTATTGGGCGTAAAGCGCGCGCATGCGGTCTTTTAAATCTGATGTCAAAGCCCACGG

gattgtct----------attgtcgattgttacctgtacagt------------aattaaGCTCAACCGTGGAGGGTCATTGGAAACTGTAAGACTTGGAGTGCAGAAGAGGATAGTGGA

Figure 3 Continued

BioMed Research International 11

lowast lowast lowast lowastlowast lowastlowast lowastlowastlowast lowast lowastlowast lowast lowast lowast lowast

lowast lowast lowastlowastlowast lowastlowast lowast lowastlowastlowast lowast lowastlowast lowastlowastlowast lowast lowastlowast lowastlowast lowast lowast lowastlowast lowast

lowastlowast lowast lowast lowastlowastlowastlowastlowast lowast lowast lowastlowast lowast lowast lowast lowast lowast lowastlowastlowast

lowastlowast lowast lowastlowast lowastlowastlowast lowastlowast lowastlowast lowast lowast lowast lowast lowastlowast lowastlowast lowast lowast lowast lowastlowast

lowastlowast lowastlowastlowastlowast

lowast lowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowast lowast lowastlowastlowastlowast lowast lowastlowast lowastlowast lowast lowastlowastlowastlowast lowast lowastlowast lowast lowast lowastlowastlowastlowastlowastlowastlowast

lowastlowast lowast lowast lowast lowast lowastlowastlowast lowastlowast lowastlowast lowast lowastlowastlowastlowastlowastlowastlowastlowast lowastlowastlowast

lowast lowast lowast lowast lowast lowast lowastlowast lowastlowast lowast lowast lowastlowastlowast lowast lowast lowastlowastlowast lowast lowast

lowastlowast lowastlowastlowastlowastlowastlowast lowastlowast lowastlowastlowast lowastlowast lowast lowastlowast lowastlowast lowastlowastlowast lowast lowast

lowastlowast lowast lowastlowastlowast lowast lowastlowastlowastlowastlowast lowast lowast lowast lowastlowastlowast lowast lowastlowast lowast

lowastlowast lowast lowast lowast lowast lowastlowast lowast lowastlowast lowast lowastlowast lowast lowastlowast lowastlowastlowastlowast lowast lowast lowastlowast

lowast lowastlowastlowast lowast lowastlowast lowast lowast lowastlowast lowast lowastlowast lowast lowastlowastlowastlowastlowastlowast lowast

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

tttatctctttcattttctgaactattaaa----tgtaagaccacc---atacatacaacATTCCACGTGTAACGGTGAAAATGAGTAGAGATGTGGAGGAACACCAGTGGAGAAACGCG

aacctccaaaattttggtttatatccggttc-----------agatttaggtattatacaACTCTCTGGTCTGTTACTGACGCTGAGGCGCGAAAGCGTGGGAGCAAACAGGATTATATA

tgtataaaaatatggaacgccaaaaatgtctacttttttatctttataattgttagctaaCCCCTGGTAGTCCAAGCCGTAAACGATGAGTGCTAGTGTTACAGGGT------------T

ttctgtattttctaattcagttttaacctcattataactcttatactcaattggaaaaatTCCGCCCTTTACTGCTGCAGCTAACG---CATTAG-GCACTCCGCCTGGGGAGTACAACT

taaatcatgagataaaaactgtctagaattaataactcctcttccttcaacaggtggagaGCAA------GGTTGAAACTCAAAGCA----TTGACCGGGGGCC---CGATAAGCTGAGG

cgtataaagattcattacat----tacccatagttcccttattgtttttataatcacttaTACATGGTGTTTAATTCCAAACCACGCCAAGAACCCTCACGAGGTCTTGACATCCTCTGG

ctttattt----------------------------------------------------ACCTCCCTAGAGAATAAGGGCTTCCCCGTCGGGGATGAAATGAACAGGTGGTGCATGGAT

-------agttcgtc---AGTCTTCAGCTCGTGCAT

Enterotoxin H (seh)

------------------------------------------------------------ATCTATGAATATAAATCTTTTAATATTGGTTTAATATAAATATTCCATTTCTAGAAACCT

------------------------------------------------------------ATATTTTATGTTTGTATAAATTCATAAATTGTTCGAAATTTATATATTAAAAGTTGTTTT

------------------------------------------------------------TGTTGTTTTATAATTAAGTTAATGAAATATATTGAGGAGTTTTAAAATGATTAATAAAAT

------------------------------------------------------------TAAAATATTATTTTCGTTTTTAGCATTATTACTTTCATTCACATCATATGCGAAAGCAGA

------------ -----------TTACGATTTAGCTTTAGCTAATGCATATGGTCAATAAGATTTACACGATAAAAGTGAGTTAACAGATTTAGCTTTAGCTAATGCATATGGTCAATA

TAATCACCCATTCATTAAAGAAAATATTAAGAGTGATGAAATAAGTGGAGAAAAAGATTTTAATCACCCATTCATTAAAGAAAATATTAAGAGTGATGAAATAAGTGGAGAAAAAGATTT

AATATTTAGAAATCAAGGTGATAGTGGCAATGATTTGAGAGTAAAGTTTGCAACTGCTGAAATATTTAGAAATCAAGGTGATAGTGGCAATGATTTGAGAGTAAAGTTTGCAACTGCTGA

TTTAGCTCAGAAGTTTAAAAATAAAAATGTAGATATATATGGGGCATCTTTTTATTATAATTTAGCTCAGAAGTTTAAAAATAAAAATGTAGATATATATGGGGCATCTTTTTATTATAA

GTGTGAAAAAATAAATGAAAATATTTCTGAATGTCTATATGGAGGTACAACACTAAATAAGTGTGAAAAAATAAGTGAAAATATTTCTGAATGTCTATATGGAGGTACAACACTAAATAG

TGAAAAATTGGCACAGGAAAGGGTGATTGGTGCTAATGTTTGGGTAGATGGTATTCAAAATGAAAAATTGGCACAGGAAAGGGTGATTGGTGCTAATGTTTGGGTAGATGGTATTCAAAA

AGAAACAGAATTAATACGAACAAATAAGAAAAATGTGACATTGCAAGAATTAGATATAAAAGAAACAGAATTAATACGAACAAATAAGAAAAATGTGACATTGCAAGAATTAGATATAAA

Enterotoxin H CNS GATCAGAAAAATATTGTCCGATAAATATAAAATTTATTATAAAGACAGCGAAATAAGGG-Enterotoxin H

Enterotoxin H CNSEnterotoxin H

Enterotoxin H CNSEnterotoxin HEnterotoxin H CNSEnterotoxin HEnterotoxin H CNSEnterotoxin HEnterotoxin H CNSEnterotoxin HEnterotoxin H CNSEnterotoxin H

Enterotoxin H CNSEnterotoxin H

Enterotoxin H CNSEnterotoxin H

Enterotoxin H CNSEnterotoxin H

Enterotoxin H CNSEnterotoxin H

Enterotoxin H CNSEnterotoxin H

GATCAGAAAAATATTGTCCGATAAATATAAAATTTATTATAAAGACAGCGAAATAAGTAA

Enterotoxin N (seln) TGATTTAGCTTTAGCTAATGCATATGGTCAATAT-AATCACCCATTCATTAAAGAAAA-----TAGATATTTATGGACTGTATTATGGAAATAAATGTGTAGGCTTAACTGAAGAAAAAA

TATTAAGAGTGATGAAATAAGTGGAGAAAAAGATTTAATATTTAGAAATCAAGGTGATAGCATCATGCTTATACGGAGGAGTTAC------GA--TATATGATGGAAATCAATTAGATGA

TGGCAATGATTTGAGAGTAAAG--------------TTTGCAA-CTGCTGATTTAGCTCAAGAGAGAGTTATAGGCGTTAATGTATTTAAAGATGGTATCCAACAAGAAGGTTTTGTTAT

GAAGTTTAAAAATAAAAATGTAGATATATATGGGGCATCTTTTTATTATAAGTGTGAAAAAAAAACTAAAAAGGCTAAAGTAACAGTACAA------GAATTAGATACTAAAGTTCGATT

AATAAGTGAAAATATTTCTGAATGTCTATATGGAGGTACAACACTAAATAATGAAAAATT------------TAAATTAGAAAATTTATATAAAATATACAATAAAGATACCGGTAACAT

Enterotoxin N CNSEnterotoxin N

Enterotoxin N CNSEnterotoxin N

Enterotoxin N CNSEnterotoxin N

Enterotoxin N CNSEnterotoxin N

Enterotoxin N CNSEnterotoxin N

Figure 3 Continued

12 BioMed Research International

lowastlowastlowast lowastlowast lowastlowast lowastlowast lowast lowastlowast lowastlowastlowast lowastlowast lowast lowastlowastlowastlowast

lowastlowast lowast lowastlowast lowast lowast lowastlowast lowastlowastlowastlowastlowast lowast lowast lowast lowastlowast lowast lowast lowastlowast lowast lowast

lowast lowast

lowast lowastlowast lowast lowastlowast lowast lowast lowast lowast lowast lowast lowast lowastlowast lowast lowastlowastlowastlowast lowast

lowast lowastlowast lowast lowast lowastlowast lowast lowastlowastlowast lowast lowastlowast lowast lowastlowast lowastlowast lowast lowast

lowastlowastlowast lowast lowast lowastlowast lowastlowast lowast lowast lowast

lowast lowast lowast lowastlowastlowast lowastlowastlowast lowastlowast lowast lowastlowastlowastlowastlowast lowastlowastlowast lowast

lowastlowast lowast lowast lowastlowast lowastlowastlowast lowastlowast lowast lowast lowastlowastlowastlowast lowastlowast

lowast lowast lowast lowastlowast lowast lowastlowast lowastlowastlowastlowastlowastlowast lowastlowast lowast lowastlowast lowastlowastlowast

lowast lowast lowast lowast lowastlowast lowastlowastlowastlowast lowast lowast lowastlowastlowast lowast lowast lowast lowast lowast

lowast lowast lowast lowast lowastlowastlowast lowast lowast lowastlowast lowastlowast lowast lowastlowastlowast lowast lowastlowastlowast lowast

lowast lowast lowastlowastlowast lowast lowastlowast lowastlowastlowast lowastlowastlowastlowastlowast lowastlowast lowast lowastlowastlowast lowast lowast lowastlowastlowast

lowastlowastlowast lowast lowastlowast lowastlowast lowastlowast lowast lowast lowast lowast lowastlowast lowastlowast lowastlowast lowast lowast

lowast lowast lowast lowast lowastlowast lowast lowast lowastlowast lowastlowast lowast lowastlowastlowast lowastlowastlowastlowast lowast lowastlowastlowastlowastlowast lowast

lowastlowast lowast lowast lowastlowastlowast lowastlowast lowast lowastlowast lowast lowastlowast lowastlowast lowastlowast lowast lowast lowast

lowastlowastlowastlowastlowast lowastlowast lowastlowast lowast lowastlowast lowast lowast lowast lowast lowastlowast lowastlowast

lowast lowast lowastlowast lowastlowast lowastlowastlowastlowastlowastlowast lowastlowast lowast lowast lowast lowast lowast lowast lowast lowast lowast lowast lowast

lowastlowast lowastlowast lowast lowastlowastlowastlowast lowast lowast lowastlowastlowast lowastlowast lowast lowastlowastlowast lowast

lowastlowastlowastlowast lowastlowast lowast lowast lowast lowast lowastlowast lowast lowast lowast lowastlowastlowastlowastlowast lowastlowast lowastlowastlowast lowast

lowast lowastlowastlowast lowastlowastlowastlowastlowast lowast lowast lowast lowastlowast lowast lowast lowastlowast lowast lowastlowastlowast lowastlowast lowast

lowastlowast lowast lowast lowast lowastlowast lowast lowastlowast lowastlowast lowastlowast lowast lowastlowastlowastlowast

ATTAATACGAACAAATAAGAAAAATGTGACATTGCAAGAATTAGATATAAAGATCAGAAA-TTAT----------TATGATTTATATAAC--------------ATAAAAGGTTCAG---

AATATTGTCCGATAAATATAAAATTTATTATAAAGACAGCGAAATAAGGTAAAAGG------TAGGAGCAGAGTTTTTTCAATTTTATAGTGATAATAGAACAGTTAGCTCATCTAATTA

Enterotoxin N CNS -TAAA---Enterotoxin N

lowast lowastlowastlowastlowastlowast lowastlowast lowast lowast lowast lowastlowast lowast lowastlowastlowastlowastlowastlowast lowastlowast lowastlowast

GGCACAGGAAAGGGTGATTGGTGCTAATGTTTGGGTAGATGGTATTCAAAAAGAAACAGAACAA----AAAGGATGCATTTTCTTT-----CATTCTAATAATCATCAAAATCAATCATT

Enterotoxin N CNSEnterotoxin N

Enterotoxin N CNSEnterotoxin N

Enterotoxin N CNSEnterotoxin N

TCATATCG

Enterotoxin M (selm)

ttgctattcgcaaaatcatatcgcaaccgctgatgtcggagtt--ttgaatcttaggaacEnterotoxin M CNSEnterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

--------------AGTATGTTTAAATCCCCGCTCACAGTTATGTTTAACTCTTCCTTTC

tattatggta-gctatccaattgaagaccaccaaagtattaatcctgaaaataatcatctTTTTCACACCGCCCCGTCGGGTGTTGCGCACGGGGTTGTTTAACCCCCCCGGAAAAAATT

ttcgcatcaattagttttttctatggataattcgacagtaacagctgaatttaagaacgtEnterotoxin M CNS

Enterotoxin M CNS

TTCCCTCCCTCCTCCTTACTC---------------CCT----------------CCCCT

tgatgatgtaaaggaattcaaaaatcatgctgtagatgtatatggtctaagttatagtggEnterotoxin M CNS TTTTCCTACAAATCCTTTCTCCCCTCCCGGAGTAGAAGTAAA------------------

atattgtttgaaaaacaaatatatatacggtggagttacattagcaggtgat-tatttagEnterotoxin M CNS -------------------TAAAACAATACCCCTACCACCTTAACAATTTTTTTATTGAG

agaaatctagacgtattcct-attaatctttgggttaatggagaacatcaaactatatctEnterotoxin M CNS CCCCTCCCCCCCCTTCTCATTTTTTCTCTTTGAC-----------GTTCCCCCCATTTCT

actgacaaagtatcaactaataaaa-agttagtaacagctcaagaaattgatactaaattEnterotoxin M CNS GCCAATGCTTAACCCACCCTTAAAGGCCATCGGCACATATTAGACACTCC-----CCACC

aagaagatatctacaagaagaatataatatttatggctttaatgatac---aaataaaggEnterotoxin M CNS CGCCCCCTCTTTTTATTAAGGACACAACTCTTTTTATTTTCCCCCAAGAACTAATTCTGA

a-agaaattatggtaataagtcaaaatttagttctgga---------tttaatgcaggaaEnterotoxin M CNS AAAAAAACGTTCCGCTTAGTTCAGAATTTCCTTTGAGCCTTTTATCCTTTTGTACGCGAA

aaatattatttca--------tttgaatgatggttcatcattttcttatgacttatttga

Enterotoxin M CNS AAACCATCAGCCATTGAATCAGTTATCCCATTGCCCTAACTAACCCAATATCTAATCTAA

tactggaacaggacaagctgaaagtttctt-aaaaatatataatgacaacaaaactgtcgEnterotoxin M CNS TTTTCGCCTACGAACGAATAATAGCTTGTGGCGAAAAATATCCAGGATTATCTACTGTAG

aaact--gaaaaattccatttagatgtagaaatatcttataaggacgaaagttgaagtatEnterotoxin M CNS CGCCTCGCCAGACGTCCTTTGAATTGA--ATATGACTCCTACTCCCCCCCGCACCGTCAA

ttcaatcataacttagtaaaggaaatgccatgaaaaaatttaaatatagtttt---atatEnterotoxin M CNS TTCAAACA---------------GGTGGAAGTTAATACTGAATAGTAGGTCTTCTTCCCG

tagtttttatattactttttaacattaaagatcttacgtatgct---caaggtgatattgEnterotoxin M CNS TGCTATTCTTAGAACTTTTAAATTATGTAAACCCAATTTTTAGTGCACTCTTTCTCCCTC

gtg-taggtaacttaagaaatttctatacaaaacatgattatatagatttaaaaggcgtcEnterotoxin M CNS GTTACTCTTAGATGGAGAAGCGTTAAGACAGAAGCTTTGTAT-------------TCCCA

acagataaaaa-cctacctattgcaaatcaacttgaattttcaac-----aggt----acEnterotoxin M CNS GCAGAGTGCAACCTTTCACAATGAAGTTTTTATCTAATTTACACGCCTTTAGGCTCCAAA

caatgatttgatctcagaatctaataattgggacgaaataagtaaatttaaaggaaagaaEnterotoxin M CNS TACGTATTAAATCTCCTCCTGT--TTATCTGACCCTAAG-ATAAAACACGAAAAATTATG

actggatatttttggcattgattataatggtccttgtaaatctaaatacatgtatggaggEnterotoxin M CNS CGTGCCCACTCATCCTAT-------TACTTTCATTATATCTATAAAA-------------

Figure 3 Alignment of the enterotoxin gene sequences found in coagulase-negative staphylococci (CNS) and in coagulase-positivestaphylococci (CPS)

BioMed Research International 13

Enterotoxin (seg) S aureus AY1587031

Enterotoxin (sec) CNS

Enterotoxin (see) S pasteuri NC0227371

Enterotoxin (see) CNS

Enterotoxin (seh) CNS

Enterotoxin (selm) S aureus AF2857601

Enterotoxin (seln) CNS

Enterotoxin (seh) S aureus AJ9375481

Enterotoxin (seln) S aureus AY1587031

Enterotoxin (sec) S aureus X516611

Enterotoxin (selm) CNS

Enterotoxin (seg) CNS

150 100

100

100

100

100

99

98

96

97

72

50 0

Figure 4 Phylogenetic tree generated from the multiple alignments of the enterotoxin sequences of CNS and CPS strains using the ClustalX20 software package The phylogenetic tree was constructed using the Mega 60 software and the unweighted pair group method (UPGMA)

MAR indices varying from 066 to 046 and the remaining04 strains showed MAR indices le026 (Table 3)

The 14 MRCNS strains were resistant to 120573-lactams(oxacillin penicillin andor cefoxitin) and to vancomycincorresponding to 73 of the total CNS strains identifiedwhile 09 strains (64) showed resistance to tetracycline andgentamicin 08 strains (57) were resistant to neomycinerythromycin and chloramphenicol 07 strains (50) wereresistant to sulfamethoprim 05 strains (36) were resistantto linezolid 03 strains (21) were resistant to rifampicin 02strains (14) were resistant to ciprofloxacin and cefepimeand 01 strain (7) was resistant to clindamycin (Table 3)

The multiresistance of CNS strains demonstrated hereinis consistent with previous studies on coagulase-negativeand coagulase-positive staphylococci that have found severalresistant and multiresistant Staphylococcus aureus strains inraw milk meat and fermented meat products [43]

Surprisingly the resistance to chloramphenicol is verysimilar to that estimated for MRCNS strains isolated fromhuman clinical samples Indeed there is no direct correla-tion between the researched features and the origin of thestaphylococci strains since different virulence factors arewidespread such as antibiotic resistance [44] reinforcing thefact that the ability of foodmatrices strains to produce SE andSEl and their multiresistance character must be consideredwhen evaluating the safety hazards of food poisoning

S epidermidis KF6005891 and S hominis JX5199881showed the highestMAR indexes (093 and 080 resp)Thesestrains found in artisanal salami are homologous to strainsisolated from human skin microbiota and should be carefullyconsidered among the potential pathogenic staphylococcifound in food from animal origins which can be caused bycontamination by poor hygienic conditions during salamimanufacturing

Depending on the conditions some species of coagulase-negative staphylococci can present health risks since theyhave shown resistance to antibiotics of therapeutic impor-tance such as beta lactams [15] However multiresistantstrains like S carnosusKJ18620021 and S xylosusCP0072081and CP0087241 were intentionally introduced in the foodmatrix since they are part of the culture starter used inItalian-salami manufacturing As previously discussed foodcan be a reservoir of multiresistant microorganisms that canspread by the consumption of an apparently safe food Dueto the intensive and indiscriminate use of antibiotics forhuman and veterinarian therapeutic purposes multiresistantstaphylococci strains are being selected and reproduced infood matrices [45]

The cefoxitin and oxacillin disk diffusion tests are rec-ommended for determining the resistance and susceptibilitybreakpoint for MRSA surveillance cultures [46] In thisstudy there was a good correlation between the disc testzone diameters for oxacillin and cefoxitin (Table 3) TheMICs for ampicillin andmethicillinoxacillincefoxitin for allsusceptible CNS strainswere determined by the diffusion disctest using 003 to 2mgmLminus1 of each antimicrobial agent

S saprophyticus HF9372521 showed an MIC of003mgmLminus1 formethicillin strain S carnosusKJ8620021 Sxylosus strains CP0087241 and CP0072081 S saprophyticusJX4901221 S hominis JX5199881 and S succinusKC3298241 were resistant to 006mgmLminus1 and two strainsS saprophyticus KJ6991511 and S saprophyticus KJ0046231showed an MIC of 05mgmLminus1 (Table 4) Two of themethicillin-resistant strains S saprophyticus KJ0046231 andS xylosus CP0072081 harbor no enterotoxin gene

Five strains S saprophyticus JX4901221 S carnosusKJ8620021 S xylosusCP0087241 S xylosusKF1980801 andS saprophyticus KJ0046231 showed an MIC for ampicillin

14 BioMed Research International

Table 3 Multiple resistance to antimicrobial as found in CNS strains from salami

Salami origin CNS strains Antimicrobial agent resistance Multiple antimicrobialresistance (MAR) indexlowast

Commercial Staphylococcus spp KF1354451 CIP CLO CPM GEN NEO OXA PENSXT TET and VAN 066

Commercial S carnosus KJ8620021 CIP GEN LZD NEO OXA SXT andTET 046

Commercial S saprophyticus AB6977171 CFO CLO ERI PEN OXA TET andVAN 046

Commercial S saprophyticus EU4309921 CFO CLO ERI GEN LZD NEO OXAPEN SXT TET and VAN 080

Commercial S saprophyticus JX4901221 CFO CLO ERI GEN NEO PEN OXASXT TET and VAN 066

Commercial S saprophyticus KJ0046231 CFO OXA and PEN 020

Commercial S sciuri JX9664361 CFO CLO GEN NEO OXA PEN andTET 046

Commercial S xylosus CP0072081 OXA LZD PEN and VAN 026

Commercial S xylosus CP0087241 CFO CLO ERI GEN NEO OXA PENTET and VAN 060

Artisanal S epidermidis KF6005891 CFO CLI CLO CPM ERI GEN LZDNEO OXA PEN RIF SXT and TET 093

Artisanal S hominis JX5199881 CIP CPM CPO ERI GEN LZD NEOOXA PEN RIF SXT and VAN 080

Artisanal S saprophyticusHF9372521 CFO OXA PEN and SXT 026Artisanal S saprophyticus KJ6991511 OXA PEN and VAN 020

Artisanal S succinus KC3298241 CFO CLO ERI GEN OXA PEN RIFSXT TET and VAN 066

Artisanal S xylosus KF1980801 CFO CLO ERI GEN NEO OXA PENTET and VAN 060

lowastTheMAR index of an isolate is defined as 119886119887 where 119886 represents the number of antimicrobials to which the isolate was resistant and 119887 represents the numberof antimicrobials to which the isolate was subjectedS aureus strains ATCC WB81 (sea) ATCC 13563 (sed) and ATCC 27664 (see) showing a MAR index of 05 and S aureus strains ATCC14458 (seb) andATCCWB72 (sec) and S xylosus ATCC 29971 showing a MAR index of 03 were used as reference strainsCPM cefepime CFO cefoxitin CLO chloramphenicol CIP ciprofloxacin CLI clindamycin ERI erythromycin GEN gentamycin NEO neomycin LZDlinezolid RIF rifampicin TET tetracycline OXA oxacillin PEN penicillin SXT sulfamethoprim and VAN vancomycin

Table 4 Minimal inhibitory concentration (MIC) of compounds used in antimicrobial therapy against staphylococci infections

Salami origin GenBank accession number and similarity () MIC mgmLminus1

Methicillin Ampicillin Vancomycin Linezolid

Commercial

S carnosus KJ8620021 006 003 mdash mdashS xylosus CP0087241 006 003 006 mdash

S saprophyticus JX4901221 006 003 05 mdashS saprophyticus AB6977171 mdash mdash 05 mdashS saprophyticus EU4309921 mdash mdash 025 0125

S succinus KC3298241 006 025 003 mdash

Artisanal

S hominis JX5199881 006 05 mdash mdashS saprophyticus HF9372521 003 mdash mdash mdash

S xylosus KF1980801 003 05 mdashS saprophyticus KJ6991511 05 025 003 mdashS saprophyticus KJ0046231 05 003 003 mdash

S xylosus CP0072081 006 mdash mdash 025Strains S epidermidis KF6005891 and Staphylococcus spp KF1354451 were not susceptible to the antimicrobial concentrations tested in the present study

BioMed Research International 15

of 003mgmLminus1 two strains S succinus KC3298241 and Ssaprophyticus KJ6991511 showed an MIC of 025mgmLminus1and S hominis JX5199881 showed an MIC of 05mgmLminus1(Table 4) S saprophyticus KJ0046231 also showed an MICof 003mgmLminus1 but does not harbor enterotoxin genes It isimportant to note that most of the CNS strains that showedresistance to ampicillin were also resistant to methicillin ashighlighted by the MIC tests

Five strains S saprophyticus HF9372521 S saprophyti-cus KJ6991511 S saprophyticus JX4901221 S saprophyticusKJ0046231 and S succinus KC3298241 showed an MICfor vancomycin of 003mgmLminus1 one strain S xylosusCP0087241 showed an MIC of 006mgmLminus1 one strain Ssaprophyticus EU4309921 showed an MIC of 025mgmLminus1and two strains S xylosus KF1980801 and S saprophyticusAB6977171 showed an MIC of 05mgmLminus1 (Table 4)

Additionally five CNS strains S xylosus CP0087241S saprophyticus JX 4901221 S succinus KC3298241 Ssaprophyticus KJ6991511 and S saprophyticus KJ0046231showed resistance tomethicillin as demonstrated by theMICdeterminations (Table 4)

Previous studies have demonstrated that linezolid isactive against Gram-positive bacteria including methicillin-resistant staphylococci [47] In the present study 05 strainsshowed linezolid resistance in the disc diffusion test but itwas impossible to establish MIC values for S epidermidisKF6005891 Staphylococcus hominis JX5199881 and Staphy-lococcus carnosus KJ8620021The remaining strains S sapro-phyticus EU4309921 and S xylosus CP0072081 presentedan MIC for linezolid of 0125mgmLminus1 and 025mgmLminus1respectively

The MIC of 003mgmLminus1 for penicillin is in accordancewith the previous values estimated for resistant S aureusstrains found in several food matrices such as meat dairyproducts and ready-to-eat food [48]

There is still a lack of information on the antimicro-bial resistance of staphylococci strains from food matricesalthough they are the most worrisome vehicles of dissemi-nation of antibiotic-resistant pathogens [49]

The high resistance found for salami staphylococci strainscan be ascribed to their inappropriate use as growth promot-ers of antimicrobial agents like oxacillin vancomycin chlo-ramphenicol neomycin and erythromycin which are com-monly used in veterinary medicine to treat infections [50]Antimicrobial therapy of infections staphylococci is basedon results of susceptibility tests in vitro [51] Methicillin-resistant coagulase-negative Staphylococcus spp found inready-to-eat products such as meats fish and dairy productsalso offer risks and although they are not classical foodpoisoning bacteria their presence in food offers significantrisks to public health due to the possible spread of antibioticresistance [52]

The standardization of salami quality should includediagnostic methods to screen and quantify the presence ofclassical and newly described enterotoxins directly in thefood matrices as a routine procedure to be conducted by themeat product industry in Brazil

The safety of salami consumption could also be enhancedby the inclusion of amicrobial barrier such as the inclusion ofprobiotic strains producing natural antibiotics or competitiveflora or even the addition of natural bioagents against spoilageor pathogenic microorganisms

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors acknowledge the financial support fromFundacao Carlos Chagas Filho de Amparo a Pesquisa doEstado do Rio de Janeiro (FAPERJ Rio de Janeiro Brazil)Conselho Nacional de Desenvolvimento Cientıfico eTecnologico (CNPq Brasılia Brazil) and Coordenacao deAperfeicoamento de Pessoal de Nıvel Superior (CAPESBrasılia Brazil)

References

[1] ICMSF International Comission on Microbiological Specifica-tions for FoodsMicroorganisms in FoodsTheir Significance andMethods of Enumerations University of Toronto Press TorontoCanada 3rd edition 1983

[2] J F Veras L S Carmo L C Tong et al ldquoA study of theenterotoxigenicity of coagulase-negative and coagulase-positivestaphylococcal isolates from food poisoning outbreaks inMinasGerais Brazilrdquo International Journal of Infectious Diseases vol12 no 4 pp 410ndash415 2008

[3] K Omoe D-L Hu H Takahashi-Omoe A Nakane and KShinagawa ldquoComprehensive analysis of classical and newlydescribed staphylococcal superantigenic toxin genes in Staphy-lococcus aureus isolatesrdquo FEMS Microbiology Letters vol 246no 2 pp 191ndash198 2005

[4] J Kadariya T C Smith and D Thapaliya ldquoStaphylococcusaureus and staphylococcal food-borne disease an ongoing chal-lenge in public healthrdquoBioMedResearch International vol 2014Article ID 827965 9 pages 2014

[5] KOmoeD-LHuH KOno et al ldquoEmetic potentials of newlyidentified staphylococcal enterotoxin-like toxinsrdquo Infection andImmunity vol 81 no 10 pp 3627ndash3631 2013

[6] J K McCormick J M Yarwood and P M SchlievertldquoToxic shock syndrome and bacterial superantigens an updaterdquoAnnual Review of Microbiology vol 55 pp 77ndash104 2001

[7] T Baba F Takeuchi M Kuroda et al ldquoGenome and virulencedeterminants of high virulence community-acquired MRSArdquoThe Lancet vol 359 no 9320 pp 1819ndash1827 2002

[8] Associacao Brasileira de Industria Produtora e Exportadorada Carne Suınas (ABIPECS) ldquoStatistics slaughter ranking2000ndash2014rdquo 2014 httpabpa-brcombrsetoressuinoculturapublicacoesrelatorios-anuais

[9] N Zdolec M Hadziosmanovic L Kozacinski et al ldquoMicrobialand physicochemical succession in fermented sausages pro-duced with bacteriocinogenic culture of Lactobacillus sakei andsemi-purified bacteriocin mesenterocin Yrdquo Meat Science vol80 no 2 pp 480ndash487 2008

16 BioMed Research International

[10] F Ravyts L D Vuyst and F Leroy ldquoBacterial diversity andfunctionalities in food fermentationsrdquo Engineering in Life Sci-ences vol 12 no 4 pp 356ndash367 2012

[11] TOhlsson andN BengtssonTheHurdle ConceptMinimal Pro-cessing Technologies in the Food IndustryWoodhead Publishing2002

[12] S Bernardi B B Golineli and C J Contreras-CastilloldquoRevisao aspectos da aplicacao de culturas starter na producaode embutidos carneos fermentadosrdquo Brazilian Journal of FoodTechnology vol 13 no 2 pp 133ndash140 2010

[13] F Irlinger ldquoSafety assessment of dairy microorganismscoagulase-negative staphylococcirdquo International Journal of FoodMicrobiology vol 126 no 3 pp 302ndash310 2008

[14] B Ghebremedhin F Layer W Konig and B Konig ldquoGeneticclassification and distinguishing of Staphylococcus species basedon different partial gap 16S rRNA hsp60 rpoB sodA and tufgene sequencesrdquo Journal of Clinical Microbiology vol 46 no 3pp 1019ndash1025 2008

[15] I Babic K Markov D Kovacevic et al ldquoIdentification andcharacterization of potential autochthonous starter culturesfrom aCroatian lsquobrandrsquo product lsquoslavonski kulenrsquordquoMeat Sciencevol 88 no 3 pp 517ndash524 2011

[16] MMehrotra GWang andWM Johnson ldquoMultiplex PCR forthe detection of genes for Staphylococcus aureus enterotoxinsexfoliative toxins toxic shock syndrome toxin and methicillinresistancerdquo Journal of Clinical Microbiology vol 38 no 3 pp1032ndash1041 2000

[17] WM Johnson SD Tyler E P Ewan F EAshtonDR Pollardand K R Rozee ldquoDetection of genes for enterotoxins exfolia-tive toxins and toxic shock syndrome toxin 1 in Staphylococcusaureus by the polymerase chain reactionrdquo Journal of ClinicalMicrobiology vol 29 no 3 pp 426ndash430 1991

[18] J Mclauchlin G L Narayanan VMithani andG OrsquoNeill ldquoThedetection of enterotoxins and toxic shock syndrome toxin genesin Staphylococcus aureus by polymerase chain reactionrdquo Journalof Food Protection vol 63 no 4 pp 479ndash488 2000

[19] J P Rosec and O Gigaud ldquoStaphylococcal enterotoxin genes ofclassical and new types detected by PCR in Francerdquo Interna-tional Journal of Food Microbiology vol 77 no 1-2 pp 61ndash702002

[20] K Omoe D-L Hu H Takahashi-Omoe A Nakane andK Shinagawa ldquoIdentification and characterization of a newstaphylococcal enterotoxin-related putative toxin encoded bytwo kinds of plasmidsrdquo Infection and Immunity vol 71 no 10pp 6088ndash6094 2003

[21] N Sergeev D Volokhov V Chizhikov and A Rasooly ldquoSimul-taneous analysis of multiple staphylococcal enterotoxin genesby an oligonucleotide microarray assayrdquo Journal of ClinicalMicrobiology vol 42 no 5 pp 2134ndash2143 2004

[22] S Holtfreter D Grumann M Schmudde et al ldquoClonal distri-bution of superantigen genes in clinical Staphylococcus aureusisolatesrdquo Journal of Clinical Microbiology vol 45 no 8 pp2669ndash2680 2007

[23] W J Mason J S Blevins K Beenken NWibowo N Ojha andM S Smeltzer ldquoMultiplex PCR protocol for the diagnosis ofstaphylococcal infectionrdquo Journal of Clinical Microbiology vol39 no 9 pp 3332ndash3338 2001

[24] E Rahimi and H G Safai ldquoDetection of classical enterotoxinsof Staphylococcus aureus strains isolated frombovine subclinicalmastitis in Isfahan Iranrdquo Veterinary Microbiology vol 141 no3-4 pp 393ndash394 2010

[25] M A Larkin G Blackshields N P Brown et al ldquoClustalW andClustal X version 20rdquo Bioinformatics vol 23 no 21 pp 2947ndash2948 2007

[26] P H A Sneath and R R Sokal Unweighted Pair Group Methodwith Arithmetic Mean Numerical Taxonomy W H FreemanSan Francisco Calif USA 1973

[27] NCCLS ldquoPerformance standards for antimicrobial susceptibil-ity testing 14th informational supplementrdquo Tech Rep M 100-S14 NCCLS 2004

[28] S Corbiere Morot-Bizot S Leroy and R Talon ldquoMonitoringof staphylococcal starters in two French processing plantsmanufacturing dry fermented sausagesrdquo Journal of AppliedMicrobiology vol 102 no 1 pp 238ndash244 2007

[29] R Talon S Leroy I Lebert et al ldquoSafety improvement andpreservation of typical sensory qualities of traditional dryfermented sausages using autochthonous starter culturesrdquo Inter-national Journal of Food Microbiology vol 126 no 1-2 pp 227ndash234 2008

[30] G Mauriello A Casaburi G Blaiotta and F Villani ldquoIsolationand technological properties of coagulase negative staphylo-cocci from fermented sausages of Southern ItalyrdquoMeat Sciencevol 67 no 1 pp 149ndash158 2004

[31] M Janssens N Myter L De Vuyst and F Leroy ldquoSpeciesdiversity and metabolic impact of the microbiota are low inspontaneously acidified Belgian sausages with an added starterculture of Staphylococcus carnosusrdquo Food Microbiology vol 29no 2 pp 167ndash177 2012

[32] G Blaiotta D Ercolini G Mauriello G Salzano and F VillanildquoRapid and reliable identification of Staphylococcus equorum bya species-specific PCR assay targeting the sodA generdquo Systematicand Applied Microbiology vol 27 no 6 pp 696ndash702 2004

[33] L Kozacinski E Drosinos F Caklovica L Cocolin J Gasparik-Reichardt and S Veskovic ldquoInvestigation of microbial associa-tion of traditionally fermented sausagesrdquo Food Technology andBiotechnology vol 46 no 1 pp 93ndash106 2008

[34] S Leroy P Giammarinaro J-P Chacornac I Lebert and RTalon ldquoBiodiversity of indigenous staphylococci of naturallyfermented dry sausages and manufacturing environments ofsmall-scale processing unitsrdquo Food Microbiology vol 27 no 2pp 294ndash301 2010

[35] E Coton M-H Desmonts S Leroy et al ldquoBiodiversity ofCoagulase-Negative Staphylococci in French cheeses dry fer-mented sausages processing environments and clinical sam-plesrdquo International Journal of Food Microbiology vol 137 no 2-3 pp 221ndash229 2010

[36] S Hosseinzadeh and H D Saei ldquoStaphylococcal species associ-ated with bovinemastitis in the NorthWest of Iran emerging ofcoagulase-negative staphylococcirdquo International Journal of Vet-erinary Science and Medicine vol 2 no 1 pp 27ndash34 2014

[37] A Piette and G Verschraegen ldquoRole of coagulase-negativestaphylococci in human diseaserdquo Veterinary Microbiology vol134 no 1-2 pp 45ndash54 2009

[38] T M Moura F S Campos P A DrsquoAzevedo et al ldquoPrevalenceof enterotoxin-encoding genes and antimicrobial resistancein coagulase-negative and coagulase-positive Staphylococcusisolates from black pudding in Southern Brazilrdquo Revista daSociedade Brasileira deMedicina Tropical vol 45 no 5 pp 579ndash585 2012

[39] H I Suzuki T Lefebure P P Bitar and M J StanhopeldquoComparative genomic analysis of the genus Staphylococcusincluding Staphylococcus aureus and its newly described sister

BioMed Research International 17

species Staphylococcus simiaerdquo BMC Genomics vol 13 article38 2012

[40] R P Lamers G Muthukrishnan T A Castoe S Tafur A MCole and C L Parkinson ldquoPhylogenetic relationships amongStaphylococcus species and refinement of cluster groups basedon multilocus datardquo BMC Evolutionary Biology vol 12 article171 2012

[41] I V Pinchuk E J Beswick and V E Reyes ldquoStaphylococcalenterotoxinsrdquo Toxins vol 2 no 8 pp 2177ndash2197 2010

[42] S R Monday and G A Bohach ldquoGenes encoding staphylo-coccal enterotoxins G and I are linked and separated by DNArelated to other staphylococcal enterotoxinsrdquo Journal of NaturalToxins vol 10 no 1 pp 1ndash8 2001

[43] D Y Thomas S Jarraud B Lemercier et al ldquoStaphylococcalenterotoxin-like toxins U2 and V two new staphylococcalsuperantigens arising from recombination within the entero-toxin gene clusterrdquo Infection and Immunity vol 74 pp 4724ndash4734 2006

[44] N Indrawattana O Sungkhachat N Sookrung et al ldquoStaphylo-coccus aureus clinical isolates antibiotic susceptibility molecu-lar characteristics and ability to form biofilmrdquo BioMed ResearchInternational vol 2013 Article ID 314654 11 pages 2013

[45] V Pereira C Lopes A Castro J Silva P Gibbs and P TeixeiraldquoCharacterization for enterotoxin production virulence factorsand antibiotic susceptibility of Staphylococcus aureus isolatesfrom various foods in Portugalrdquo Food Microbiology vol 26 no3 pp 278ndash282 2009

[46] I Anacarso C Condo C Sabia and et al ldquoAntimicrobialresistance and other related virulence factors in staphylococcusspp isolated from food environmental and humans in ItalyrdquoUniversal Journal Microbiology Research vol 1 no 1 pp 1ndash92013

[47] B Martın M Garriga M Hugas S Bover-Cid M T Veciana-Nogues and T Aymerich ldquoMolecular technological and safetycharacterization of Gram-positive catalase-positive cocci fromslightly fermented sausagesrdquo International Journal of FoodMicrobiology vol 107 no 2 pp 148ndash158 2006

[48] NM Broekema T Van Tam T AMonson S AMarshall andD M Warshauer ldquoComparison of cefoxitin and oxacillin diskdiffusionmethods for detection ofmecA-mediated resistance inStaphylococcus aureus in a large-scale studyrdquo Journal of ClinicalMicrobiology vol 47 no 1 pp 217ndash219 2009

[49] E Cercenado F Garcıa-Garrote and E Bouza ldquoIn vitro activityof linezolid against multiply resistant Gram-positive clinicalisolatesrdquo Journal of Antimicrobial Chemotherapy vol 47 no 1pp 77ndash81 2001

[50] A Aydin K Muratoglu M Sudagidan K Bostan B Okukluand SHarsa ldquoPrevalence and antibiotic resistance of foodborneStaphylococcus aureus isolates in Turkeyrdquo Foodborne Pathogensand Disease vol 8 no 1 pp 63ndash69 2011

[51] D Sergelidis A Abrahim T Papadopoulos et al ldquoIsolation ofmethicillin-resistant Staphylococcus spp from ready-to-eat fishproductsrdquo Letters in Applied Microbiology vol 59 no 5 pp500ndash506 2014

[52] I PhillipsM Casewell T Cox et al ldquoDoes the use of antibioticsin food animals pose a risk to human health A critical reviewof published datardquo Journal of Antimicrobial Chemotherapy vol53 no 1 pp 28ndash52 2004

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Page 10: Research Article Safety Evaluation of the Coagulase ...downloads.hindawi.com/journals/bmri/2015/483548.pdf · Research Article Safety Evaluation of the Coagulase-Negative Staphylococci

10 BioMed Research International

lowast lowastlowastlowastlowastlowast lowast lowast lowastlowast lowast lowast lowastlowast lowastlowastlowastlowast lowastlowastlowast lowast

lowastlowast lowast lowast lowastlowastlowastlowast lowast lowast lowastlowast lowast

lowastlowastlowastlowast lowast lowast lowast

lowastlowast lowast lowast lowastlowast lowastlowast lowast lowastlowast lowastlowastlowastlowastlowast lowastlowast lowast lowastlowast lowastlowast

lowastlowast lowastlowastlowast lowast lowast lowast lowast lowast lowastlowastlowastlowastlowast

lowastlowast lowastlowast lowastlowast lowastlowast lowast lowast lowastlowast lowast lowast lowastlowastlowast lowast lowastlowast lowastlowastlowast

lowast lowast lowast lowast lowast lowast lowastlowast lowast lowastlowastlowast lowast lowastlowastlowast lowast lowast lowastlowastlowast lowast lowastlowastlowast lowast

lowast lowast lowast lowastlowastlowastlowast lowast lowastlowastlowast lowast lowast lowastlowastlowast lowast lowast lowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast lowast lowast lowast lowast lowastlowast lowast lowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

Enterotoxin C (sec) GATAAAAATGAATAAGAGTCGATTTATTTCATGCGTAATTTTGATATTCGCACTTATACT------------------------------------------------------------

AGTTCTTTTTACACCCAACGTATTAGCAGAGAGCCAACCAGACCCTACGCCAGATGAGTT------------------------------------------------------------

GCACAAAGCGAGTAAATTCACTGGTTTGATGGAAAATATGAAAGTTTTATATGATGATCA------------------------------------------------------------

TTATGTATCAGCAACTAAAGTTAAGTCTGTAGATAAATTTTTGGCACATGATTTAATTTA------------------------------------------------------------

TAACATTAGTGATAAAAAACTGAAAAATTATGACAAAGTGAAAACAGAGTTATTAAATGA------------------------------------------------------------

AGGTTTAGCAAAGAAGTACAAAGATGAAGTAGTTGATGTGTATGGATCAAATTACTATGT------------------------------------------------------------

AAACTGCTATTTTTCATCCAAAGATAATGTAGGTAAAGTTACAGGTGGCAAAACTTGTAT------------------------------------------------------------

GTATGGAGGAATAACAAAACATGAAGGAAACCACTTTGATAATGGGAACTTACAAAATGT------------------------------------------------------------

ACTTATAAGAGTTTATGAAAATAAAAGAAACACAATTTCTTTTGAAGTGCAAACTGATAA------------------------------------------------------------

GAAAAGTGTAACAGCTCAAGAACTAGACATAAAAGCTAGGAATTTTTTAATTAATAAAAA------------------------------------------------------------

AAATTTGTATGAGTTTAACAGTTCACCATATGAAACAGGATATATAAAATTTATTGAAAA----TTGTATGAGTTTAACAGTTCACCATATGAAACAGGATATATAAAATTTATTGAAAA

TAACGGCAATACTTTTTGGTATGATATGATGCCTGCACCAGGCGATAAGTTTGACCAATCTAACGGCAATACTTTTTGGTATGATATGATGCCTGCACCAGGCGATAAGTTTGACCAATC

TAAATATTTAATGATGTACAACGACAATAAAACGGTTGATTCTAAAAGTGTGAAGATAGATAAATATTTAATGATGTACAACGACAATAAAACAGTTGATTCTAAAAGTGTGAAGATAGA

AGTCCACCTTACAACAAAGAATGGATAATGTTAATCCGATTTTGATATAAAAAGTGAAAGAGTCCACCTTACAACAAAGAATGGATAATGTAATCCGATTATGGACAAGTGTGCTTTTTT

Enterotoxin C TATTAGATATATTTGAAAGGTAAGTACTTCGGTGCTTGCCTTTTTAGGATGCATATATATEnterotoxin C CNS

Enterotoxin CEnterotoxin C CNS

Enterotoxin CEnterotoxin C CNSEnterotoxin CEnterotoxin C CNSEnterotoxin CEnterotoxin C CNSEnterotoxin CEnterotoxin C CNSEnterotoxin CEnterotoxin C CNSEnterotoxin CEnterotoxin C CNSEnterotoxin CEnterotoxin C CNSEnterotoxin CEnterotoxin C CNS

Enterotoxin CEnterotoxin C CNSEnterotoxin CEnterotoxin C CNS

Enterotoxin CEnterotoxin C CNS

Enterotoxin CEnterotoxin C CNS

Enterotoxin CEnterotoxin C CNS

TTTGTGATCGTCGGCCA-----ACCGATACCGTCGTATTCTTGTACCGCGCAAGGCGAAGlowast lowast lowastlowastlowast lowast lowast lowast lowast lowastlowast lowast lowastlowastlowast lowast lowast lowast lowast

Enterotoxin E (see) Enterotoxin E TGCTTTAGGCGGGGTCTTACAGCATATTATCGTAGAAGATGCACAAGCTGCACGTAAAGCS pasteuri TGCTTTAGGCGCATCGTTACAACATGTCATCGTAGAAGATGAAAAAGATGGTCGTCAGGC

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast lowastlowastlowastlowast lowastlowastlowast lowast lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast lowast lowastlowastlowast lowastlowast lowastlowastlowast lowast lowastlowastlowast

Enterotoxin E ATTAGAATTCTTGAAATCAAAACA-AGCAGGACGTGCTACATTTCTACCATTAAATGTCAS pasteuri

Enterotoxin ES pasteuri

GATACAATT-TTTAAAACAACGTAGTTTAGGACGTGCCACATTCCTACCTTTAAATGTTA lowastlowast lowastlowastlowastlowast lowastlowast lowastlowastlowast lowastlowastlowast lowast lowastlowastlowastlowastlowastlowastlowastlowastlowast lowastlowastlowastlowastlowast lowastlowastlowastlowastlowast lowastlowastlowastlowastlowastlowastlowastlowast lowast

TCAAACCTAGAAATATTTCAACCAAGATATATlowast lowastlowastlowastlowast lowastlowastlowast lowastlowastlowastlowastlowast

Enterotoxin G (seg) Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

---------------------------attaagaaatactt--ccattttaatactcttaGGGGATATTTCCGGGAAACCGGGGCTAATACTGGATAACTTTTCTCTCCGAACGAAGAGA

gaatcaacaactttattatctccg--taaat-----atttaaaaacttatatggaacaaaGATTCAACGATGGTCTTGGTTTTCAATTAAGATGAACCCGCGGCGCTTAGCTGGTGGGAC

aggtactagtt-----------------------------------------cttttttaAGGTCACGGCTCACCCAAGCCCCCATGCGTATACGACCTGGAAGGGTGATCGGCCACATT

ggaaataagtcaaaccaaaaacttgtattgttcttttcagtaaat---------------GGGACTGAGACACAGCACAAACTCCTACGGGAGGCAGCAATAGGGAATCTTCCGCAATGG

------------------------------------------------------------CCGAAAGTCTGACGGAGCAACGCCGCGAGACTGAAGAATGTCTTCGGATCGTAAAACTCT

------------------------tttatatatccagattcaaa-----tgcagaaccatGTTGTTAGGGAAGAACAAGTATCGTTCGAATAGGGCGGTACCTTGACGGTACCTAACCAG

caaactcgtatagctttttttct---------ttagtgagccagtgtcttgctttg----AAAGCTTTGCTAACTACCTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCG

-taatctagttcctgaatagtaaccatattcttatttgtagttattgta--aatccaagtGAATTATTGGGCGTAAAGCGCGCGCATGCGGTCTTTTAAATCTGATGTCAAAGCCCACGG

gattgtct----------attgtcgattgttacctgtacagt------------aattaaGCTCAACCGTGGAGGGTCATTGGAAACTGTAAGACTTGGAGTGCAGAAGAGGATAGTGGA

Figure 3 Continued

BioMed Research International 11

lowast lowast lowast lowastlowast lowastlowast lowastlowastlowast lowast lowastlowast lowast lowast lowast lowast

lowast lowast lowastlowastlowast lowastlowast lowast lowastlowastlowast lowast lowastlowast lowastlowastlowast lowast lowastlowast lowastlowast lowast lowast lowastlowast lowast

lowastlowast lowast lowast lowastlowastlowastlowastlowast lowast lowast lowastlowast lowast lowast lowast lowast lowast lowastlowastlowast

lowastlowast lowast lowastlowast lowastlowastlowast lowastlowast lowastlowast lowast lowast lowast lowast lowastlowast lowastlowast lowast lowast lowast lowastlowast

lowastlowast lowastlowastlowastlowast

lowast lowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowast lowast lowastlowastlowastlowast lowast lowastlowast lowastlowast lowast lowastlowastlowastlowast lowast lowastlowast lowast lowast lowastlowastlowastlowastlowastlowastlowast

lowastlowast lowast lowast lowast lowast lowastlowastlowast lowastlowast lowastlowast lowast lowastlowastlowastlowastlowastlowastlowastlowast lowastlowastlowast

lowast lowast lowast lowast lowast lowast lowastlowast lowastlowast lowast lowast lowastlowastlowast lowast lowast lowastlowastlowast lowast lowast

lowastlowast lowastlowastlowastlowastlowastlowast lowastlowast lowastlowastlowast lowastlowast lowast lowastlowast lowastlowast lowastlowastlowast lowast lowast

lowastlowast lowast lowastlowastlowast lowast lowastlowastlowastlowastlowast lowast lowast lowast lowastlowastlowast lowast lowastlowast lowast

lowastlowast lowast lowast lowast lowast lowastlowast lowast lowastlowast lowast lowastlowast lowast lowastlowast lowastlowastlowastlowast lowast lowast lowastlowast

lowast lowastlowastlowast lowast lowastlowast lowast lowast lowastlowast lowast lowastlowast lowast lowastlowastlowastlowastlowastlowast lowast

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

tttatctctttcattttctgaactattaaa----tgtaagaccacc---atacatacaacATTCCACGTGTAACGGTGAAAATGAGTAGAGATGTGGAGGAACACCAGTGGAGAAACGCG

aacctccaaaattttggtttatatccggttc-----------agatttaggtattatacaACTCTCTGGTCTGTTACTGACGCTGAGGCGCGAAAGCGTGGGAGCAAACAGGATTATATA

tgtataaaaatatggaacgccaaaaatgtctacttttttatctttataattgttagctaaCCCCTGGTAGTCCAAGCCGTAAACGATGAGTGCTAGTGTTACAGGGT------------T

ttctgtattttctaattcagttttaacctcattataactcttatactcaattggaaaaatTCCGCCCTTTACTGCTGCAGCTAACG---CATTAG-GCACTCCGCCTGGGGAGTACAACT

taaatcatgagataaaaactgtctagaattaataactcctcttccttcaacaggtggagaGCAA------GGTTGAAACTCAAAGCA----TTGACCGGGGGCC---CGATAAGCTGAGG

cgtataaagattcattacat----tacccatagttcccttattgtttttataatcacttaTACATGGTGTTTAATTCCAAACCACGCCAAGAACCCTCACGAGGTCTTGACATCCTCTGG

ctttattt----------------------------------------------------ACCTCCCTAGAGAATAAGGGCTTCCCCGTCGGGGATGAAATGAACAGGTGGTGCATGGAT

-------agttcgtc---AGTCTTCAGCTCGTGCAT

Enterotoxin H (seh)

------------------------------------------------------------ATCTATGAATATAAATCTTTTAATATTGGTTTAATATAAATATTCCATTTCTAGAAACCT

------------------------------------------------------------ATATTTTATGTTTGTATAAATTCATAAATTGTTCGAAATTTATATATTAAAAGTTGTTTT

------------------------------------------------------------TGTTGTTTTATAATTAAGTTAATGAAATATATTGAGGAGTTTTAAAATGATTAATAAAAT

------------------------------------------------------------TAAAATATTATTTTCGTTTTTAGCATTATTACTTTCATTCACATCATATGCGAAAGCAGA

------------ -----------TTACGATTTAGCTTTAGCTAATGCATATGGTCAATAAGATTTACACGATAAAAGTGAGTTAACAGATTTAGCTTTAGCTAATGCATATGGTCAATA

TAATCACCCATTCATTAAAGAAAATATTAAGAGTGATGAAATAAGTGGAGAAAAAGATTTTAATCACCCATTCATTAAAGAAAATATTAAGAGTGATGAAATAAGTGGAGAAAAAGATTT

AATATTTAGAAATCAAGGTGATAGTGGCAATGATTTGAGAGTAAAGTTTGCAACTGCTGAAATATTTAGAAATCAAGGTGATAGTGGCAATGATTTGAGAGTAAAGTTTGCAACTGCTGA

TTTAGCTCAGAAGTTTAAAAATAAAAATGTAGATATATATGGGGCATCTTTTTATTATAATTTAGCTCAGAAGTTTAAAAATAAAAATGTAGATATATATGGGGCATCTTTTTATTATAA

GTGTGAAAAAATAAATGAAAATATTTCTGAATGTCTATATGGAGGTACAACACTAAATAAGTGTGAAAAAATAAGTGAAAATATTTCTGAATGTCTATATGGAGGTACAACACTAAATAG

TGAAAAATTGGCACAGGAAAGGGTGATTGGTGCTAATGTTTGGGTAGATGGTATTCAAAATGAAAAATTGGCACAGGAAAGGGTGATTGGTGCTAATGTTTGGGTAGATGGTATTCAAAA

AGAAACAGAATTAATACGAACAAATAAGAAAAATGTGACATTGCAAGAATTAGATATAAAAGAAACAGAATTAATACGAACAAATAAGAAAAATGTGACATTGCAAGAATTAGATATAAA

Enterotoxin H CNS GATCAGAAAAATATTGTCCGATAAATATAAAATTTATTATAAAGACAGCGAAATAAGGG-Enterotoxin H

Enterotoxin H CNSEnterotoxin H

Enterotoxin H CNSEnterotoxin HEnterotoxin H CNSEnterotoxin HEnterotoxin H CNSEnterotoxin HEnterotoxin H CNSEnterotoxin HEnterotoxin H CNSEnterotoxin H

Enterotoxin H CNSEnterotoxin H

Enterotoxin H CNSEnterotoxin H

Enterotoxin H CNSEnterotoxin H

Enterotoxin H CNSEnterotoxin H

Enterotoxin H CNSEnterotoxin H

GATCAGAAAAATATTGTCCGATAAATATAAAATTTATTATAAAGACAGCGAAATAAGTAA

Enterotoxin N (seln) TGATTTAGCTTTAGCTAATGCATATGGTCAATAT-AATCACCCATTCATTAAAGAAAA-----TAGATATTTATGGACTGTATTATGGAAATAAATGTGTAGGCTTAACTGAAGAAAAAA

TATTAAGAGTGATGAAATAAGTGGAGAAAAAGATTTAATATTTAGAAATCAAGGTGATAGCATCATGCTTATACGGAGGAGTTAC------GA--TATATGATGGAAATCAATTAGATGA

TGGCAATGATTTGAGAGTAAAG--------------TTTGCAA-CTGCTGATTTAGCTCAAGAGAGAGTTATAGGCGTTAATGTATTTAAAGATGGTATCCAACAAGAAGGTTTTGTTAT

GAAGTTTAAAAATAAAAATGTAGATATATATGGGGCATCTTTTTATTATAAGTGTGAAAAAAAAACTAAAAAGGCTAAAGTAACAGTACAA------GAATTAGATACTAAAGTTCGATT

AATAAGTGAAAATATTTCTGAATGTCTATATGGAGGTACAACACTAAATAATGAAAAATT------------TAAATTAGAAAATTTATATAAAATATACAATAAAGATACCGGTAACAT

Enterotoxin N CNSEnterotoxin N

Enterotoxin N CNSEnterotoxin N

Enterotoxin N CNSEnterotoxin N

Enterotoxin N CNSEnterotoxin N

Enterotoxin N CNSEnterotoxin N

Figure 3 Continued

12 BioMed Research International

lowastlowastlowast lowastlowast lowastlowast lowastlowast lowast lowastlowast lowastlowastlowast lowastlowast lowast lowastlowastlowastlowast

lowastlowast lowast lowastlowast lowast lowast lowastlowast lowastlowastlowastlowastlowast lowast lowast lowast lowastlowast lowast lowast lowastlowast lowast lowast

lowast lowast

lowast lowastlowast lowast lowastlowast lowast lowast lowast lowast lowast lowast lowast lowastlowast lowast lowastlowastlowastlowast lowast

lowast lowastlowast lowast lowast lowastlowast lowast lowastlowastlowast lowast lowastlowast lowast lowastlowast lowastlowast lowast lowast

lowastlowastlowast lowast lowast lowastlowast lowastlowast lowast lowast lowast

lowast lowast lowast lowastlowastlowast lowastlowastlowast lowastlowast lowast lowastlowastlowastlowastlowast lowastlowastlowast lowast

lowastlowast lowast lowast lowastlowast lowastlowastlowast lowastlowast lowast lowast lowastlowastlowastlowast lowastlowast

lowast lowast lowast lowastlowast lowast lowastlowast lowastlowastlowastlowastlowastlowast lowastlowast lowast lowastlowast lowastlowastlowast

lowast lowast lowast lowast lowastlowast lowastlowastlowastlowast lowast lowast lowastlowastlowast lowast lowast lowast lowast lowast

lowast lowast lowast lowast lowastlowastlowast lowast lowast lowastlowast lowastlowast lowast lowastlowastlowast lowast lowastlowastlowast lowast

lowast lowast lowastlowastlowast lowast lowastlowast lowastlowastlowast lowastlowastlowastlowastlowast lowastlowast lowast lowastlowastlowast lowast lowast lowastlowastlowast

lowastlowastlowast lowast lowastlowast lowastlowast lowastlowast lowast lowast lowast lowast lowastlowast lowastlowast lowastlowast lowast lowast

lowast lowast lowast lowast lowastlowast lowast lowast lowastlowast lowastlowast lowast lowastlowastlowast lowastlowastlowastlowast lowast lowastlowastlowastlowastlowast lowast

lowastlowast lowast lowast lowastlowastlowast lowastlowast lowast lowastlowast lowast lowastlowast lowastlowast lowastlowast lowast lowast lowast

lowastlowastlowastlowastlowast lowastlowast lowastlowast lowast lowastlowast lowast lowast lowast lowast lowastlowast lowastlowast

lowast lowast lowastlowast lowastlowast lowastlowastlowastlowastlowastlowast lowastlowast lowast lowast lowast lowast lowast lowast lowast lowast lowast lowast lowast

lowastlowast lowastlowast lowast lowastlowastlowastlowast lowast lowast lowastlowastlowast lowastlowast lowast lowastlowastlowast lowast

lowastlowastlowastlowast lowastlowast lowast lowast lowast lowast lowastlowast lowast lowast lowast lowastlowastlowastlowastlowast lowastlowast lowastlowastlowast lowast

lowast lowastlowastlowast lowastlowastlowastlowastlowast lowast lowast lowast lowastlowast lowast lowast lowastlowast lowast lowastlowastlowast lowastlowast lowast

lowastlowast lowast lowast lowast lowastlowast lowast lowastlowast lowastlowast lowastlowast lowast lowastlowastlowastlowast

ATTAATACGAACAAATAAGAAAAATGTGACATTGCAAGAATTAGATATAAAGATCAGAAA-TTAT----------TATGATTTATATAAC--------------ATAAAAGGTTCAG---

AATATTGTCCGATAAATATAAAATTTATTATAAAGACAGCGAAATAAGGTAAAAGG------TAGGAGCAGAGTTTTTTCAATTTTATAGTGATAATAGAACAGTTAGCTCATCTAATTA

Enterotoxin N CNS -TAAA---Enterotoxin N

lowast lowastlowastlowastlowastlowast lowastlowast lowast lowast lowast lowastlowast lowast lowastlowastlowastlowastlowastlowast lowastlowast lowastlowast

GGCACAGGAAAGGGTGATTGGTGCTAATGTTTGGGTAGATGGTATTCAAAAAGAAACAGAACAA----AAAGGATGCATTTTCTTT-----CATTCTAATAATCATCAAAATCAATCATT

Enterotoxin N CNSEnterotoxin N

Enterotoxin N CNSEnterotoxin N

Enterotoxin N CNSEnterotoxin N

TCATATCG

Enterotoxin M (selm)

ttgctattcgcaaaatcatatcgcaaccgctgatgtcggagtt--ttgaatcttaggaacEnterotoxin M CNSEnterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

--------------AGTATGTTTAAATCCCCGCTCACAGTTATGTTTAACTCTTCCTTTC

tattatggta-gctatccaattgaagaccaccaaagtattaatcctgaaaataatcatctTTTTCACACCGCCCCGTCGGGTGTTGCGCACGGGGTTGTTTAACCCCCCCGGAAAAAATT

ttcgcatcaattagttttttctatggataattcgacagtaacagctgaatttaagaacgtEnterotoxin M CNS

Enterotoxin M CNS

TTCCCTCCCTCCTCCTTACTC---------------CCT----------------CCCCT

tgatgatgtaaaggaattcaaaaatcatgctgtagatgtatatggtctaagttatagtggEnterotoxin M CNS TTTTCCTACAAATCCTTTCTCCCCTCCCGGAGTAGAAGTAAA------------------

atattgtttgaaaaacaaatatatatacggtggagttacattagcaggtgat-tatttagEnterotoxin M CNS -------------------TAAAACAATACCCCTACCACCTTAACAATTTTTTTATTGAG

agaaatctagacgtattcct-attaatctttgggttaatggagaacatcaaactatatctEnterotoxin M CNS CCCCTCCCCCCCCTTCTCATTTTTTCTCTTTGAC-----------GTTCCCCCCATTTCT

actgacaaagtatcaactaataaaa-agttagtaacagctcaagaaattgatactaaattEnterotoxin M CNS GCCAATGCTTAACCCACCCTTAAAGGCCATCGGCACATATTAGACACTCC-----CCACC

aagaagatatctacaagaagaatataatatttatggctttaatgatac---aaataaaggEnterotoxin M CNS CGCCCCCTCTTTTTATTAAGGACACAACTCTTTTTATTTTCCCCCAAGAACTAATTCTGA

a-agaaattatggtaataagtcaaaatttagttctgga---------tttaatgcaggaaEnterotoxin M CNS AAAAAAACGTTCCGCTTAGTTCAGAATTTCCTTTGAGCCTTTTATCCTTTTGTACGCGAA

aaatattatttca--------tttgaatgatggttcatcattttcttatgacttatttga

Enterotoxin M CNS AAACCATCAGCCATTGAATCAGTTATCCCATTGCCCTAACTAACCCAATATCTAATCTAA

tactggaacaggacaagctgaaagtttctt-aaaaatatataatgacaacaaaactgtcgEnterotoxin M CNS TTTTCGCCTACGAACGAATAATAGCTTGTGGCGAAAAATATCCAGGATTATCTACTGTAG

aaact--gaaaaattccatttagatgtagaaatatcttataaggacgaaagttgaagtatEnterotoxin M CNS CGCCTCGCCAGACGTCCTTTGAATTGA--ATATGACTCCTACTCCCCCCCGCACCGTCAA

ttcaatcataacttagtaaaggaaatgccatgaaaaaatttaaatatagtttt---atatEnterotoxin M CNS TTCAAACA---------------GGTGGAAGTTAATACTGAATAGTAGGTCTTCTTCCCG

tagtttttatattactttttaacattaaagatcttacgtatgct---caaggtgatattgEnterotoxin M CNS TGCTATTCTTAGAACTTTTAAATTATGTAAACCCAATTTTTAGTGCACTCTTTCTCCCTC

gtg-taggtaacttaagaaatttctatacaaaacatgattatatagatttaaaaggcgtcEnterotoxin M CNS GTTACTCTTAGATGGAGAAGCGTTAAGACAGAAGCTTTGTAT-------------TCCCA

acagataaaaa-cctacctattgcaaatcaacttgaattttcaac-----aggt----acEnterotoxin M CNS GCAGAGTGCAACCTTTCACAATGAAGTTTTTATCTAATTTACACGCCTTTAGGCTCCAAA

caatgatttgatctcagaatctaataattgggacgaaataagtaaatttaaaggaaagaaEnterotoxin M CNS TACGTATTAAATCTCCTCCTGT--TTATCTGACCCTAAG-ATAAAACACGAAAAATTATG

actggatatttttggcattgattataatggtccttgtaaatctaaatacatgtatggaggEnterotoxin M CNS CGTGCCCACTCATCCTAT-------TACTTTCATTATATCTATAAAA-------------

Figure 3 Alignment of the enterotoxin gene sequences found in coagulase-negative staphylococci (CNS) and in coagulase-positivestaphylococci (CPS)

BioMed Research International 13

Enterotoxin (seg) S aureus AY1587031

Enterotoxin (sec) CNS

Enterotoxin (see) S pasteuri NC0227371

Enterotoxin (see) CNS

Enterotoxin (seh) CNS

Enterotoxin (selm) S aureus AF2857601

Enterotoxin (seln) CNS

Enterotoxin (seh) S aureus AJ9375481

Enterotoxin (seln) S aureus AY1587031

Enterotoxin (sec) S aureus X516611

Enterotoxin (selm) CNS

Enterotoxin (seg) CNS

150 100

100

100

100

100

99

98

96

97

72

50 0

Figure 4 Phylogenetic tree generated from the multiple alignments of the enterotoxin sequences of CNS and CPS strains using the ClustalX20 software package The phylogenetic tree was constructed using the Mega 60 software and the unweighted pair group method (UPGMA)

MAR indices varying from 066 to 046 and the remaining04 strains showed MAR indices le026 (Table 3)

The 14 MRCNS strains were resistant to 120573-lactams(oxacillin penicillin andor cefoxitin) and to vancomycincorresponding to 73 of the total CNS strains identifiedwhile 09 strains (64) showed resistance to tetracycline andgentamicin 08 strains (57) were resistant to neomycinerythromycin and chloramphenicol 07 strains (50) wereresistant to sulfamethoprim 05 strains (36) were resistantto linezolid 03 strains (21) were resistant to rifampicin 02strains (14) were resistant to ciprofloxacin and cefepimeand 01 strain (7) was resistant to clindamycin (Table 3)

The multiresistance of CNS strains demonstrated hereinis consistent with previous studies on coagulase-negativeand coagulase-positive staphylococci that have found severalresistant and multiresistant Staphylococcus aureus strains inraw milk meat and fermented meat products [43]

Surprisingly the resistance to chloramphenicol is verysimilar to that estimated for MRCNS strains isolated fromhuman clinical samples Indeed there is no direct correla-tion between the researched features and the origin of thestaphylococci strains since different virulence factors arewidespread such as antibiotic resistance [44] reinforcing thefact that the ability of foodmatrices strains to produce SE andSEl and their multiresistance character must be consideredwhen evaluating the safety hazards of food poisoning

S epidermidis KF6005891 and S hominis JX5199881showed the highestMAR indexes (093 and 080 resp)Thesestrains found in artisanal salami are homologous to strainsisolated from human skin microbiota and should be carefullyconsidered among the potential pathogenic staphylococcifound in food from animal origins which can be caused bycontamination by poor hygienic conditions during salamimanufacturing

Depending on the conditions some species of coagulase-negative staphylococci can present health risks since theyhave shown resistance to antibiotics of therapeutic impor-tance such as beta lactams [15] However multiresistantstrains like S carnosusKJ18620021 and S xylosusCP0072081and CP0087241 were intentionally introduced in the foodmatrix since they are part of the culture starter used inItalian-salami manufacturing As previously discussed foodcan be a reservoir of multiresistant microorganisms that canspread by the consumption of an apparently safe food Dueto the intensive and indiscriminate use of antibiotics forhuman and veterinarian therapeutic purposes multiresistantstaphylococci strains are being selected and reproduced infood matrices [45]

The cefoxitin and oxacillin disk diffusion tests are rec-ommended for determining the resistance and susceptibilitybreakpoint for MRSA surveillance cultures [46] In thisstudy there was a good correlation between the disc testzone diameters for oxacillin and cefoxitin (Table 3) TheMICs for ampicillin andmethicillinoxacillincefoxitin for allsusceptible CNS strainswere determined by the diffusion disctest using 003 to 2mgmLminus1 of each antimicrobial agent

S saprophyticus HF9372521 showed an MIC of003mgmLminus1 formethicillin strain S carnosusKJ8620021 Sxylosus strains CP0087241 and CP0072081 S saprophyticusJX4901221 S hominis JX5199881 and S succinusKC3298241 were resistant to 006mgmLminus1 and two strainsS saprophyticus KJ6991511 and S saprophyticus KJ0046231showed an MIC of 05mgmLminus1 (Table 4) Two of themethicillin-resistant strains S saprophyticus KJ0046231 andS xylosus CP0072081 harbor no enterotoxin gene

Five strains S saprophyticus JX4901221 S carnosusKJ8620021 S xylosusCP0087241 S xylosusKF1980801 andS saprophyticus KJ0046231 showed an MIC for ampicillin

14 BioMed Research International

Table 3 Multiple resistance to antimicrobial as found in CNS strains from salami

Salami origin CNS strains Antimicrobial agent resistance Multiple antimicrobialresistance (MAR) indexlowast

Commercial Staphylococcus spp KF1354451 CIP CLO CPM GEN NEO OXA PENSXT TET and VAN 066

Commercial S carnosus KJ8620021 CIP GEN LZD NEO OXA SXT andTET 046

Commercial S saprophyticus AB6977171 CFO CLO ERI PEN OXA TET andVAN 046

Commercial S saprophyticus EU4309921 CFO CLO ERI GEN LZD NEO OXAPEN SXT TET and VAN 080

Commercial S saprophyticus JX4901221 CFO CLO ERI GEN NEO PEN OXASXT TET and VAN 066

Commercial S saprophyticus KJ0046231 CFO OXA and PEN 020

Commercial S sciuri JX9664361 CFO CLO GEN NEO OXA PEN andTET 046

Commercial S xylosus CP0072081 OXA LZD PEN and VAN 026

Commercial S xylosus CP0087241 CFO CLO ERI GEN NEO OXA PENTET and VAN 060

Artisanal S epidermidis KF6005891 CFO CLI CLO CPM ERI GEN LZDNEO OXA PEN RIF SXT and TET 093

Artisanal S hominis JX5199881 CIP CPM CPO ERI GEN LZD NEOOXA PEN RIF SXT and VAN 080

Artisanal S saprophyticusHF9372521 CFO OXA PEN and SXT 026Artisanal S saprophyticus KJ6991511 OXA PEN and VAN 020

Artisanal S succinus KC3298241 CFO CLO ERI GEN OXA PEN RIFSXT TET and VAN 066

Artisanal S xylosus KF1980801 CFO CLO ERI GEN NEO OXA PENTET and VAN 060

lowastTheMAR index of an isolate is defined as 119886119887 where 119886 represents the number of antimicrobials to which the isolate was resistant and 119887 represents the numberof antimicrobials to which the isolate was subjectedS aureus strains ATCC WB81 (sea) ATCC 13563 (sed) and ATCC 27664 (see) showing a MAR index of 05 and S aureus strains ATCC14458 (seb) andATCCWB72 (sec) and S xylosus ATCC 29971 showing a MAR index of 03 were used as reference strainsCPM cefepime CFO cefoxitin CLO chloramphenicol CIP ciprofloxacin CLI clindamycin ERI erythromycin GEN gentamycin NEO neomycin LZDlinezolid RIF rifampicin TET tetracycline OXA oxacillin PEN penicillin SXT sulfamethoprim and VAN vancomycin

Table 4 Minimal inhibitory concentration (MIC) of compounds used in antimicrobial therapy against staphylococci infections

Salami origin GenBank accession number and similarity () MIC mgmLminus1

Methicillin Ampicillin Vancomycin Linezolid

Commercial

S carnosus KJ8620021 006 003 mdash mdashS xylosus CP0087241 006 003 006 mdash

S saprophyticus JX4901221 006 003 05 mdashS saprophyticus AB6977171 mdash mdash 05 mdashS saprophyticus EU4309921 mdash mdash 025 0125

S succinus KC3298241 006 025 003 mdash

Artisanal

S hominis JX5199881 006 05 mdash mdashS saprophyticus HF9372521 003 mdash mdash mdash

S xylosus KF1980801 003 05 mdashS saprophyticus KJ6991511 05 025 003 mdashS saprophyticus KJ0046231 05 003 003 mdash

S xylosus CP0072081 006 mdash mdash 025Strains S epidermidis KF6005891 and Staphylococcus spp KF1354451 were not susceptible to the antimicrobial concentrations tested in the present study

BioMed Research International 15

of 003mgmLminus1 two strains S succinus KC3298241 and Ssaprophyticus KJ6991511 showed an MIC of 025mgmLminus1and S hominis JX5199881 showed an MIC of 05mgmLminus1(Table 4) S saprophyticus KJ0046231 also showed an MICof 003mgmLminus1 but does not harbor enterotoxin genes It isimportant to note that most of the CNS strains that showedresistance to ampicillin were also resistant to methicillin ashighlighted by the MIC tests

Five strains S saprophyticus HF9372521 S saprophyti-cus KJ6991511 S saprophyticus JX4901221 S saprophyticusKJ0046231 and S succinus KC3298241 showed an MICfor vancomycin of 003mgmLminus1 one strain S xylosusCP0087241 showed an MIC of 006mgmLminus1 one strain Ssaprophyticus EU4309921 showed an MIC of 025mgmLminus1and two strains S xylosus KF1980801 and S saprophyticusAB6977171 showed an MIC of 05mgmLminus1 (Table 4)

Additionally five CNS strains S xylosus CP0087241S saprophyticus JX 4901221 S succinus KC3298241 Ssaprophyticus KJ6991511 and S saprophyticus KJ0046231showed resistance tomethicillin as demonstrated by theMICdeterminations (Table 4)

Previous studies have demonstrated that linezolid isactive against Gram-positive bacteria including methicillin-resistant staphylococci [47] In the present study 05 strainsshowed linezolid resistance in the disc diffusion test but itwas impossible to establish MIC values for S epidermidisKF6005891 Staphylococcus hominis JX5199881 and Staphy-lococcus carnosus KJ8620021The remaining strains S sapro-phyticus EU4309921 and S xylosus CP0072081 presentedan MIC for linezolid of 0125mgmLminus1 and 025mgmLminus1respectively

The MIC of 003mgmLminus1 for penicillin is in accordancewith the previous values estimated for resistant S aureusstrains found in several food matrices such as meat dairyproducts and ready-to-eat food [48]

There is still a lack of information on the antimicro-bial resistance of staphylococci strains from food matricesalthough they are the most worrisome vehicles of dissemi-nation of antibiotic-resistant pathogens [49]

The high resistance found for salami staphylococci strainscan be ascribed to their inappropriate use as growth promot-ers of antimicrobial agents like oxacillin vancomycin chlo-ramphenicol neomycin and erythromycin which are com-monly used in veterinary medicine to treat infections [50]Antimicrobial therapy of infections staphylococci is basedon results of susceptibility tests in vitro [51] Methicillin-resistant coagulase-negative Staphylococcus spp found inready-to-eat products such as meats fish and dairy productsalso offer risks and although they are not classical foodpoisoning bacteria their presence in food offers significantrisks to public health due to the possible spread of antibioticresistance [52]

The standardization of salami quality should includediagnostic methods to screen and quantify the presence ofclassical and newly described enterotoxins directly in thefood matrices as a routine procedure to be conducted by themeat product industry in Brazil

The safety of salami consumption could also be enhancedby the inclusion of amicrobial barrier such as the inclusion ofprobiotic strains producing natural antibiotics or competitiveflora or even the addition of natural bioagents against spoilageor pathogenic microorganisms

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors acknowledge the financial support fromFundacao Carlos Chagas Filho de Amparo a Pesquisa doEstado do Rio de Janeiro (FAPERJ Rio de Janeiro Brazil)Conselho Nacional de Desenvolvimento Cientıfico eTecnologico (CNPq Brasılia Brazil) and Coordenacao deAperfeicoamento de Pessoal de Nıvel Superior (CAPESBrasılia Brazil)

References

[1] ICMSF International Comission on Microbiological Specifica-tions for FoodsMicroorganisms in FoodsTheir Significance andMethods of Enumerations University of Toronto Press TorontoCanada 3rd edition 1983

[2] J F Veras L S Carmo L C Tong et al ldquoA study of theenterotoxigenicity of coagulase-negative and coagulase-positivestaphylococcal isolates from food poisoning outbreaks inMinasGerais Brazilrdquo International Journal of Infectious Diseases vol12 no 4 pp 410ndash415 2008

[3] K Omoe D-L Hu H Takahashi-Omoe A Nakane and KShinagawa ldquoComprehensive analysis of classical and newlydescribed staphylococcal superantigenic toxin genes in Staphy-lococcus aureus isolatesrdquo FEMS Microbiology Letters vol 246no 2 pp 191ndash198 2005

[4] J Kadariya T C Smith and D Thapaliya ldquoStaphylococcusaureus and staphylococcal food-borne disease an ongoing chal-lenge in public healthrdquoBioMedResearch International vol 2014Article ID 827965 9 pages 2014

[5] KOmoeD-LHuH KOno et al ldquoEmetic potentials of newlyidentified staphylococcal enterotoxin-like toxinsrdquo Infection andImmunity vol 81 no 10 pp 3627ndash3631 2013

[6] J K McCormick J M Yarwood and P M SchlievertldquoToxic shock syndrome and bacterial superantigens an updaterdquoAnnual Review of Microbiology vol 55 pp 77ndash104 2001

[7] T Baba F Takeuchi M Kuroda et al ldquoGenome and virulencedeterminants of high virulence community-acquired MRSArdquoThe Lancet vol 359 no 9320 pp 1819ndash1827 2002

[8] Associacao Brasileira de Industria Produtora e Exportadorada Carne Suınas (ABIPECS) ldquoStatistics slaughter ranking2000ndash2014rdquo 2014 httpabpa-brcombrsetoressuinoculturapublicacoesrelatorios-anuais

[9] N Zdolec M Hadziosmanovic L Kozacinski et al ldquoMicrobialand physicochemical succession in fermented sausages pro-duced with bacteriocinogenic culture of Lactobacillus sakei andsemi-purified bacteriocin mesenterocin Yrdquo Meat Science vol80 no 2 pp 480ndash487 2008

16 BioMed Research International

[10] F Ravyts L D Vuyst and F Leroy ldquoBacterial diversity andfunctionalities in food fermentationsrdquo Engineering in Life Sci-ences vol 12 no 4 pp 356ndash367 2012

[11] TOhlsson andN BengtssonTheHurdle ConceptMinimal Pro-cessing Technologies in the Food IndustryWoodhead Publishing2002

[12] S Bernardi B B Golineli and C J Contreras-CastilloldquoRevisao aspectos da aplicacao de culturas starter na producaode embutidos carneos fermentadosrdquo Brazilian Journal of FoodTechnology vol 13 no 2 pp 133ndash140 2010

[13] F Irlinger ldquoSafety assessment of dairy microorganismscoagulase-negative staphylococcirdquo International Journal of FoodMicrobiology vol 126 no 3 pp 302ndash310 2008

[14] B Ghebremedhin F Layer W Konig and B Konig ldquoGeneticclassification and distinguishing of Staphylococcus species basedon different partial gap 16S rRNA hsp60 rpoB sodA and tufgene sequencesrdquo Journal of Clinical Microbiology vol 46 no 3pp 1019ndash1025 2008

[15] I Babic K Markov D Kovacevic et al ldquoIdentification andcharacterization of potential autochthonous starter culturesfrom aCroatian lsquobrandrsquo product lsquoslavonski kulenrsquordquoMeat Sciencevol 88 no 3 pp 517ndash524 2011

[16] MMehrotra GWang andWM Johnson ldquoMultiplex PCR forthe detection of genes for Staphylococcus aureus enterotoxinsexfoliative toxins toxic shock syndrome toxin and methicillinresistancerdquo Journal of Clinical Microbiology vol 38 no 3 pp1032ndash1041 2000

[17] WM Johnson SD Tyler E P Ewan F EAshtonDR Pollardand K R Rozee ldquoDetection of genes for enterotoxins exfolia-tive toxins and toxic shock syndrome toxin 1 in Staphylococcusaureus by the polymerase chain reactionrdquo Journal of ClinicalMicrobiology vol 29 no 3 pp 426ndash430 1991

[18] J Mclauchlin G L Narayanan VMithani andG OrsquoNeill ldquoThedetection of enterotoxins and toxic shock syndrome toxin genesin Staphylococcus aureus by polymerase chain reactionrdquo Journalof Food Protection vol 63 no 4 pp 479ndash488 2000

[19] J P Rosec and O Gigaud ldquoStaphylococcal enterotoxin genes ofclassical and new types detected by PCR in Francerdquo Interna-tional Journal of Food Microbiology vol 77 no 1-2 pp 61ndash702002

[20] K Omoe D-L Hu H Takahashi-Omoe A Nakane andK Shinagawa ldquoIdentification and characterization of a newstaphylococcal enterotoxin-related putative toxin encoded bytwo kinds of plasmidsrdquo Infection and Immunity vol 71 no 10pp 6088ndash6094 2003

[21] N Sergeev D Volokhov V Chizhikov and A Rasooly ldquoSimul-taneous analysis of multiple staphylococcal enterotoxin genesby an oligonucleotide microarray assayrdquo Journal of ClinicalMicrobiology vol 42 no 5 pp 2134ndash2143 2004

[22] S Holtfreter D Grumann M Schmudde et al ldquoClonal distri-bution of superantigen genes in clinical Staphylococcus aureusisolatesrdquo Journal of Clinical Microbiology vol 45 no 8 pp2669ndash2680 2007

[23] W J Mason J S Blevins K Beenken NWibowo N Ojha andM S Smeltzer ldquoMultiplex PCR protocol for the diagnosis ofstaphylococcal infectionrdquo Journal of Clinical Microbiology vol39 no 9 pp 3332ndash3338 2001

[24] E Rahimi and H G Safai ldquoDetection of classical enterotoxinsof Staphylococcus aureus strains isolated frombovine subclinicalmastitis in Isfahan Iranrdquo Veterinary Microbiology vol 141 no3-4 pp 393ndash394 2010

[25] M A Larkin G Blackshields N P Brown et al ldquoClustalW andClustal X version 20rdquo Bioinformatics vol 23 no 21 pp 2947ndash2948 2007

[26] P H A Sneath and R R Sokal Unweighted Pair Group Methodwith Arithmetic Mean Numerical Taxonomy W H FreemanSan Francisco Calif USA 1973

[27] NCCLS ldquoPerformance standards for antimicrobial susceptibil-ity testing 14th informational supplementrdquo Tech Rep M 100-S14 NCCLS 2004

[28] S Corbiere Morot-Bizot S Leroy and R Talon ldquoMonitoringof staphylococcal starters in two French processing plantsmanufacturing dry fermented sausagesrdquo Journal of AppliedMicrobiology vol 102 no 1 pp 238ndash244 2007

[29] R Talon S Leroy I Lebert et al ldquoSafety improvement andpreservation of typical sensory qualities of traditional dryfermented sausages using autochthonous starter culturesrdquo Inter-national Journal of Food Microbiology vol 126 no 1-2 pp 227ndash234 2008

[30] G Mauriello A Casaburi G Blaiotta and F Villani ldquoIsolationand technological properties of coagulase negative staphylo-cocci from fermented sausages of Southern ItalyrdquoMeat Sciencevol 67 no 1 pp 149ndash158 2004

[31] M Janssens N Myter L De Vuyst and F Leroy ldquoSpeciesdiversity and metabolic impact of the microbiota are low inspontaneously acidified Belgian sausages with an added starterculture of Staphylococcus carnosusrdquo Food Microbiology vol 29no 2 pp 167ndash177 2012

[32] G Blaiotta D Ercolini G Mauriello G Salzano and F VillanildquoRapid and reliable identification of Staphylococcus equorum bya species-specific PCR assay targeting the sodA generdquo Systematicand Applied Microbiology vol 27 no 6 pp 696ndash702 2004

[33] L Kozacinski E Drosinos F Caklovica L Cocolin J Gasparik-Reichardt and S Veskovic ldquoInvestigation of microbial associa-tion of traditionally fermented sausagesrdquo Food Technology andBiotechnology vol 46 no 1 pp 93ndash106 2008

[34] S Leroy P Giammarinaro J-P Chacornac I Lebert and RTalon ldquoBiodiversity of indigenous staphylococci of naturallyfermented dry sausages and manufacturing environments ofsmall-scale processing unitsrdquo Food Microbiology vol 27 no 2pp 294ndash301 2010

[35] E Coton M-H Desmonts S Leroy et al ldquoBiodiversity ofCoagulase-Negative Staphylococci in French cheeses dry fer-mented sausages processing environments and clinical sam-plesrdquo International Journal of Food Microbiology vol 137 no 2-3 pp 221ndash229 2010

[36] S Hosseinzadeh and H D Saei ldquoStaphylococcal species associ-ated with bovinemastitis in the NorthWest of Iran emerging ofcoagulase-negative staphylococcirdquo International Journal of Vet-erinary Science and Medicine vol 2 no 1 pp 27ndash34 2014

[37] A Piette and G Verschraegen ldquoRole of coagulase-negativestaphylococci in human diseaserdquo Veterinary Microbiology vol134 no 1-2 pp 45ndash54 2009

[38] T M Moura F S Campos P A DrsquoAzevedo et al ldquoPrevalenceof enterotoxin-encoding genes and antimicrobial resistancein coagulase-negative and coagulase-positive Staphylococcusisolates from black pudding in Southern Brazilrdquo Revista daSociedade Brasileira deMedicina Tropical vol 45 no 5 pp 579ndash585 2012

[39] H I Suzuki T Lefebure P P Bitar and M J StanhopeldquoComparative genomic analysis of the genus Staphylococcusincluding Staphylococcus aureus and its newly described sister

BioMed Research International 17

species Staphylococcus simiaerdquo BMC Genomics vol 13 article38 2012

[40] R P Lamers G Muthukrishnan T A Castoe S Tafur A MCole and C L Parkinson ldquoPhylogenetic relationships amongStaphylococcus species and refinement of cluster groups basedon multilocus datardquo BMC Evolutionary Biology vol 12 article171 2012

[41] I V Pinchuk E J Beswick and V E Reyes ldquoStaphylococcalenterotoxinsrdquo Toxins vol 2 no 8 pp 2177ndash2197 2010

[42] S R Monday and G A Bohach ldquoGenes encoding staphylo-coccal enterotoxins G and I are linked and separated by DNArelated to other staphylococcal enterotoxinsrdquo Journal of NaturalToxins vol 10 no 1 pp 1ndash8 2001

[43] D Y Thomas S Jarraud B Lemercier et al ldquoStaphylococcalenterotoxin-like toxins U2 and V two new staphylococcalsuperantigens arising from recombination within the entero-toxin gene clusterrdquo Infection and Immunity vol 74 pp 4724ndash4734 2006

[44] N Indrawattana O Sungkhachat N Sookrung et al ldquoStaphylo-coccus aureus clinical isolates antibiotic susceptibility molecu-lar characteristics and ability to form biofilmrdquo BioMed ResearchInternational vol 2013 Article ID 314654 11 pages 2013

[45] V Pereira C Lopes A Castro J Silva P Gibbs and P TeixeiraldquoCharacterization for enterotoxin production virulence factorsand antibiotic susceptibility of Staphylococcus aureus isolatesfrom various foods in Portugalrdquo Food Microbiology vol 26 no3 pp 278ndash282 2009

[46] I Anacarso C Condo C Sabia and et al ldquoAntimicrobialresistance and other related virulence factors in staphylococcusspp isolated from food environmental and humans in ItalyrdquoUniversal Journal Microbiology Research vol 1 no 1 pp 1ndash92013

[47] B Martın M Garriga M Hugas S Bover-Cid M T Veciana-Nogues and T Aymerich ldquoMolecular technological and safetycharacterization of Gram-positive catalase-positive cocci fromslightly fermented sausagesrdquo International Journal of FoodMicrobiology vol 107 no 2 pp 148ndash158 2006

[48] NM Broekema T Van Tam T AMonson S AMarshall andD M Warshauer ldquoComparison of cefoxitin and oxacillin diskdiffusionmethods for detection ofmecA-mediated resistance inStaphylococcus aureus in a large-scale studyrdquo Journal of ClinicalMicrobiology vol 47 no 1 pp 217ndash219 2009

[49] E Cercenado F Garcıa-Garrote and E Bouza ldquoIn vitro activityof linezolid against multiply resistant Gram-positive clinicalisolatesrdquo Journal of Antimicrobial Chemotherapy vol 47 no 1pp 77ndash81 2001

[50] A Aydin K Muratoglu M Sudagidan K Bostan B Okukluand SHarsa ldquoPrevalence and antibiotic resistance of foodborneStaphylococcus aureus isolates in Turkeyrdquo Foodborne Pathogensand Disease vol 8 no 1 pp 63ndash69 2011

[51] D Sergelidis A Abrahim T Papadopoulos et al ldquoIsolation ofmethicillin-resistant Staphylococcus spp from ready-to-eat fishproductsrdquo Letters in Applied Microbiology vol 59 no 5 pp500ndash506 2014

[52] I PhillipsM Casewell T Cox et al ldquoDoes the use of antibioticsin food animals pose a risk to human health A critical reviewof published datardquo Journal of Antimicrobial Chemotherapy vol53 no 1 pp 28ndash52 2004

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Page 11: Research Article Safety Evaluation of the Coagulase ...downloads.hindawi.com/journals/bmri/2015/483548.pdf · Research Article Safety Evaluation of the Coagulase-Negative Staphylococci

BioMed Research International 11

lowast lowast lowast lowastlowast lowastlowast lowastlowastlowast lowast lowastlowast lowast lowast lowast lowast

lowast lowast lowastlowastlowast lowastlowast lowast lowastlowastlowast lowast lowastlowast lowastlowastlowast lowast lowastlowast lowastlowast lowast lowast lowastlowast lowast

lowastlowast lowast lowast lowastlowastlowastlowastlowast lowast lowast lowastlowast lowast lowast lowast lowast lowast lowastlowastlowast

lowastlowast lowast lowastlowast lowastlowastlowast lowastlowast lowastlowast lowast lowast lowast lowast lowastlowast lowastlowast lowast lowast lowast lowastlowast

lowastlowast lowastlowastlowastlowast

lowast lowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowast lowast lowastlowastlowastlowast lowast lowastlowast lowastlowast lowast lowastlowastlowastlowast lowast lowastlowast lowast lowast lowastlowastlowastlowastlowastlowastlowast

lowastlowast lowast lowast lowast lowast lowastlowastlowast lowastlowast lowastlowast lowast lowastlowastlowastlowastlowastlowastlowastlowast lowastlowastlowast

lowast lowast lowast lowast lowast lowast lowastlowast lowastlowast lowast lowast lowastlowastlowast lowast lowast lowastlowastlowast lowast lowast

lowastlowast lowastlowastlowastlowastlowastlowast lowastlowast lowastlowastlowast lowastlowast lowast lowastlowast lowastlowast lowastlowastlowast lowast lowast

lowastlowast lowast lowastlowastlowast lowast lowastlowastlowastlowastlowast lowast lowast lowast lowastlowastlowast lowast lowastlowast lowast

lowastlowast lowast lowast lowast lowast lowastlowast lowast lowastlowast lowast lowastlowast lowast lowastlowast lowastlowastlowastlowast lowast lowast lowastlowast

lowast lowastlowastlowast lowast lowastlowast lowast lowast lowastlowast lowast lowastlowast lowast lowastlowastlowastlowastlowastlowast lowast

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

Enterotoxin G Enterotoxin G CNS

tttatctctttcattttctgaactattaaa----tgtaagaccacc---atacatacaacATTCCACGTGTAACGGTGAAAATGAGTAGAGATGTGGAGGAACACCAGTGGAGAAACGCG

aacctccaaaattttggtttatatccggttc-----------agatttaggtattatacaACTCTCTGGTCTGTTACTGACGCTGAGGCGCGAAAGCGTGGGAGCAAACAGGATTATATA

tgtataaaaatatggaacgccaaaaatgtctacttttttatctttataattgttagctaaCCCCTGGTAGTCCAAGCCGTAAACGATGAGTGCTAGTGTTACAGGGT------------T

ttctgtattttctaattcagttttaacctcattataactcttatactcaattggaaaaatTCCGCCCTTTACTGCTGCAGCTAACG---CATTAG-GCACTCCGCCTGGGGAGTACAACT

taaatcatgagataaaaactgtctagaattaataactcctcttccttcaacaggtggagaGCAA------GGTTGAAACTCAAAGCA----TTGACCGGGGGCC---CGATAAGCTGAGG

cgtataaagattcattacat----tacccatagttcccttattgtttttataatcacttaTACATGGTGTTTAATTCCAAACCACGCCAAGAACCCTCACGAGGTCTTGACATCCTCTGG

ctttattt----------------------------------------------------ACCTCCCTAGAGAATAAGGGCTTCCCCGTCGGGGATGAAATGAACAGGTGGTGCATGGAT

-------agttcgtc---AGTCTTCAGCTCGTGCAT

Enterotoxin H (seh)

------------------------------------------------------------ATCTATGAATATAAATCTTTTAATATTGGTTTAATATAAATATTCCATTTCTAGAAACCT

------------------------------------------------------------ATATTTTATGTTTGTATAAATTCATAAATTGTTCGAAATTTATATATTAAAAGTTGTTTT

------------------------------------------------------------TGTTGTTTTATAATTAAGTTAATGAAATATATTGAGGAGTTTTAAAATGATTAATAAAAT

------------------------------------------------------------TAAAATATTATTTTCGTTTTTAGCATTATTACTTTCATTCACATCATATGCGAAAGCAGA

------------ -----------TTACGATTTAGCTTTAGCTAATGCATATGGTCAATAAGATTTACACGATAAAAGTGAGTTAACAGATTTAGCTTTAGCTAATGCATATGGTCAATA

TAATCACCCATTCATTAAAGAAAATATTAAGAGTGATGAAATAAGTGGAGAAAAAGATTTTAATCACCCATTCATTAAAGAAAATATTAAGAGTGATGAAATAAGTGGAGAAAAAGATTT

AATATTTAGAAATCAAGGTGATAGTGGCAATGATTTGAGAGTAAAGTTTGCAACTGCTGAAATATTTAGAAATCAAGGTGATAGTGGCAATGATTTGAGAGTAAAGTTTGCAACTGCTGA

TTTAGCTCAGAAGTTTAAAAATAAAAATGTAGATATATATGGGGCATCTTTTTATTATAATTTAGCTCAGAAGTTTAAAAATAAAAATGTAGATATATATGGGGCATCTTTTTATTATAA

GTGTGAAAAAATAAATGAAAATATTTCTGAATGTCTATATGGAGGTACAACACTAAATAAGTGTGAAAAAATAAGTGAAAATATTTCTGAATGTCTATATGGAGGTACAACACTAAATAG

TGAAAAATTGGCACAGGAAAGGGTGATTGGTGCTAATGTTTGGGTAGATGGTATTCAAAATGAAAAATTGGCACAGGAAAGGGTGATTGGTGCTAATGTTTGGGTAGATGGTATTCAAAA

AGAAACAGAATTAATACGAACAAATAAGAAAAATGTGACATTGCAAGAATTAGATATAAAAGAAACAGAATTAATACGAACAAATAAGAAAAATGTGACATTGCAAGAATTAGATATAAA

Enterotoxin H CNS GATCAGAAAAATATTGTCCGATAAATATAAAATTTATTATAAAGACAGCGAAATAAGGG-Enterotoxin H

Enterotoxin H CNSEnterotoxin H

Enterotoxin H CNSEnterotoxin HEnterotoxin H CNSEnterotoxin HEnterotoxin H CNSEnterotoxin HEnterotoxin H CNSEnterotoxin HEnterotoxin H CNSEnterotoxin H

Enterotoxin H CNSEnterotoxin H

Enterotoxin H CNSEnterotoxin H

Enterotoxin H CNSEnterotoxin H

Enterotoxin H CNSEnterotoxin H

Enterotoxin H CNSEnterotoxin H

GATCAGAAAAATATTGTCCGATAAATATAAAATTTATTATAAAGACAGCGAAATAAGTAA

Enterotoxin N (seln) TGATTTAGCTTTAGCTAATGCATATGGTCAATAT-AATCACCCATTCATTAAAGAAAA-----TAGATATTTATGGACTGTATTATGGAAATAAATGTGTAGGCTTAACTGAAGAAAAAA

TATTAAGAGTGATGAAATAAGTGGAGAAAAAGATTTAATATTTAGAAATCAAGGTGATAGCATCATGCTTATACGGAGGAGTTAC------GA--TATATGATGGAAATCAATTAGATGA

TGGCAATGATTTGAGAGTAAAG--------------TTTGCAA-CTGCTGATTTAGCTCAAGAGAGAGTTATAGGCGTTAATGTATTTAAAGATGGTATCCAACAAGAAGGTTTTGTTAT

GAAGTTTAAAAATAAAAATGTAGATATATATGGGGCATCTTTTTATTATAAGTGTGAAAAAAAAACTAAAAAGGCTAAAGTAACAGTACAA------GAATTAGATACTAAAGTTCGATT

AATAAGTGAAAATATTTCTGAATGTCTATATGGAGGTACAACACTAAATAATGAAAAATT------------TAAATTAGAAAATTTATATAAAATATACAATAAAGATACCGGTAACAT

Enterotoxin N CNSEnterotoxin N

Enterotoxin N CNSEnterotoxin N

Enterotoxin N CNSEnterotoxin N

Enterotoxin N CNSEnterotoxin N

Enterotoxin N CNSEnterotoxin N

Figure 3 Continued

12 BioMed Research International

lowastlowastlowast lowastlowast lowastlowast lowastlowast lowast lowastlowast lowastlowastlowast lowastlowast lowast lowastlowastlowastlowast

lowastlowast lowast lowastlowast lowast lowast lowastlowast lowastlowastlowastlowastlowast lowast lowast lowast lowastlowast lowast lowast lowastlowast lowast lowast

lowast lowast

lowast lowastlowast lowast lowastlowast lowast lowast lowast lowast lowast lowast lowast lowastlowast lowast lowastlowastlowastlowast lowast

lowast lowastlowast lowast lowast lowastlowast lowast lowastlowastlowast lowast lowastlowast lowast lowastlowast lowastlowast lowast lowast

lowastlowastlowast lowast lowast lowastlowast lowastlowast lowast lowast lowast

lowast lowast lowast lowastlowastlowast lowastlowastlowast lowastlowast lowast lowastlowastlowastlowastlowast lowastlowastlowast lowast

lowastlowast lowast lowast lowastlowast lowastlowastlowast lowastlowast lowast lowast lowastlowastlowastlowast lowastlowast

lowast lowast lowast lowastlowast lowast lowastlowast lowastlowastlowastlowastlowastlowast lowastlowast lowast lowastlowast lowastlowastlowast

lowast lowast lowast lowast lowastlowast lowastlowastlowastlowast lowast lowast lowastlowastlowast lowast lowast lowast lowast lowast

lowast lowast lowast lowast lowastlowastlowast lowast lowast lowastlowast lowastlowast lowast lowastlowastlowast lowast lowastlowastlowast lowast

lowast lowast lowastlowastlowast lowast lowastlowast lowastlowastlowast lowastlowastlowastlowastlowast lowastlowast lowast lowastlowastlowast lowast lowast lowastlowastlowast

lowastlowastlowast lowast lowastlowast lowastlowast lowastlowast lowast lowast lowast lowast lowastlowast lowastlowast lowastlowast lowast lowast

lowast lowast lowast lowast lowastlowast lowast lowast lowastlowast lowastlowast lowast lowastlowastlowast lowastlowastlowastlowast lowast lowastlowastlowastlowastlowast lowast

lowastlowast lowast lowast lowastlowastlowast lowastlowast lowast lowastlowast lowast lowastlowast lowastlowast lowastlowast lowast lowast lowast

lowastlowastlowastlowastlowast lowastlowast lowastlowast lowast lowastlowast lowast lowast lowast lowast lowastlowast lowastlowast

lowast lowast lowastlowast lowastlowast lowastlowastlowastlowastlowastlowast lowastlowast lowast lowast lowast lowast lowast lowast lowast lowast lowast lowast lowast

lowastlowast lowastlowast lowast lowastlowastlowastlowast lowast lowast lowastlowastlowast lowastlowast lowast lowastlowastlowast lowast

lowastlowastlowastlowast lowastlowast lowast lowast lowast lowast lowastlowast lowast lowast lowast lowastlowastlowastlowastlowast lowastlowast lowastlowastlowast lowast

lowast lowastlowastlowast lowastlowastlowastlowastlowast lowast lowast lowast lowastlowast lowast lowast lowastlowast lowast lowastlowastlowast lowastlowast lowast

lowastlowast lowast lowast lowast lowastlowast lowast lowastlowast lowastlowast lowastlowast lowast lowastlowastlowastlowast

ATTAATACGAACAAATAAGAAAAATGTGACATTGCAAGAATTAGATATAAAGATCAGAAA-TTAT----------TATGATTTATATAAC--------------ATAAAAGGTTCAG---

AATATTGTCCGATAAATATAAAATTTATTATAAAGACAGCGAAATAAGGTAAAAGG------TAGGAGCAGAGTTTTTTCAATTTTATAGTGATAATAGAACAGTTAGCTCATCTAATTA

Enterotoxin N CNS -TAAA---Enterotoxin N

lowast lowastlowastlowastlowastlowast lowastlowast lowast lowast lowast lowastlowast lowast lowastlowastlowastlowastlowastlowast lowastlowast lowastlowast

GGCACAGGAAAGGGTGATTGGTGCTAATGTTTGGGTAGATGGTATTCAAAAAGAAACAGAACAA----AAAGGATGCATTTTCTTT-----CATTCTAATAATCATCAAAATCAATCATT

Enterotoxin N CNSEnterotoxin N

Enterotoxin N CNSEnterotoxin N

Enterotoxin N CNSEnterotoxin N

TCATATCG

Enterotoxin M (selm)

ttgctattcgcaaaatcatatcgcaaccgctgatgtcggagtt--ttgaatcttaggaacEnterotoxin M CNSEnterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

--------------AGTATGTTTAAATCCCCGCTCACAGTTATGTTTAACTCTTCCTTTC

tattatggta-gctatccaattgaagaccaccaaagtattaatcctgaaaataatcatctTTTTCACACCGCCCCGTCGGGTGTTGCGCACGGGGTTGTTTAACCCCCCCGGAAAAAATT

ttcgcatcaattagttttttctatggataattcgacagtaacagctgaatttaagaacgtEnterotoxin M CNS

Enterotoxin M CNS

TTCCCTCCCTCCTCCTTACTC---------------CCT----------------CCCCT

tgatgatgtaaaggaattcaaaaatcatgctgtagatgtatatggtctaagttatagtggEnterotoxin M CNS TTTTCCTACAAATCCTTTCTCCCCTCCCGGAGTAGAAGTAAA------------------

atattgtttgaaaaacaaatatatatacggtggagttacattagcaggtgat-tatttagEnterotoxin M CNS -------------------TAAAACAATACCCCTACCACCTTAACAATTTTTTTATTGAG

agaaatctagacgtattcct-attaatctttgggttaatggagaacatcaaactatatctEnterotoxin M CNS CCCCTCCCCCCCCTTCTCATTTTTTCTCTTTGAC-----------GTTCCCCCCATTTCT

actgacaaagtatcaactaataaaa-agttagtaacagctcaagaaattgatactaaattEnterotoxin M CNS GCCAATGCTTAACCCACCCTTAAAGGCCATCGGCACATATTAGACACTCC-----CCACC

aagaagatatctacaagaagaatataatatttatggctttaatgatac---aaataaaggEnterotoxin M CNS CGCCCCCTCTTTTTATTAAGGACACAACTCTTTTTATTTTCCCCCAAGAACTAATTCTGA

a-agaaattatggtaataagtcaaaatttagttctgga---------tttaatgcaggaaEnterotoxin M CNS AAAAAAACGTTCCGCTTAGTTCAGAATTTCCTTTGAGCCTTTTATCCTTTTGTACGCGAA

aaatattatttca--------tttgaatgatggttcatcattttcttatgacttatttga

Enterotoxin M CNS AAACCATCAGCCATTGAATCAGTTATCCCATTGCCCTAACTAACCCAATATCTAATCTAA

tactggaacaggacaagctgaaagtttctt-aaaaatatataatgacaacaaaactgtcgEnterotoxin M CNS TTTTCGCCTACGAACGAATAATAGCTTGTGGCGAAAAATATCCAGGATTATCTACTGTAG

aaact--gaaaaattccatttagatgtagaaatatcttataaggacgaaagttgaagtatEnterotoxin M CNS CGCCTCGCCAGACGTCCTTTGAATTGA--ATATGACTCCTACTCCCCCCCGCACCGTCAA

ttcaatcataacttagtaaaggaaatgccatgaaaaaatttaaatatagtttt---atatEnterotoxin M CNS TTCAAACA---------------GGTGGAAGTTAATACTGAATAGTAGGTCTTCTTCCCG

tagtttttatattactttttaacattaaagatcttacgtatgct---caaggtgatattgEnterotoxin M CNS TGCTATTCTTAGAACTTTTAAATTATGTAAACCCAATTTTTAGTGCACTCTTTCTCCCTC

gtg-taggtaacttaagaaatttctatacaaaacatgattatatagatttaaaaggcgtcEnterotoxin M CNS GTTACTCTTAGATGGAGAAGCGTTAAGACAGAAGCTTTGTAT-------------TCCCA

acagataaaaa-cctacctattgcaaatcaacttgaattttcaac-----aggt----acEnterotoxin M CNS GCAGAGTGCAACCTTTCACAATGAAGTTTTTATCTAATTTACACGCCTTTAGGCTCCAAA

caatgatttgatctcagaatctaataattgggacgaaataagtaaatttaaaggaaagaaEnterotoxin M CNS TACGTATTAAATCTCCTCCTGT--TTATCTGACCCTAAG-ATAAAACACGAAAAATTATG

actggatatttttggcattgattataatggtccttgtaaatctaaatacatgtatggaggEnterotoxin M CNS CGTGCCCACTCATCCTAT-------TACTTTCATTATATCTATAAAA-------------

Figure 3 Alignment of the enterotoxin gene sequences found in coagulase-negative staphylococci (CNS) and in coagulase-positivestaphylococci (CPS)

BioMed Research International 13

Enterotoxin (seg) S aureus AY1587031

Enterotoxin (sec) CNS

Enterotoxin (see) S pasteuri NC0227371

Enterotoxin (see) CNS

Enterotoxin (seh) CNS

Enterotoxin (selm) S aureus AF2857601

Enterotoxin (seln) CNS

Enterotoxin (seh) S aureus AJ9375481

Enterotoxin (seln) S aureus AY1587031

Enterotoxin (sec) S aureus X516611

Enterotoxin (selm) CNS

Enterotoxin (seg) CNS

150 100

100

100

100

100

99

98

96

97

72

50 0

Figure 4 Phylogenetic tree generated from the multiple alignments of the enterotoxin sequences of CNS and CPS strains using the ClustalX20 software package The phylogenetic tree was constructed using the Mega 60 software and the unweighted pair group method (UPGMA)

MAR indices varying from 066 to 046 and the remaining04 strains showed MAR indices le026 (Table 3)

The 14 MRCNS strains were resistant to 120573-lactams(oxacillin penicillin andor cefoxitin) and to vancomycincorresponding to 73 of the total CNS strains identifiedwhile 09 strains (64) showed resistance to tetracycline andgentamicin 08 strains (57) were resistant to neomycinerythromycin and chloramphenicol 07 strains (50) wereresistant to sulfamethoprim 05 strains (36) were resistantto linezolid 03 strains (21) were resistant to rifampicin 02strains (14) were resistant to ciprofloxacin and cefepimeand 01 strain (7) was resistant to clindamycin (Table 3)

The multiresistance of CNS strains demonstrated hereinis consistent with previous studies on coagulase-negativeand coagulase-positive staphylococci that have found severalresistant and multiresistant Staphylococcus aureus strains inraw milk meat and fermented meat products [43]

Surprisingly the resistance to chloramphenicol is verysimilar to that estimated for MRCNS strains isolated fromhuman clinical samples Indeed there is no direct correla-tion between the researched features and the origin of thestaphylococci strains since different virulence factors arewidespread such as antibiotic resistance [44] reinforcing thefact that the ability of foodmatrices strains to produce SE andSEl and their multiresistance character must be consideredwhen evaluating the safety hazards of food poisoning

S epidermidis KF6005891 and S hominis JX5199881showed the highestMAR indexes (093 and 080 resp)Thesestrains found in artisanal salami are homologous to strainsisolated from human skin microbiota and should be carefullyconsidered among the potential pathogenic staphylococcifound in food from animal origins which can be caused bycontamination by poor hygienic conditions during salamimanufacturing

Depending on the conditions some species of coagulase-negative staphylococci can present health risks since theyhave shown resistance to antibiotics of therapeutic impor-tance such as beta lactams [15] However multiresistantstrains like S carnosusKJ18620021 and S xylosusCP0072081and CP0087241 were intentionally introduced in the foodmatrix since they are part of the culture starter used inItalian-salami manufacturing As previously discussed foodcan be a reservoir of multiresistant microorganisms that canspread by the consumption of an apparently safe food Dueto the intensive and indiscriminate use of antibiotics forhuman and veterinarian therapeutic purposes multiresistantstaphylococci strains are being selected and reproduced infood matrices [45]

The cefoxitin and oxacillin disk diffusion tests are rec-ommended for determining the resistance and susceptibilitybreakpoint for MRSA surveillance cultures [46] In thisstudy there was a good correlation between the disc testzone diameters for oxacillin and cefoxitin (Table 3) TheMICs for ampicillin andmethicillinoxacillincefoxitin for allsusceptible CNS strainswere determined by the diffusion disctest using 003 to 2mgmLminus1 of each antimicrobial agent

S saprophyticus HF9372521 showed an MIC of003mgmLminus1 formethicillin strain S carnosusKJ8620021 Sxylosus strains CP0087241 and CP0072081 S saprophyticusJX4901221 S hominis JX5199881 and S succinusKC3298241 were resistant to 006mgmLminus1 and two strainsS saprophyticus KJ6991511 and S saprophyticus KJ0046231showed an MIC of 05mgmLminus1 (Table 4) Two of themethicillin-resistant strains S saprophyticus KJ0046231 andS xylosus CP0072081 harbor no enterotoxin gene

Five strains S saprophyticus JX4901221 S carnosusKJ8620021 S xylosusCP0087241 S xylosusKF1980801 andS saprophyticus KJ0046231 showed an MIC for ampicillin

14 BioMed Research International

Table 3 Multiple resistance to antimicrobial as found in CNS strains from salami

Salami origin CNS strains Antimicrobial agent resistance Multiple antimicrobialresistance (MAR) indexlowast

Commercial Staphylococcus spp KF1354451 CIP CLO CPM GEN NEO OXA PENSXT TET and VAN 066

Commercial S carnosus KJ8620021 CIP GEN LZD NEO OXA SXT andTET 046

Commercial S saprophyticus AB6977171 CFO CLO ERI PEN OXA TET andVAN 046

Commercial S saprophyticus EU4309921 CFO CLO ERI GEN LZD NEO OXAPEN SXT TET and VAN 080

Commercial S saprophyticus JX4901221 CFO CLO ERI GEN NEO PEN OXASXT TET and VAN 066

Commercial S saprophyticus KJ0046231 CFO OXA and PEN 020

Commercial S sciuri JX9664361 CFO CLO GEN NEO OXA PEN andTET 046

Commercial S xylosus CP0072081 OXA LZD PEN and VAN 026

Commercial S xylosus CP0087241 CFO CLO ERI GEN NEO OXA PENTET and VAN 060

Artisanal S epidermidis KF6005891 CFO CLI CLO CPM ERI GEN LZDNEO OXA PEN RIF SXT and TET 093

Artisanal S hominis JX5199881 CIP CPM CPO ERI GEN LZD NEOOXA PEN RIF SXT and VAN 080

Artisanal S saprophyticusHF9372521 CFO OXA PEN and SXT 026Artisanal S saprophyticus KJ6991511 OXA PEN and VAN 020

Artisanal S succinus KC3298241 CFO CLO ERI GEN OXA PEN RIFSXT TET and VAN 066

Artisanal S xylosus KF1980801 CFO CLO ERI GEN NEO OXA PENTET and VAN 060

lowastTheMAR index of an isolate is defined as 119886119887 where 119886 represents the number of antimicrobials to which the isolate was resistant and 119887 represents the numberof antimicrobials to which the isolate was subjectedS aureus strains ATCC WB81 (sea) ATCC 13563 (sed) and ATCC 27664 (see) showing a MAR index of 05 and S aureus strains ATCC14458 (seb) andATCCWB72 (sec) and S xylosus ATCC 29971 showing a MAR index of 03 were used as reference strainsCPM cefepime CFO cefoxitin CLO chloramphenicol CIP ciprofloxacin CLI clindamycin ERI erythromycin GEN gentamycin NEO neomycin LZDlinezolid RIF rifampicin TET tetracycline OXA oxacillin PEN penicillin SXT sulfamethoprim and VAN vancomycin

Table 4 Minimal inhibitory concentration (MIC) of compounds used in antimicrobial therapy against staphylococci infections

Salami origin GenBank accession number and similarity () MIC mgmLminus1

Methicillin Ampicillin Vancomycin Linezolid

Commercial

S carnosus KJ8620021 006 003 mdash mdashS xylosus CP0087241 006 003 006 mdash

S saprophyticus JX4901221 006 003 05 mdashS saprophyticus AB6977171 mdash mdash 05 mdashS saprophyticus EU4309921 mdash mdash 025 0125

S succinus KC3298241 006 025 003 mdash

Artisanal

S hominis JX5199881 006 05 mdash mdashS saprophyticus HF9372521 003 mdash mdash mdash

S xylosus KF1980801 003 05 mdashS saprophyticus KJ6991511 05 025 003 mdashS saprophyticus KJ0046231 05 003 003 mdash

S xylosus CP0072081 006 mdash mdash 025Strains S epidermidis KF6005891 and Staphylococcus spp KF1354451 were not susceptible to the antimicrobial concentrations tested in the present study

BioMed Research International 15

of 003mgmLminus1 two strains S succinus KC3298241 and Ssaprophyticus KJ6991511 showed an MIC of 025mgmLminus1and S hominis JX5199881 showed an MIC of 05mgmLminus1(Table 4) S saprophyticus KJ0046231 also showed an MICof 003mgmLminus1 but does not harbor enterotoxin genes It isimportant to note that most of the CNS strains that showedresistance to ampicillin were also resistant to methicillin ashighlighted by the MIC tests

Five strains S saprophyticus HF9372521 S saprophyti-cus KJ6991511 S saprophyticus JX4901221 S saprophyticusKJ0046231 and S succinus KC3298241 showed an MICfor vancomycin of 003mgmLminus1 one strain S xylosusCP0087241 showed an MIC of 006mgmLminus1 one strain Ssaprophyticus EU4309921 showed an MIC of 025mgmLminus1and two strains S xylosus KF1980801 and S saprophyticusAB6977171 showed an MIC of 05mgmLminus1 (Table 4)

Additionally five CNS strains S xylosus CP0087241S saprophyticus JX 4901221 S succinus KC3298241 Ssaprophyticus KJ6991511 and S saprophyticus KJ0046231showed resistance tomethicillin as demonstrated by theMICdeterminations (Table 4)

Previous studies have demonstrated that linezolid isactive against Gram-positive bacteria including methicillin-resistant staphylococci [47] In the present study 05 strainsshowed linezolid resistance in the disc diffusion test but itwas impossible to establish MIC values for S epidermidisKF6005891 Staphylococcus hominis JX5199881 and Staphy-lococcus carnosus KJ8620021The remaining strains S sapro-phyticus EU4309921 and S xylosus CP0072081 presentedan MIC for linezolid of 0125mgmLminus1 and 025mgmLminus1respectively

The MIC of 003mgmLminus1 for penicillin is in accordancewith the previous values estimated for resistant S aureusstrains found in several food matrices such as meat dairyproducts and ready-to-eat food [48]

There is still a lack of information on the antimicro-bial resistance of staphylococci strains from food matricesalthough they are the most worrisome vehicles of dissemi-nation of antibiotic-resistant pathogens [49]

The high resistance found for salami staphylococci strainscan be ascribed to their inappropriate use as growth promot-ers of antimicrobial agents like oxacillin vancomycin chlo-ramphenicol neomycin and erythromycin which are com-monly used in veterinary medicine to treat infections [50]Antimicrobial therapy of infections staphylococci is basedon results of susceptibility tests in vitro [51] Methicillin-resistant coagulase-negative Staphylococcus spp found inready-to-eat products such as meats fish and dairy productsalso offer risks and although they are not classical foodpoisoning bacteria their presence in food offers significantrisks to public health due to the possible spread of antibioticresistance [52]

The standardization of salami quality should includediagnostic methods to screen and quantify the presence ofclassical and newly described enterotoxins directly in thefood matrices as a routine procedure to be conducted by themeat product industry in Brazil

The safety of salami consumption could also be enhancedby the inclusion of amicrobial barrier such as the inclusion ofprobiotic strains producing natural antibiotics or competitiveflora or even the addition of natural bioagents against spoilageor pathogenic microorganisms

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors acknowledge the financial support fromFundacao Carlos Chagas Filho de Amparo a Pesquisa doEstado do Rio de Janeiro (FAPERJ Rio de Janeiro Brazil)Conselho Nacional de Desenvolvimento Cientıfico eTecnologico (CNPq Brasılia Brazil) and Coordenacao deAperfeicoamento de Pessoal de Nıvel Superior (CAPESBrasılia Brazil)

References

[1] ICMSF International Comission on Microbiological Specifica-tions for FoodsMicroorganisms in FoodsTheir Significance andMethods of Enumerations University of Toronto Press TorontoCanada 3rd edition 1983

[2] J F Veras L S Carmo L C Tong et al ldquoA study of theenterotoxigenicity of coagulase-negative and coagulase-positivestaphylococcal isolates from food poisoning outbreaks inMinasGerais Brazilrdquo International Journal of Infectious Diseases vol12 no 4 pp 410ndash415 2008

[3] K Omoe D-L Hu H Takahashi-Omoe A Nakane and KShinagawa ldquoComprehensive analysis of classical and newlydescribed staphylococcal superantigenic toxin genes in Staphy-lococcus aureus isolatesrdquo FEMS Microbiology Letters vol 246no 2 pp 191ndash198 2005

[4] J Kadariya T C Smith and D Thapaliya ldquoStaphylococcusaureus and staphylococcal food-borne disease an ongoing chal-lenge in public healthrdquoBioMedResearch International vol 2014Article ID 827965 9 pages 2014

[5] KOmoeD-LHuH KOno et al ldquoEmetic potentials of newlyidentified staphylococcal enterotoxin-like toxinsrdquo Infection andImmunity vol 81 no 10 pp 3627ndash3631 2013

[6] J K McCormick J M Yarwood and P M SchlievertldquoToxic shock syndrome and bacterial superantigens an updaterdquoAnnual Review of Microbiology vol 55 pp 77ndash104 2001

[7] T Baba F Takeuchi M Kuroda et al ldquoGenome and virulencedeterminants of high virulence community-acquired MRSArdquoThe Lancet vol 359 no 9320 pp 1819ndash1827 2002

[8] Associacao Brasileira de Industria Produtora e Exportadorada Carne Suınas (ABIPECS) ldquoStatistics slaughter ranking2000ndash2014rdquo 2014 httpabpa-brcombrsetoressuinoculturapublicacoesrelatorios-anuais

[9] N Zdolec M Hadziosmanovic L Kozacinski et al ldquoMicrobialand physicochemical succession in fermented sausages pro-duced with bacteriocinogenic culture of Lactobacillus sakei andsemi-purified bacteriocin mesenterocin Yrdquo Meat Science vol80 no 2 pp 480ndash487 2008

16 BioMed Research International

[10] F Ravyts L D Vuyst and F Leroy ldquoBacterial diversity andfunctionalities in food fermentationsrdquo Engineering in Life Sci-ences vol 12 no 4 pp 356ndash367 2012

[11] TOhlsson andN BengtssonTheHurdle ConceptMinimal Pro-cessing Technologies in the Food IndustryWoodhead Publishing2002

[12] S Bernardi B B Golineli and C J Contreras-CastilloldquoRevisao aspectos da aplicacao de culturas starter na producaode embutidos carneos fermentadosrdquo Brazilian Journal of FoodTechnology vol 13 no 2 pp 133ndash140 2010

[13] F Irlinger ldquoSafety assessment of dairy microorganismscoagulase-negative staphylococcirdquo International Journal of FoodMicrobiology vol 126 no 3 pp 302ndash310 2008

[14] B Ghebremedhin F Layer W Konig and B Konig ldquoGeneticclassification and distinguishing of Staphylococcus species basedon different partial gap 16S rRNA hsp60 rpoB sodA and tufgene sequencesrdquo Journal of Clinical Microbiology vol 46 no 3pp 1019ndash1025 2008

[15] I Babic K Markov D Kovacevic et al ldquoIdentification andcharacterization of potential autochthonous starter culturesfrom aCroatian lsquobrandrsquo product lsquoslavonski kulenrsquordquoMeat Sciencevol 88 no 3 pp 517ndash524 2011

[16] MMehrotra GWang andWM Johnson ldquoMultiplex PCR forthe detection of genes for Staphylococcus aureus enterotoxinsexfoliative toxins toxic shock syndrome toxin and methicillinresistancerdquo Journal of Clinical Microbiology vol 38 no 3 pp1032ndash1041 2000

[17] WM Johnson SD Tyler E P Ewan F EAshtonDR Pollardand K R Rozee ldquoDetection of genes for enterotoxins exfolia-tive toxins and toxic shock syndrome toxin 1 in Staphylococcusaureus by the polymerase chain reactionrdquo Journal of ClinicalMicrobiology vol 29 no 3 pp 426ndash430 1991

[18] J Mclauchlin G L Narayanan VMithani andG OrsquoNeill ldquoThedetection of enterotoxins and toxic shock syndrome toxin genesin Staphylococcus aureus by polymerase chain reactionrdquo Journalof Food Protection vol 63 no 4 pp 479ndash488 2000

[19] J P Rosec and O Gigaud ldquoStaphylococcal enterotoxin genes ofclassical and new types detected by PCR in Francerdquo Interna-tional Journal of Food Microbiology vol 77 no 1-2 pp 61ndash702002

[20] K Omoe D-L Hu H Takahashi-Omoe A Nakane andK Shinagawa ldquoIdentification and characterization of a newstaphylococcal enterotoxin-related putative toxin encoded bytwo kinds of plasmidsrdquo Infection and Immunity vol 71 no 10pp 6088ndash6094 2003

[21] N Sergeev D Volokhov V Chizhikov and A Rasooly ldquoSimul-taneous analysis of multiple staphylococcal enterotoxin genesby an oligonucleotide microarray assayrdquo Journal of ClinicalMicrobiology vol 42 no 5 pp 2134ndash2143 2004

[22] S Holtfreter D Grumann M Schmudde et al ldquoClonal distri-bution of superantigen genes in clinical Staphylococcus aureusisolatesrdquo Journal of Clinical Microbiology vol 45 no 8 pp2669ndash2680 2007

[23] W J Mason J S Blevins K Beenken NWibowo N Ojha andM S Smeltzer ldquoMultiplex PCR protocol for the diagnosis ofstaphylococcal infectionrdquo Journal of Clinical Microbiology vol39 no 9 pp 3332ndash3338 2001

[24] E Rahimi and H G Safai ldquoDetection of classical enterotoxinsof Staphylococcus aureus strains isolated frombovine subclinicalmastitis in Isfahan Iranrdquo Veterinary Microbiology vol 141 no3-4 pp 393ndash394 2010

[25] M A Larkin G Blackshields N P Brown et al ldquoClustalW andClustal X version 20rdquo Bioinformatics vol 23 no 21 pp 2947ndash2948 2007

[26] P H A Sneath and R R Sokal Unweighted Pair Group Methodwith Arithmetic Mean Numerical Taxonomy W H FreemanSan Francisco Calif USA 1973

[27] NCCLS ldquoPerformance standards for antimicrobial susceptibil-ity testing 14th informational supplementrdquo Tech Rep M 100-S14 NCCLS 2004

[28] S Corbiere Morot-Bizot S Leroy and R Talon ldquoMonitoringof staphylococcal starters in two French processing plantsmanufacturing dry fermented sausagesrdquo Journal of AppliedMicrobiology vol 102 no 1 pp 238ndash244 2007

[29] R Talon S Leroy I Lebert et al ldquoSafety improvement andpreservation of typical sensory qualities of traditional dryfermented sausages using autochthonous starter culturesrdquo Inter-national Journal of Food Microbiology vol 126 no 1-2 pp 227ndash234 2008

[30] G Mauriello A Casaburi G Blaiotta and F Villani ldquoIsolationand technological properties of coagulase negative staphylo-cocci from fermented sausages of Southern ItalyrdquoMeat Sciencevol 67 no 1 pp 149ndash158 2004

[31] M Janssens N Myter L De Vuyst and F Leroy ldquoSpeciesdiversity and metabolic impact of the microbiota are low inspontaneously acidified Belgian sausages with an added starterculture of Staphylococcus carnosusrdquo Food Microbiology vol 29no 2 pp 167ndash177 2012

[32] G Blaiotta D Ercolini G Mauriello G Salzano and F VillanildquoRapid and reliable identification of Staphylococcus equorum bya species-specific PCR assay targeting the sodA generdquo Systematicand Applied Microbiology vol 27 no 6 pp 696ndash702 2004

[33] L Kozacinski E Drosinos F Caklovica L Cocolin J Gasparik-Reichardt and S Veskovic ldquoInvestigation of microbial associa-tion of traditionally fermented sausagesrdquo Food Technology andBiotechnology vol 46 no 1 pp 93ndash106 2008

[34] S Leroy P Giammarinaro J-P Chacornac I Lebert and RTalon ldquoBiodiversity of indigenous staphylococci of naturallyfermented dry sausages and manufacturing environments ofsmall-scale processing unitsrdquo Food Microbiology vol 27 no 2pp 294ndash301 2010

[35] E Coton M-H Desmonts S Leroy et al ldquoBiodiversity ofCoagulase-Negative Staphylococci in French cheeses dry fer-mented sausages processing environments and clinical sam-plesrdquo International Journal of Food Microbiology vol 137 no 2-3 pp 221ndash229 2010

[36] S Hosseinzadeh and H D Saei ldquoStaphylococcal species associ-ated with bovinemastitis in the NorthWest of Iran emerging ofcoagulase-negative staphylococcirdquo International Journal of Vet-erinary Science and Medicine vol 2 no 1 pp 27ndash34 2014

[37] A Piette and G Verschraegen ldquoRole of coagulase-negativestaphylococci in human diseaserdquo Veterinary Microbiology vol134 no 1-2 pp 45ndash54 2009

[38] T M Moura F S Campos P A DrsquoAzevedo et al ldquoPrevalenceof enterotoxin-encoding genes and antimicrobial resistancein coagulase-negative and coagulase-positive Staphylococcusisolates from black pudding in Southern Brazilrdquo Revista daSociedade Brasileira deMedicina Tropical vol 45 no 5 pp 579ndash585 2012

[39] H I Suzuki T Lefebure P P Bitar and M J StanhopeldquoComparative genomic analysis of the genus Staphylococcusincluding Staphylococcus aureus and its newly described sister

BioMed Research International 17

species Staphylococcus simiaerdquo BMC Genomics vol 13 article38 2012

[40] R P Lamers G Muthukrishnan T A Castoe S Tafur A MCole and C L Parkinson ldquoPhylogenetic relationships amongStaphylococcus species and refinement of cluster groups basedon multilocus datardquo BMC Evolutionary Biology vol 12 article171 2012

[41] I V Pinchuk E J Beswick and V E Reyes ldquoStaphylococcalenterotoxinsrdquo Toxins vol 2 no 8 pp 2177ndash2197 2010

[42] S R Monday and G A Bohach ldquoGenes encoding staphylo-coccal enterotoxins G and I are linked and separated by DNArelated to other staphylococcal enterotoxinsrdquo Journal of NaturalToxins vol 10 no 1 pp 1ndash8 2001

[43] D Y Thomas S Jarraud B Lemercier et al ldquoStaphylococcalenterotoxin-like toxins U2 and V two new staphylococcalsuperantigens arising from recombination within the entero-toxin gene clusterrdquo Infection and Immunity vol 74 pp 4724ndash4734 2006

[44] N Indrawattana O Sungkhachat N Sookrung et al ldquoStaphylo-coccus aureus clinical isolates antibiotic susceptibility molecu-lar characteristics and ability to form biofilmrdquo BioMed ResearchInternational vol 2013 Article ID 314654 11 pages 2013

[45] V Pereira C Lopes A Castro J Silva P Gibbs and P TeixeiraldquoCharacterization for enterotoxin production virulence factorsand antibiotic susceptibility of Staphylococcus aureus isolatesfrom various foods in Portugalrdquo Food Microbiology vol 26 no3 pp 278ndash282 2009

[46] I Anacarso C Condo C Sabia and et al ldquoAntimicrobialresistance and other related virulence factors in staphylococcusspp isolated from food environmental and humans in ItalyrdquoUniversal Journal Microbiology Research vol 1 no 1 pp 1ndash92013

[47] B Martın M Garriga M Hugas S Bover-Cid M T Veciana-Nogues and T Aymerich ldquoMolecular technological and safetycharacterization of Gram-positive catalase-positive cocci fromslightly fermented sausagesrdquo International Journal of FoodMicrobiology vol 107 no 2 pp 148ndash158 2006

[48] NM Broekema T Van Tam T AMonson S AMarshall andD M Warshauer ldquoComparison of cefoxitin and oxacillin diskdiffusionmethods for detection ofmecA-mediated resistance inStaphylococcus aureus in a large-scale studyrdquo Journal of ClinicalMicrobiology vol 47 no 1 pp 217ndash219 2009

[49] E Cercenado F Garcıa-Garrote and E Bouza ldquoIn vitro activityof linezolid against multiply resistant Gram-positive clinicalisolatesrdquo Journal of Antimicrobial Chemotherapy vol 47 no 1pp 77ndash81 2001

[50] A Aydin K Muratoglu M Sudagidan K Bostan B Okukluand SHarsa ldquoPrevalence and antibiotic resistance of foodborneStaphylococcus aureus isolates in Turkeyrdquo Foodborne Pathogensand Disease vol 8 no 1 pp 63ndash69 2011

[51] D Sergelidis A Abrahim T Papadopoulos et al ldquoIsolation ofmethicillin-resistant Staphylococcus spp from ready-to-eat fishproductsrdquo Letters in Applied Microbiology vol 59 no 5 pp500ndash506 2014

[52] I PhillipsM Casewell T Cox et al ldquoDoes the use of antibioticsin food animals pose a risk to human health A critical reviewof published datardquo Journal of Antimicrobial Chemotherapy vol53 no 1 pp 28ndash52 2004

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Page 12: Research Article Safety Evaluation of the Coagulase ...downloads.hindawi.com/journals/bmri/2015/483548.pdf · Research Article Safety Evaluation of the Coagulase-Negative Staphylococci

12 BioMed Research International

lowastlowastlowast lowastlowast lowastlowast lowastlowast lowast lowastlowast lowastlowastlowast lowastlowast lowast lowastlowastlowastlowast

lowastlowast lowast lowastlowast lowast lowast lowastlowast lowastlowastlowastlowastlowast lowast lowast lowast lowastlowast lowast lowast lowastlowast lowast lowast

lowast lowast

lowast lowastlowast lowast lowastlowast lowast lowast lowast lowast lowast lowast lowast lowastlowast lowast lowastlowastlowastlowast lowast

lowast lowastlowast lowast lowast lowastlowast lowast lowastlowastlowast lowast lowastlowast lowast lowastlowast lowastlowast lowast lowast

lowastlowastlowast lowast lowast lowastlowast lowastlowast lowast lowast lowast

lowast lowast lowast lowastlowastlowast lowastlowastlowast lowastlowast lowast lowastlowastlowastlowastlowast lowastlowastlowast lowast

lowastlowast lowast lowast lowastlowast lowastlowastlowast lowastlowast lowast lowast lowastlowastlowastlowast lowastlowast

lowast lowast lowast lowastlowast lowast lowastlowast lowastlowastlowastlowastlowastlowast lowastlowast lowast lowastlowast lowastlowastlowast

lowast lowast lowast lowast lowastlowast lowastlowastlowastlowast lowast lowast lowastlowastlowast lowast lowast lowast lowast lowast

lowast lowast lowast lowast lowastlowastlowast lowast lowast lowastlowast lowastlowast lowast lowastlowastlowast lowast lowastlowastlowast lowast

lowast lowast lowastlowastlowast lowast lowastlowast lowastlowastlowast lowastlowastlowastlowastlowast lowastlowast lowast lowastlowastlowast lowast lowast lowastlowastlowast

lowastlowastlowast lowast lowastlowast lowastlowast lowastlowast lowast lowast lowast lowast lowastlowast lowastlowast lowastlowast lowast lowast

lowast lowast lowast lowast lowastlowast lowast lowast lowastlowast lowastlowast lowast lowastlowastlowast lowastlowastlowastlowast lowast lowastlowastlowastlowastlowast lowast

lowastlowast lowast lowast lowastlowastlowast lowastlowast lowast lowastlowast lowast lowastlowast lowastlowast lowastlowast lowast lowast lowast

lowastlowastlowastlowastlowast lowastlowast lowastlowast lowast lowastlowast lowast lowast lowast lowast lowastlowast lowastlowast

lowast lowast lowastlowast lowastlowast lowastlowastlowastlowastlowastlowast lowastlowast lowast lowast lowast lowast lowast lowast lowast lowast lowast lowast lowast

lowastlowast lowastlowast lowast lowastlowastlowastlowast lowast lowast lowastlowastlowast lowastlowast lowast lowastlowastlowast lowast

lowastlowastlowastlowast lowastlowast lowast lowast lowast lowast lowastlowast lowast lowast lowast lowastlowastlowastlowastlowast lowastlowast lowastlowastlowast lowast

lowast lowastlowastlowast lowastlowastlowastlowastlowast lowast lowast lowast lowastlowast lowast lowast lowastlowast lowast lowastlowastlowast lowastlowast lowast

lowastlowast lowast lowast lowast lowastlowast lowast lowastlowast lowastlowast lowastlowast lowast lowastlowastlowastlowast

ATTAATACGAACAAATAAGAAAAATGTGACATTGCAAGAATTAGATATAAAGATCAGAAA-TTAT----------TATGATTTATATAAC--------------ATAAAAGGTTCAG---

AATATTGTCCGATAAATATAAAATTTATTATAAAGACAGCGAAATAAGGTAAAAGG------TAGGAGCAGAGTTTTTTCAATTTTATAGTGATAATAGAACAGTTAGCTCATCTAATTA

Enterotoxin N CNS -TAAA---Enterotoxin N

lowast lowastlowastlowastlowastlowast lowastlowast lowast lowast lowast lowastlowast lowast lowastlowastlowastlowastlowastlowast lowastlowast lowastlowast

GGCACAGGAAAGGGTGATTGGTGCTAATGTTTGGGTAGATGGTATTCAAAAAGAAACAGAACAA----AAAGGATGCATTTTCTTT-----CATTCTAATAATCATCAAAATCAATCATT

Enterotoxin N CNSEnterotoxin N

Enterotoxin N CNSEnterotoxin N

Enterotoxin N CNSEnterotoxin N

TCATATCG

Enterotoxin M (selm)

ttgctattcgcaaaatcatatcgcaaccgctgatgtcggagtt--ttgaatcttaggaacEnterotoxin M CNSEnterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

Enterotoxin M

--------------AGTATGTTTAAATCCCCGCTCACAGTTATGTTTAACTCTTCCTTTC

tattatggta-gctatccaattgaagaccaccaaagtattaatcctgaaaataatcatctTTTTCACACCGCCCCGTCGGGTGTTGCGCACGGGGTTGTTTAACCCCCCCGGAAAAAATT

ttcgcatcaattagttttttctatggataattcgacagtaacagctgaatttaagaacgtEnterotoxin M CNS

Enterotoxin M CNS

TTCCCTCCCTCCTCCTTACTC---------------CCT----------------CCCCT

tgatgatgtaaaggaattcaaaaatcatgctgtagatgtatatggtctaagttatagtggEnterotoxin M CNS TTTTCCTACAAATCCTTTCTCCCCTCCCGGAGTAGAAGTAAA------------------

atattgtttgaaaaacaaatatatatacggtggagttacattagcaggtgat-tatttagEnterotoxin M CNS -------------------TAAAACAATACCCCTACCACCTTAACAATTTTTTTATTGAG

agaaatctagacgtattcct-attaatctttgggttaatggagaacatcaaactatatctEnterotoxin M CNS CCCCTCCCCCCCCTTCTCATTTTTTCTCTTTGAC-----------GTTCCCCCCATTTCT

actgacaaagtatcaactaataaaa-agttagtaacagctcaagaaattgatactaaattEnterotoxin M CNS GCCAATGCTTAACCCACCCTTAAAGGCCATCGGCACATATTAGACACTCC-----CCACC

aagaagatatctacaagaagaatataatatttatggctttaatgatac---aaataaaggEnterotoxin M CNS CGCCCCCTCTTTTTATTAAGGACACAACTCTTTTTATTTTCCCCCAAGAACTAATTCTGA

a-agaaattatggtaataagtcaaaatttagttctgga---------tttaatgcaggaaEnterotoxin M CNS AAAAAAACGTTCCGCTTAGTTCAGAATTTCCTTTGAGCCTTTTATCCTTTTGTACGCGAA

aaatattatttca--------tttgaatgatggttcatcattttcttatgacttatttga

Enterotoxin M CNS AAACCATCAGCCATTGAATCAGTTATCCCATTGCCCTAACTAACCCAATATCTAATCTAA

tactggaacaggacaagctgaaagtttctt-aaaaatatataatgacaacaaaactgtcgEnterotoxin M CNS TTTTCGCCTACGAACGAATAATAGCTTGTGGCGAAAAATATCCAGGATTATCTACTGTAG

aaact--gaaaaattccatttagatgtagaaatatcttataaggacgaaagttgaagtatEnterotoxin M CNS CGCCTCGCCAGACGTCCTTTGAATTGA--ATATGACTCCTACTCCCCCCCGCACCGTCAA

ttcaatcataacttagtaaaggaaatgccatgaaaaaatttaaatatagtttt---atatEnterotoxin M CNS TTCAAACA---------------GGTGGAAGTTAATACTGAATAGTAGGTCTTCTTCCCG

tagtttttatattactttttaacattaaagatcttacgtatgct---caaggtgatattgEnterotoxin M CNS TGCTATTCTTAGAACTTTTAAATTATGTAAACCCAATTTTTAGTGCACTCTTTCTCCCTC

gtg-taggtaacttaagaaatttctatacaaaacatgattatatagatttaaaaggcgtcEnterotoxin M CNS GTTACTCTTAGATGGAGAAGCGTTAAGACAGAAGCTTTGTAT-------------TCCCA

acagataaaaa-cctacctattgcaaatcaacttgaattttcaac-----aggt----acEnterotoxin M CNS GCAGAGTGCAACCTTTCACAATGAAGTTTTTATCTAATTTACACGCCTTTAGGCTCCAAA

caatgatttgatctcagaatctaataattgggacgaaataagtaaatttaaaggaaagaaEnterotoxin M CNS TACGTATTAAATCTCCTCCTGT--TTATCTGACCCTAAG-ATAAAACACGAAAAATTATG

actggatatttttggcattgattataatggtccttgtaaatctaaatacatgtatggaggEnterotoxin M CNS CGTGCCCACTCATCCTAT-------TACTTTCATTATATCTATAAAA-------------

Figure 3 Alignment of the enterotoxin gene sequences found in coagulase-negative staphylococci (CNS) and in coagulase-positivestaphylococci (CPS)

BioMed Research International 13

Enterotoxin (seg) S aureus AY1587031

Enterotoxin (sec) CNS

Enterotoxin (see) S pasteuri NC0227371

Enterotoxin (see) CNS

Enterotoxin (seh) CNS

Enterotoxin (selm) S aureus AF2857601

Enterotoxin (seln) CNS

Enterotoxin (seh) S aureus AJ9375481

Enterotoxin (seln) S aureus AY1587031

Enterotoxin (sec) S aureus X516611

Enterotoxin (selm) CNS

Enterotoxin (seg) CNS

150 100

100

100

100

100

99

98

96

97

72

50 0

Figure 4 Phylogenetic tree generated from the multiple alignments of the enterotoxin sequences of CNS and CPS strains using the ClustalX20 software package The phylogenetic tree was constructed using the Mega 60 software and the unweighted pair group method (UPGMA)

MAR indices varying from 066 to 046 and the remaining04 strains showed MAR indices le026 (Table 3)

The 14 MRCNS strains were resistant to 120573-lactams(oxacillin penicillin andor cefoxitin) and to vancomycincorresponding to 73 of the total CNS strains identifiedwhile 09 strains (64) showed resistance to tetracycline andgentamicin 08 strains (57) were resistant to neomycinerythromycin and chloramphenicol 07 strains (50) wereresistant to sulfamethoprim 05 strains (36) were resistantto linezolid 03 strains (21) were resistant to rifampicin 02strains (14) were resistant to ciprofloxacin and cefepimeand 01 strain (7) was resistant to clindamycin (Table 3)

The multiresistance of CNS strains demonstrated hereinis consistent with previous studies on coagulase-negativeand coagulase-positive staphylococci that have found severalresistant and multiresistant Staphylococcus aureus strains inraw milk meat and fermented meat products [43]

Surprisingly the resistance to chloramphenicol is verysimilar to that estimated for MRCNS strains isolated fromhuman clinical samples Indeed there is no direct correla-tion between the researched features and the origin of thestaphylococci strains since different virulence factors arewidespread such as antibiotic resistance [44] reinforcing thefact that the ability of foodmatrices strains to produce SE andSEl and their multiresistance character must be consideredwhen evaluating the safety hazards of food poisoning

S epidermidis KF6005891 and S hominis JX5199881showed the highestMAR indexes (093 and 080 resp)Thesestrains found in artisanal salami are homologous to strainsisolated from human skin microbiota and should be carefullyconsidered among the potential pathogenic staphylococcifound in food from animal origins which can be caused bycontamination by poor hygienic conditions during salamimanufacturing

Depending on the conditions some species of coagulase-negative staphylococci can present health risks since theyhave shown resistance to antibiotics of therapeutic impor-tance such as beta lactams [15] However multiresistantstrains like S carnosusKJ18620021 and S xylosusCP0072081and CP0087241 were intentionally introduced in the foodmatrix since they are part of the culture starter used inItalian-salami manufacturing As previously discussed foodcan be a reservoir of multiresistant microorganisms that canspread by the consumption of an apparently safe food Dueto the intensive and indiscriminate use of antibiotics forhuman and veterinarian therapeutic purposes multiresistantstaphylococci strains are being selected and reproduced infood matrices [45]

The cefoxitin and oxacillin disk diffusion tests are rec-ommended for determining the resistance and susceptibilitybreakpoint for MRSA surveillance cultures [46] In thisstudy there was a good correlation between the disc testzone diameters for oxacillin and cefoxitin (Table 3) TheMICs for ampicillin andmethicillinoxacillincefoxitin for allsusceptible CNS strainswere determined by the diffusion disctest using 003 to 2mgmLminus1 of each antimicrobial agent

S saprophyticus HF9372521 showed an MIC of003mgmLminus1 formethicillin strain S carnosusKJ8620021 Sxylosus strains CP0087241 and CP0072081 S saprophyticusJX4901221 S hominis JX5199881 and S succinusKC3298241 were resistant to 006mgmLminus1 and two strainsS saprophyticus KJ6991511 and S saprophyticus KJ0046231showed an MIC of 05mgmLminus1 (Table 4) Two of themethicillin-resistant strains S saprophyticus KJ0046231 andS xylosus CP0072081 harbor no enterotoxin gene

Five strains S saprophyticus JX4901221 S carnosusKJ8620021 S xylosusCP0087241 S xylosusKF1980801 andS saprophyticus KJ0046231 showed an MIC for ampicillin

14 BioMed Research International

Table 3 Multiple resistance to antimicrobial as found in CNS strains from salami

Salami origin CNS strains Antimicrobial agent resistance Multiple antimicrobialresistance (MAR) indexlowast

Commercial Staphylococcus spp KF1354451 CIP CLO CPM GEN NEO OXA PENSXT TET and VAN 066

Commercial S carnosus KJ8620021 CIP GEN LZD NEO OXA SXT andTET 046

Commercial S saprophyticus AB6977171 CFO CLO ERI PEN OXA TET andVAN 046

Commercial S saprophyticus EU4309921 CFO CLO ERI GEN LZD NEO OXAPEN SXT TET and VAN 080

Commercial S saprophyticus JX4901221 CFO CLO ERI GEN NEO PEN OXASXT TET and VAN 066

Commercial S saprophyticus KJ0046231 CFO OXA and PEN 020

Commercial S sciuri JX9664361 CFO CLO GEN NEO OXA PEN andTET 046

Commercial S xylosus CP0072081 OXA LZD PEN and VAN 026

Commercial S xylosus CP0087241 CFO CLO ERI GEN NEO OXA PENTET and VAN 060

Artisanal S epidermidis KF6005891 CFO CLI CLO CPM ERI GEN LZDNEO OXA PEN RIF SXT and TET 093

Artisanal S hominis JX5199881 CIP CPM CPO ERI GEN LZD NEOOXA PEN RIF SXT and VAN 080

Artisanal S saprophyticusHF9372521 CFO OXA PEN and SXT 026Artisanal S saprophyticus KJ6991511 OXA PEN and VAN 020

Artisanal S succinus KC3298241 CFO CLO ERI GEN OXA PEN RIFSXT TET and VAN 066

Artisanal S xylosus KF1980801 CFO CLO ERI GEN NEO OXA PENTET and VAN 060

lowastTheMAR index of an isolate is defined as 119886119887 where 119886 represents the number of antimicrobials to which the isolate was resistant and 119887 represents the numberof antimicrobials to which the isolate was subjectedS aureus strains ATCC WB81 (sea) ATCC 13563 (sed) and ATCC 27664 (see) showing a MAR index of 05 and S aureus strains ATCC14458 (seb) andATCCWB72 (sec) and S xylosus ATCC 29971 showing a MAR index of 03 were used as reference strainsCPM cefepime CFO cefoxitin CLO chloramphenicol CIP ciprofloxacin CLI clindamycin ERI erythromycin GEN gentamycin NEO neomycin LZDlinezolid RIF rifampicin TET tetracycline OXA oxacillin PEN penicillin SXT sulfamethoprim and VAN vancomycin

Table 4 Minimal inhibitory concentration (MIC) of compounds used in antimicrobial therapy against staphylococci infections

Salami origin GenBank accession number and similarity () MIC mgmLminus1

Methicillin Ampicillin Vancomycin Linezolid

Commercial

S carnosus KJ8620021 006 003 mdash mdashS xylosus CP0087241 006 003 006 mdash

S saprophyticus JX4901221 006 003 05 mdashS saprophyticus AB6977171 mdash mdash 05 mdashS saprophyticus EU4309921 mdash mdash 025 0125

S succinus KC3298241 006 025 003 mdash

Artisanal

S hominis JX5199881 006 05 mdash mdashS saprophyticus HF9372521 003 mdash mdash mdash

S xylosus KF1980801 003 05 mdashS saprophyticus KJ6991511 05 025 003 mdashS saprophyticus KJ0046231 05 003 003 mdash

S xylosus CP0072081 006 mdash mdash 025Strains S epidermidis KF6005891 and Staphylococcus spp KF1354451 were not susceptible to the antimicrobial concentrations tested in the present study

BioMed Research International 15

of 003mgmLminus1 two strains S succinus KC3298241 and Ssaprophyticus KJ6991511 showed an MIC of 025mgmLminus1and S hominis JX5199881 showed an MIC of 05mgmLminus1(Table 4) S saprophyticus KJ0046231 also showed an MICof 003mgmLminus1 but does not harbor enterotoxin genes It isimportant to note that most of the CNS strains that showedresistance to ampicillin were also resistant to methicillin ashighlighted by the MIC tests

Five strains S saprophyticus HF9372521 S saprophyti-cus KJ6991511 S saprophyticus JX4901221 S saprophyticusKJ0046231 and S succinus KC3298241 showed an MICfor vancomycin of 003mgmLminus1 one strain S xylosusCP0087241 showed an MIC of 006mgmLminus1 one strain Ssaprophyticus EU4309921 showed an MIC of 025mgmLminus1and two strains S xylosus KF1980801 and S saprophyticusAB6977171 showed an MIC of 05mgmLminus1 (Table 4)

Additionally five CNS strains S xylosus CP0087241S saprophyticus JX 4901221 S succinus KC3298241 Ssaprophyticus KJ6991511 and S saprophyticus KJ0046231showed resistance tomethicillin as demonstrated by theMICdeterminations (Table 4)

Previous studies have demonstrated that linezolid isactive against Gram-positive bacteria including methicillin-resistant staphylococci [47] In the present study 05 strainsshowed linezolid resistance in the disc diffusion test but itwas impossible to establish MIC values for S epidermidisKF6005891 Staphylococcus hominis JX5199881 and Staphy-lococcus carnosus KJ8620021The remaining strains S sapro-phyticus EU4309921 and S xylosus CP0072081 presentedan MIC for linezolid of 0125mgmLminus1 and 025mgmLminus1respectively

The MIC of 003mgmLminus1 for penicillin is in accordancewith the previous values estimated for resistant S aureusstrains found in several food matrices such as meat dairyproducts and ready-to-eat food [48]

There is still a lack of information on the antimicro-bial resistance of staphylococci strains from food matricesalthough they are the most worrisome vehicles of dissemi-nation of antibiotic-resistant pathogens [49]

The high resistance found for salami staphylococci strainscan be ascribed to their inappropriate use as growth promot-ers of antimicrobial agents like oxacillin vancomycin chlo-ramphenicol neomycin and erythromycin which are com-monly used in veterinary medicine to treat infections [50]Antimicrobial therapy of infections staphylococci is basedon results of susceptibility tests in vitro [51] Methicillin-resistant coagulase-negative Staphylococcus spp found inready-to-eat products such as meats fish and dairy productsalso offer risks and although they are not classical foodpoisoning bacteria their presence in food offers significantrisks to public health due to the possible spread of antibioticresistance [52]

The standardization of salami quality should includediagnostic methods to screen and quantify the presence ofclassical and newly described enterotoxins directly in thefood matrices as a routine procedure to be conducted by themeat product industry in Brazil

The safety of salami consumption could also be enhancedby the inclusion of amicrobial barrier such as the inclusion ofprobiotic strains producing natural antibiotics or competitiveflora or even the addition of natural bioagents against spoilageor pathogenic microorganisms

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors acknowledge the financial support fromFundacao Carlos Chagas Filho de Amparo a Pesquisa doEstado do Rio de Janeiro (FAPERJ Rio de Janeiro Brazil)Conselho Nacional de Desenvolvimento Cientıfico eTecnologico (CNPq Brasılia Brazil) and Coordenacao deAperfeicoamento de Pessoal de Nıvel Superior (CAPESBrasılia Brazil)

References

[1] ICMSF International Comission on Microbiological Specifica-tions for FoodsMicroorganisms in FoodsTheir Significance andMethods of Enumerations University of Toronto Press TorontoCanada 3rd edition 1983

[2] J F Veras L S Carmo L C Tong et al ldquoA study of theenterotoxigenicity of coagulase-negative and coagulase-positivestaphylococcal isolates from food poisoning outbreaks inMinasGerais Brazilrdquo International Journal of Infectious Diseases vol12 no 4 pp 410ndash415 2008

[3] K Omoe D-L Hu H Takahashi-Omoe A Nakane and KShinagawa ldquoComprehensive analysis of classical and newlydescribed staphylococcal superantigenic toxin genes in Staphy-lococcus aureus isolatesrdquo FEMS Microbiology Letters vol 246no 2 pp 191ndash198 2005

[4] J Kadariya T C Smith and D Thapaliya ldquoStaphylococcusaureus and staphylococcal food-borne disease an ongoing chal-lenge in public healthrdquoBioMedResearch International vol 2014Article ID 827965 9 pages 2014

[5] KOmoeD-LHuH KOno et al ldquoEmetic potentials of newlyidentified staphylococcal enterotoxin-like toxinsrdquo Infection andImmunity vol 81 no 10 pp 3627ndash3631 2013

[6] J K McCormick J M Yarwood and P M SchlievertldquoToxic shock syndrome and bacterial superantigens an updaterdquoAnnual Review of Microbiology vol 55 pp 77ndash104 2001

[7] T Baba F Takeuchi M Kuroda et al ldquoGenome and virulencedeterminants of high virulence community-acquired MRSArdquoThe Lancet vol 359 no 9320 pp 1819ndash1827 2002

[8] Associacao Brasileira de Industria Produtora e Exportadorada Carne Suınas (ABIPECS) ldquoStatistics slaughter ranking2000ndash2014rdquo 2014 httpabpa-brcombrsetoressuinoculturapublicacoesrelatorios-anuais

[9] N Zdolec M Hadziosmanovic L Kozacinski et al ldquoMicrobialand physicochemical succession in fermented sausages pro-duced with bacteriocinogenic culture of Lactobacillus sakei andsemi-purified bacteriocin mesenterocin Yrdquo Meat Science vol80 no 2 pp 480ndash487 2008

16 BioMed Research International

[10] F Ravyts L D Vuyst and F Leroy ldquoBacterial diversity andfunctionalities in food fermentationsrdquo Engineering in Life Sci-ences vol 12 no 4 pp 356ndash367 2012

[11] TOhlsson andN BengtssonTheHurdle ConceptMinimal Pro-cessing Technologies in the Food IndustryWoodhead Publishing2002

[12] S Bernardi B B Golineli and C J Contreras-CastilloldquoRevisao aspectos da aplicacao de culturas starter na producaode embutidos carneos fermentadosrdquo Brazilian Journal of FoodTechnology vol 13 no 2 pp 133ndash140 2010

[13] F Irlinger ldquoSafety assessment of dairy microorganismscoagulase-negative staphylococcirdquo International Journal of FoodMicrobiology vol 126 no 3 pp 302ndash310 2008

[14] B Ghebremedhin F Layer W Konig and B Konig ldquoGeneticclassification and distinguishing of Staphylococcus species basedon different partial gap 16S rRNA hsp60 rpoB sodA and tufgene sequencesrdquo Journal of Clinical Microbiology vol 46 no 3pp 1019ndash1025 2008

[15] I Babic K Markov D Kovacevic et al ldquoIdentification andcharacterization of potential autochthonous starter culturesfrom aCroatian lsquobrandrsquo product lsquoslavonski kulenrsquordquoMeat Sciencevol 88 no 3 pp 517ndash524 2011

[16] MMehrotra GWang andWM Johnson ldquoMultiplex PCR forthe detection of genes for Staphylococcus aureus enterotoxinsexfoliative toxins toxic shock syndrome toxin and methicillinresistancerdquo Journal of Clinical Microbiology vol 38 no 3 pp1032ndash1041 2000

[17] WM Johnson SD Tyler E P Ewan F EAshtonDR Pollardand K R Rozee ldquoDetection of genes for enterotoxins exfolia-tive toxins and toxic shock syndrome toxin 1 in Staphylococcusaureus by the polymerase chain reactionrdquo Journal of ClinicalMicrobiology vol 29 no 3 pp 426ndash430 1991

[18] J Mclauchlin G L Narayanan VMithani andG OrsquoNeill ldquoThedetection of enterotoxins and toxic shock syndrome toxin genesin Staphylococcus aureus by polymerase chain reactionrdquo Journalof Food Protection vol 63 no 4 pp 479ndash488 2000

[19] J P Rosec and O Gigaud ldquoStaphylococcal enterotoxin genes ofclassical and new types detected by PCR in Francerdquo Interna-tional Journal of Food Microbiology vol 77 no 1-2 pp 61ndash702002

[20] K Omoe D-L Hu H Takahashi-Omoe A Nakane andK Shinagawa ldquoIdentification and characterization of a newstaphylococcal enterotoxin-related putative toxin encoded bytwo kinds of plasmidsrdquo Infection and Immunity vol 71 no 10pp 6088ndash6094 2003

[21] N Sergeev D Volokhov V Chizhikov and A Rasooly ldquoSimul-taneous analysis of multiple staphylococcal enterotoxin genesby an oligonucleotide microarray assayrdquo Journal of ClinicalMicrobiology vol 42 no 5 pp 2134ndash2143 2004

[22] S Holtfreter D Grumann M Schmudde et al ldquoClonal distri-bution of superantigen genes in clinical Staphylococcus aureusisolatesrdquo Journal of Clinical Microbiology vol 45 no 8 pp2669ndash2680 2007

[23] W J Mason J S Blevins K Beenken NWibowo N Ojha andM S Smeltzer ldquoMultiplex PCR protocol for the diagnosis ofstaphylococcal infectionrdquo Journal of Clinical Microbiology vol39 no 9 pp 3332ndash3338 2001

[24] E Rahimi and H G Safai ldquoDetection of classical enterotoxinsof Staphylococcus aureus strains isolated frombovine subclinicalmastitis in Isfahan Iranrdquo Veterinary Microbiology vol 141 no3-4 pp 393ndash394 2010

[25] M A Larkin G Blackshields N P Brown et al ldquoClustalW andClustal X version 20rdquo Bioinformatics vol 23 no 21 pp 2947ndash2948 2007

[26] P H A Sneath and R R Sokal Unweighted Pair Group Methodwith Arithmetic Mean Numerical Taxonomy W H FreemanSan Francisco Calif USA 1973

[27] NCCLS ldquoPerformance standards for antimicrobial susceptibil-ity testing 14th informational supplementrdquo Tech Rep M 100-S14 NCCLS 2004

[28] S Corbiere Morot-Bizot S Leroy and R Talon ldquoMonitoringof staphylococcal starters in two French processing plantsmanufacturing dry fermented sausagesrdquo Journal of AppliedMicrobiology vol 102 no 1 pp 238ndash244 2007

[29] R Talon S Leroy I Lebert et al ldquoSafety improvement andpreservation of typical sensory qualities of traditional dryfermented sausages using autochthonous starter culturesrdquo Inter-national Journal of Food Microbiology vol 126 no 1-2 pp 227ndash234 2008

[30] G Mauriello A Casaburi G Blaiotta and F Villani ldquoIsolationand technological properties of coagulase negative staphylo-cocci from fermented sausages of Southern ItalyrdquoMeat Sciencevol 67 no 1 pp 149ndash158 2004

[31] M Janssens N Myter L De Vuyst and F Leroy ldquoSpeciesdiversity and metabolic impact of the microbiota are low inspontaneously acidified Belgian sausages with an added starterculture of Staphylococcus carnosusrdquo Food Microbiology vol 29no 2 pp 167ndash177 2012

[32] G Blaiotta D Ercolini G Mauriello G Salzano and F VillanildquoRapid and reliable identification of Staphylococcus equorum bya species-specific PCR assay targeting the sodA generdquo Systematicand Applied Microbiology vol 27 no 6 pp 696ndash702 2004

[33] L Kozacinski E Drosinos F Caklovica L Cocolin J Gasparik-Reichardt and S Veskovic ldquoInvestigation of microbial associa-tion of traditionally fermented sausagesrdquo Food Technology andBiotechnology vol 46 no 1 pp 93ndash106 2008

[34] S Leroy P Giammarinaro J-P Chacornac I Lebert and RTalon ldquoBiodiversity of indigenous staphylococci of naturallyfermented dry sausages and manufacturing environments ofsmall-scale processing unitsrdquo Food Microbiology vol 27 no 2pp 294ndash301 2010

[35] E Coton M-H Desmonts S Leroy et al ldquoBiodiversity ofCoagulase-Negative Staphylococci in French cheeses dry fer-mented sausages processing environments and clinical sam-plesrdquo International Journal of Food Microbiology vol 137 no 2-3 pp 221ndash229 2010

[36] S Hosseinzadeh and H D Saei ldquoStaphylococcal species associ-ated with bovinemastitis in the NorthWest of Iran emerging ofcoagulase-negative staphylococcirdquo International Journal of Vet-erinary Science and Medicine vol 2 no 1 pp 27ndash34 2014

[37] A Piette and G Verschraegen ldquoRole of coagulase-negativestaphylococci in human diseaserdquo Veterinary Microbiology vol134 no 1-2 pp 45ndash54 2009

[38] T M Moura F S Campos P A DrsquoAzevedo et al ldquoPrevalenceof enterotoxin-encoding genes and antimicrobial resistancein coagulase-negative and coagulase-positive Staphylococcusisolates from black pudding in Southern Brazilrdquo Revista daSociedade Brasileira deMedicina Tropical vol 45 no 5 pp 579ndash585 2012

[39] H I Suzuki T Lefebure P P Bitar and M J StanhopeldquoComparative genomic analysis of the genus Staphylococcusincluding Staphylococcus aureus and its newly described sister

BioMed Research International 17

species Staphylococcus simiaerdquo BMC Genomics vol 13 article38 2012

[40] R P Lamers G Muthukrishnan T A Castoe S Tafur A MCole and C L Parkinson ldquoPhylogenetic relationships amongStaphylococcus species and refinement of cluster groups basedon multilocus datardquo BMC Evolutionary Biology vol 12 article171 2012

[41] I V Pinchuk E J Beswick and V E Reyes ldquoStaphylococcalenterotoxinsrdquo Toxins vol 2 no 8 pp 2177ndash2197 2010

[42] S R Monday and G A Bohach ldquoGenes encoding staphylo-coccal enterotoxins G and I are linked and separated by DNArelated to other staphylococcal enterotoxinsrdquo Journal of NaturalToxins vol 10 no 1 pp 1ndash8 2001

[43] D Y Thomas S Jarraud B Lemercier et al ldquoStaphylococcalenterotoxin-like toxins U2 and V two new staphylococcalsuperantigens arising from recombination within the entero-toxin gene clusterrdquo Infection and Immunity vol 74 pp 4724ndash4734 2006

[44] N Indrawattana O Sungkhachat N Sookrung et al ldquoStaphylo-coccus aureus clinical isolates antibiotic susceptibility molecu-lar characteristics and ability to form biofilmrdquo BioMed ResearchInternational vol 2013 Article ID 314654 11 pages 2013

[45] V Pereira C Lopes A Castro J Silva P Gibbs and P TeixeiraldquoCharacterization for enterotoxin production virulence factorsand antibiotic susceptibility of Staphylococcus aureus isolatesfrom various foods in Portugalrdquo Food Microbiology vol 26 no3 pp 278ndash282 2009

[46] I Anacarso C Condo C Sabia and et al ldquoAntimicrobialresistance and other related virulence factors in staphylococcusspp isolated from food environmental and humans in ItalyrdquoUniversal Journal Microbiology Research vol 1 no 1 pp 1ndash92013

[47] B Martın M Garriga M Hugas S Bover-Cid M T Veciana-Nogues and T Aymerich ldquoMolecular technological and safetycharacterization of Gram-positive catalase-positive cocci fromslightly fermented sausagesrdquo International Journal of FoodMicrobiology vol 107 no 2 pp 148ndash158 2006

[48] NM Broekema T Van Tam T AMonson S AMarshall andD M Warshauer ldquoComparison of cefoxitin and oxacillin diskdiffusionmethods for detection ofmecA-mediated resistance inStaphylococcus aureus in a large-scale studyrdquo Journal of ClinicalMicrobiology vol 47 no 1 pp 217ndash219 2009

[49] E Cercenado F Garcıa-Garrote and E Bouza ldquoIn vitro activityof linezolid against multiply resistant Gram-positive clinicalisolatesrdquo Journal of Antimicrobial Chemotherapy vol 47 no 1pp 77ndash81 2001

[50] A Aydin K Muratoglu M Sudagidan K Bostan B Okukluand SHarsa ldquoPrevalence and antibiotic resistance of foodborneStaphylococcus aureus isolates in Turkeyrdquo Foodborne Pathogensand Disease vol 8 no 1 pp 63ndash69 2011

[51] D Sergelidis A Abrahim T Papadopoulos et al ldquoIsolation ofmethicillin-resistant Staphylococcus spp from ready-to-eat fishproductsrdquo Letters in Applied Microbiology vol 59 no 5 pp500ndash506 2014

[52] I PhillipsM Casewell T Cox et al ldquoDoes the use of antibioticsin food animals pose a risk to human health A critical reviewof published datardquo Journal of Antimicrobial Chemotherapy vol53 no 1 pp 28ndash52 2004

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Page 13: Research Article Safety Evaluation of the Coagulase ...downloads.hindawi.com/journals/bmri/2015/483548.pdf · Research Article Safety Evaluation of the Coagulase-Negative Staphylococci

BioMed Research International 13

Enterotoxin (seg) S aureus AY1587031

Enterotoxin (sec) CNS

Enterotoxin (see) S pasteuri NC0227371

Enterotoxin (see) CNS

Enterotoxin (seh) CNS

Enterotoxin (selm) S aureus AF2857601

Enterotoxin (seln) CNS

Enterotoxin (seh) S aureus AJ9375481

Enterotoxin (seln) S aureus AY1587031

Enterotoxin (sec) S aureus X516611

Enterotoxin (selm) CNS

Enterotoxin (seg) CNS

150 100

100

100

100

100

99

98

96

97

72

50 0

Figure 4 Phylogenetic tree generated from the multiple alignments of the enterotoxin sequences of CNS and CPS strains using the ClustalX20 software package The phylogenetic tree was constructed using the Mega 60 software and the unweighted pair group method (UPGMA)

MAR indices varying from 066 to 046 and the remaining04 strains showed MAR indices le026 (Table 3)

The 14 MRCNS strains were resistant to 120573-lactams(oxacillin penicillin andor cefoxitin) and to vancomycincorresponding to 73 of the total CNS strains identifiedwhile 09 strains (64) showed resistance to tetracycline andgentamicin 08 strains (57) were resistant to neomycinerythromycin and chloramphenicol 07 strains (50) wereresistant to sulfamethoprim 05 strains (36) were resistantto linezolid 03 strains (21) were resistant to rifampicin 02strains (14) were resistant to ciprofloxacin and cefepimeand 01 strain (7) was resistant to clindamycin (Table 3)

The multiresistance of CNS strains demonstrated hereinis consistent with previous studies on coagulase-negativeand coagulase-positive staphylococci that have found severalresistant and multiresistant Staphylococcus aureus strains inraw milk meat and fermented meat products [43]

Surprisingly the resistance to chloramphenicol is verysimilar to that estimated for MRCNS strains isolated fromhuman clinical samples Indeed there is no direct correla-tion between the researched features and the origin of thestaphylococci strains since different virulence factors arewidespread such as antibiotic resistance [44] reinforcing thefact that the ability of foodmatrices strains to produce SE andSEl and their multiresistance character must be consideredwhen evaluating the safety hazards of food poisoning

S epidermidis KF6005891 and S hominis JX5199881showed the highestMAR indexes (093 and 080 resp)Thesestrains found in artisanal salami are homologous to strainsisolated from human skin microbiota and should be carefullyconsidered among the potential pathogenic staphylococcifound in food from animal origins which can be caused bycontamination by poor hygienic conditions during salamimanufacturing

Depending on the conditions some species of coagulase-negative staphylococci can present health risks since theyhave shown resistance to antibiotics of therapeutic impor-tance such as beta lactams [15] However multiresistantstrains like S carnosusKJ18620021 and S xylosusCP0072081and CP0087241 were intentionally introduced in the foodmatrix since they are part of the culture starter used inItalian-salami manufacturing As previously discussed foodcan be a reservoir of multiresistant microorganisms that canspread by the consumption of an apparently safe food Dueto the intensive and indiscriminate use of antibiotics forhuman and veterinarian therapeutic purposes multiresistantstaphylococci strains are being selected and reproduced infood matrices [45]

The cefoxitin and oxacillin disk diffusion tests are rec-ommended for determining the resistance and susceptibilitybreakpoint for MRSA surveillance cultures [46] In thisstudy there was a good correlation between the disc testzone diameters for oxacillin and cefoxitin (Table 3) TheMICs for ampicillin andmethicillinoxacillincefoxitin for allsusceptible CNS strainswere determined by the diffusion disctest using 003 to 2mgmLminus1 of each antimicrobial agent

S saprophyticus HF9372521 showed an MIC of003mgmLminus1 formethicillin strain S carnosusKJ8620021 Sxylosus strains CP0087241 and CP0072081 S saprophyticusJX4901221 S hominis JX5199881 and S succinusKC3298241 were resistant to 006mgmLminus1 and two strainsS saprophyticus KJ6991511 and S saprophyticus KJ0046231showed an MIC of 05mgmLminus1 (Table 4) Two of themethicillin-resistant strains S saprophyticus KJ0046231 andS xylosus CP0072081 harbor no enterotoxin gene

Five strains S saprophyticus JX4901221 S carnosusKJ8620021 S xylosusCP0087241 S xylosusKF1980801 andS saprophyticus KJ0046231 showed an MIC for ampicillin

14 BioMed Research International

Table 3 Multiple resistance to antimicrobial as found in CNS strains from salami

Salami origin CNS strains Antimicrobial agent resistance Multiple antimicrobialresistance (MAR) indexlowast

Commercial Staphylococcus spp KF1354451 CIP CLO CPM GEN NEO OXA PENSXT TET and VAN 066

Commercial S carnosus KJ8620021 CIP GEN LZD NEO OXA SXT andTET 046

Commercial S saprophyticus AB6977171 CFO CLO ERI PEN OXA TET andVAN 046

Commercial S saprophyticus EU4309921 CFO CLO ERI GEN LZD NEO OXAPEN SXT TET and VAN 080

Commercial S saprophyticus JX4901221 CFO CLO ERI GEN NEO PEN OXASXT TET and VAN 066

Commercial S saprophyticus KJ0046231 CFO OXA and PEN 020

Commercial S sciuri JX9664361 CFO CLO GEN NEO OXA PEN andTET 046

Commercial S xylosus CP0072081 OXA LZD PEN and VAN 026

Commercial S xylosus CP0087241 CFO CLO ERI GEN NEO OXA PENTET and VAN 060

Artisanal S epidermidis KF6005891 CFO CLI CLO CPM ERI GEN LZDNEO OXA PEN RIF SXT and TET 093

Artisanal S hominis JX5199881 CIP CPM CPO ERI GEN LZD NEOOXA PEN RIF SXT and VAN 080

Artisanal S saprophyticusHF9372521 CFO OXA PEN and SXT 026Artisanal S saprophyticus KJ6991511 OXA PEN and VAN 020

Artisanal S succinus KC3298241 CFO CLO ERI GEN OXA PEN RIFSXT TET and VAN 066

Artisanal S xylosus KF1980801 CFO CLO ERI GEN NEO OXA PENTET and VAN 060

lowastTheMAR index of an isolate is defined as 119886119887 where 119886 represents the number of antimicrobials to which the isolate was resistant and 119887 represents the numberof antimicrobials to which the isolate was subjectedS aureus strains ATCC WB81 (sea) ATCC 13563 (sed) and ATCC 27664 (see) showing a MAR index of 05 and S aureus strains ATCC14458 (seb) andATCCWB72 (sec) and S xylosus ATCC 29971 showing a MAR index of 03 were used as reference strainsCPM cefepime CFO cefoxitin CLO chloramphenicol CIP ciprofloxacin CLI clindamycin ERI erythromycin GEN gentamycin NEO neomycin LZDlinezolid RIF rifampicin TET tetracycline OXA oxacillin PEN penicillin SXT sulfamethoprim and VAN vancomycin

Table 4 Minimal inhibitory concentration (MIC) of compounds used in antimicrobial therapy against staphylococci infections

Salami origin GenBank accession number and similarity () MIC mgmLminus1

Methicillin Ampicillin Vancomycin Linezolid

Commercial

S carnosus KJ8620021 006 003 mdash mdashS xylosus CP0087241 006 003 006 mdash

S saprophyticus JX4901221 006 003 05 mdashS saprophyticus AB6977171 mdash mdash 05 mdashS saprophyticus EU4309921 mdash mdash 025 0125

S succinus KC3298241 006 025 003 mdash

Artisanal

S hominis JX5199881 006 05 mdash mdashS saprophyticus HF9372521 003 mdash mdash mdash

S xylosus KF1980801 003 05 mdashS saprophyticus KJ6991511 05 025 003 mdashS saprophyticus KJ0046231 05 003 003 mdash

S xylosus CP0072081 006 mdash mdash 025Strains S epidermidis KF6005891 and Staphylococcus spp KF1354451 were not susceptible to the antimicrobial concentrations tested in the present study

BioMed Research International 15

of 003mgmLminus1 two strains S succinus KC3298241 and Ssaprophyticus KJ6991511 showed an MIC of 025mgmLminus1and S hominis JX5199881 showed an MIC of 05mgmLminus1(Table 4) S saprophyticus KJ0046231 also showed an MICof 003mgmLminus1 but does not harbor enterotoxin genes It isimportant to note that most of the CNS strains that showedresistance to ampicillin were also resistant to methicillin ashighlighted by the MIC tests

Five strains S saprophyticus HF9372521 S saprophyti-cus KJ6991511 S saprophyticus JX4901221 S saprophyticusKJ0046231 and S succinus KC3298241 showed an MICfor vancomycin of 003mgmLminus1 one strain S xylosusCP0087241 showed an MIC of 006mgmLminus1 one strain Ssaprophyticus EU4309921 showed an MIC of 025mgmLminus1and two strains S xylosus KF1980801 and S saprophyticusAB6977171 showed an MIC of 05mgmLminus1 (Table 4)

Additionally five CNS strains S xylosus CP0087241S saprophyticus JX 4901221 S succinus KC3298241 Ssaprophyticus KJ6991511 and S saprophyticus KJ0046231showed resistance tomethicillin as demonstrated by theMICdeterminations (Table 4)

Previous studies have demonstrated that linezolid isactive against Gram-positive bacteria including methicillin-resistant staphylococci [47] In the present study 05 strainsshowed linezolid resistance in the disc diffusion test but itwas impossible to establish MIC values for S epidermidisKF6005891 Staphylococcus hominis JX5199881 and Staphy-lococcus carnosus KJ8620021The remaining strains S sapro-phyticus EU4309921 and S xylosus CP0072081 presentedan MIC for linezolid of 0125mgmLminus1 and 025mgmLminus1respectively

The MIC of 003mgmLminus1 for penicillin is in accordancewith the previous values estimated for resistant S aureusstrains found in several food matrices such as meat dairyproducts and ready-to-eat food [48]

There is still a lack of information on the antimicro-bial resistance of staphylococci strains from food matricesalthough they are the most worrisome vehicles of dissemi-nation of antibiotic-resistant pathogens [49]

The high resistance found for salami staphylococci strainscan be ascribed to their inappropriate use as growth promot-ers of antimicrobial agents like oxacillin vancomycin chlo-ramphenicol neomycin and erythromycin which are com-monly used in veterinary medicine to treat infections [50]Antimicrobial therapy of infections staphylococci is basedon results of susceptibility tests in vitro [51] Methicillin-resistant coagulase-negative Staphylococcus spp found inready-to-eat products such as meats fish and dairy productsalso offer risks and although they are not classical foodpoisoning bacteria their presence in food offers significantrisks to public health due to the possible spread of antibioticresistance [52]

The standardization of salami quality should includediagnostic methods to screen and quantify the presence ofclassical and newly described enterotoxins directly in thefood matrices as a routine procedure to be conducted by themeat product industry in Brazil

The safety of salami consumption could also be enhancedby the inclusion of amicrobial barrier such as the inclusion ofprobiotic strains producing natural antibiotics or competitiveflora or even the addition of natural bioagents against spoilageor pathogenic microorganisms

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors acknowledge the financial support fromFundacao Carlos Chagas Filho de Amparo a Pesquisa doEstado do Rio de Janeiro (FAPERJ Rio de Janeiro Brazil)Conselho Nacional de Desenvolvimento Cientıfico eTecnologico (CNPq Brasılia Brazil) and Coordenacao deAperfeicoamento de Pessoal de Nıvel Superior (CAPESBrasılia Brazil)

References

[1] ICMSF International Comission on Microbiological Specifica-tions for FoodsMicroorganisms in FoodsTheir Significance andMethods of Enumerations University of Toronto Press TorontoCanada 3rd edition 1983

[2] J F Veras L S Carmo L C Tong et al ldquoA study of theenterotoxigenicity of coagulase-negative and coagulase-positivestaphylococcal isolates from food poisoning outbreaks inMinasGerais Brazilrdquo International Journal of Infectious Diseases vol12 no 4 pp 410ndash415 2008

[3] K Omoe D-L Hu H Takahashi-Omoe A Nakane and KShinagawa ldquoComprehensive analysis of classical and newlydescribed staphylococcal superantigenic toxin genes in Staphy-lococcus aureus isolatesrdquo FEMS Microbiology Letters vol 246no 2 pp 191ndash198 2005

[4] J Kadariya T C Smith and D Thapaliya ldquoStaphylococcusaureus and staphylococcal food-borne disease an ongoing chal-lenge in public healthrdquoBioMedResearch International vol 2014Article ID 827965 9 pages 2014

[5] KOmoeD-LHuH KOno et al ldquoEmetic potentials of newlyidentified staphylococcal enterotoxin-like toxinsrdquo Infection andImmunity vol 81 no 10 pp 3627ndash3631 2013

[6] J K McCormick J M Yarwood and P M SchlievertldquoToxic shock syndrome and bacterial superantigens an updaterdquoAnnual Review of Microbiology vol 55 pp 77ndash104 2001

[7] T Baba F Takeuchi M Kuroda et al ldquoGenome and virulencedeterminants of high virulence community-acquired MRSArdquoThe Lancet vol 359 no 9320 pp 1819ndash1827 2002

[8] Associacao Brasileira de Industria Produtora e Exportadorada Carne Suınas (ABIPECS) ldquoStatistics slaughter ranking2000ndash2014rdquo 2014 httpabpa-brcombrsetoressuinoculturapublicacoesrelatorios-anuais

[9] N Zdolec M Hadziosmanovic L Kozacinski et al ldquoMicrobialand physicochemical succession in fermented sausages pro-duced with bacteriocinogenic culture of Lactobacillus sakei andsemi-purified bacteriocin mesenterocin Yrdquo Meat Science vol80 no 2 pp 480ndash487 2008

16 BioMed Research International

[10] F Ravyts L D Vuyst and F Leroy ldquoBacterial diversity andfunctionalities in food fermentationsrdquo Engineering in Life Sci-ences vol 12 no 4 pp 356ndash367 2012

[11] TOhlsson andN BengtssonTheHurdle ConceptMinimal Pro-cessing Technologies in the Food IndustryWoodhead Publishing2002

[12] S Bernardi B B Golineli and C J Contreras-CastilloldquoRevisao aspectos da aplicacao de culturas starter na producaode embutidos carneos fermentadosrdquo Brazilian Journal of FoodTechnology vol 13 no 2 pp 133ndash140 2010

[13] F Irlinger ldquoSafety assessment of dairy microorganismscoagulase-negative staphylococcirdquo International Journal of FoodMicrobiology vol 126 no 3 pp 302ndash310 2008

[14] B Ghebremedhin F Layer W Konig and B Konig ldquoGeneticclassification and distinguishing of Staphylococcus species basedon different partial gap 16S rRNA hsp60 rpoB sodA and tufgene sequencesrdquo Journal of Clinical Microbiology vol 46 no 3pp 1019ndash1025 2008

[15] I Babic K Markov D Kovacevic et al ldquoIdentification andcharacterization of potential autochthonous starter culturesfrom aCroatian lsquobrandrsquo product lsquoslavonski kulenrsquordquoMeat Sciencevol 88 no 3 pp 517ndash524 2011

[16] MMehrotra GWang andWM Johnson ldquoMultiplex PCR forthe detection of genes for Staphylococcus aureus enterotoxinsexfoliative toxins toxic shock syndrome toxin and methicillinresistancerdquo Journal of Clinical Microbiology vol 38 no 3 pp1032ndash1041 2000

[17] WM Johnson SD Tyler E P Ewan F EAshtonDR Pollardand K R Rozee ldquoDetection of genes for enterotoxins exfolia-tive toxins and toxic shock syndrome toxin 1 in Staphylococcusaureus by the polymerase chain reactionrdquo Journal of ClinicalMicrobiology vol 29 no 3 pp 426ndash430 1991

[18] J Mclauchlin G L Narayanan VMithani andG OrsquoNeill ldquoThedetection of enterotoxins and toxic shock syndrome toxin genesin Staphylococcus aureus by polymerase chain reactionrdquo Journalof Food Protection vol 63 no 4 pp 479ndash488 2000

[19] J P Rosec and O Gigaud ldquoStaphylococcal enterotoxin genes ofclassical and new types detected by PCR in Francerdquo Interna-tional Journal of Food Microbiology vol 77 no 1-2 pp 61ndash702002

[20] K Omoe D-L Hu H Takahashi-Omoe A Nakane andK Shinagawa ldquoIdentification and characterization of a newstaphylococcal enterotoxin-related putative toxin encoded bytwo kinds of plasmidsrdquo Infection and Immunity vol 71 no 10pp 6088ndash6094 2003

[21] N Sergeev D Volokhov V Chizhikov and A Rasooly ldquoSimul-taneous analysis of multiple staphylococcal enterotoxin genesby an oligonucleotide microarray assayrdquo Journal of ClinicalMicrobiology vol 42 no 5 pp 2134ndash2143 2004

[22] S Holtfreter D Grumann M Schmudde et al ldquoClonal distri-bution of superantigen genes in clinical Staphylococcus aureusisolatesrdquo Journal of Clinical Microbiology vol 45 no 8 pp2669ndash2680 2007

[23] W J Mason J S Blevins K Beenken NWibowo N Ojha andM S Smeltzer ldquoMultiplex PCR protocol for the diagnosis ofstaphylococcal infectionrdquo Journal of Clinical Microbiology vol39 no 9 pp 3332ndash3338 2001

[24] E Rahimi and H G Safai ldquoDetection of classical enterotoxinsof Staphylococcus aureus strains isolated frombovine subclinicalmastitis in Isfahan Iranrdquo Veterinary Microbiology vol 141 no3-4 pp 393ndash394 2010

[25] M A Larkin G Blackshields N P Brown et al ldquoClustalW andClustal X version 20rdquo Bioinformatics vol 23 no 21 pp 2947ndash2948 2007

[26] P H A Sneath and R R Sokal Unweighted Pair Group Methodwith Arithmetic Mean Numerical Taxonomy W H FreemanSan Francisco Calif USA 1973

[27] NCCLS ldquoPerformance standards for antimicrobial susceptibil-ity testing 14th informational supplementrdquo Tech Rep M 100-S14 NCCLS 2004

[28] S Corbiere Morot-Bizot S Leroy and R Talon ldquoMonitoringof staphylococcal starters in two French processing plantsmanufacturing dry fermented sausagesrdquo Journal of AppliedMicrobiology vol 102 no 1 pp 238ndash244 2007

[29] R Talon S Leroy I Lebert et al ldquoSafety improvement andpreservation of typical sensory qualities of traditional dryfermented sausages using autochthonous starter culturesrdquo Inter-national Journal of Food Microbiology vol 126 no 1-2 pp 227ndash234 2008

[30] G Mauriello A Casaburi G Blaiotta and F Villani ldquoIsolationand technological properties of coagulase negative staphylo-cocci from fermented sausages of Southern ItalyrdquoMeat Sciencevol 67 no 1 pp 149ndash158 2004

[31] M Janssens N Myter L De Vuyst and F Leroy ldquoSpeciesdiversity and metabolic impact of the microbiota are low inspontaneously acidified Belgian sausages with an added starterculture of Staphylococcus carnosusrdquo Food Microbiology vol 29no 2 pp 167ndash177 2012

[32] G Blaiotta D Ercolini G Mauriello G Salzano and F VillanildquoRapid and reliable identification of Staphylococcus equorum bya species-specific PCR assay targeting the sodA generdquo Systematicand Applied Microbiology vol 27 no 6 pp 696ndash702 2004

[33] L Kozacinski E Drosinos F Caklovica L Cocolin J Gasparik-Reichardt and S Veskovic ldquoInvestigation of microbial associa-tion of traditionally fermented sausagesrdquo Food Technology andBiotechnology vol 46 no 1 pp 93ndash106 2008

[34] S Leroy P Giammarinaro J-P Chacornac I Lebert and RTalon ldquoBiodiversity of indigenous staphylococci of naturallyfermented dry sausages and manufacturing environments ofsmall-scale processing unitsrdquo Food Microbiology vol 27 no 2pp 294ndash301 2010

[35] E Coton M-H Desmonts S Leroy et al ldquoBiodiversity ofCoagulase-Negative Staphylococci in French cheeses dry fer-mented sausages processing environments and clinical sam-plesrdquo International Journal of Food Microbiology vol 137 no 2-3 pp 221ndash229 2010

[36] S Hosseinzadeh and H D Saei ldquoStaphylococcal species associ-ated with bovinemastitis in the NorthWest of Iran emerging ofcoagulase-negative staphylococcirdquo International Journal of Vet-erinary Science and Medicine vol 2 no 1 pp 27ndash34 2014

[37] A Piette and G Verschraegen ldquoRole of coagulase-negativestaphylococci in human diseaserdquo Veterinary Microbiology vol134 no 1-2 pp 45ndash54 2009

[38] T M Moura F S Campos P A DrsquoAzevedo et al ldquoPrevalenceof enterotoxin-encoding genes and antimicrobial resistancein coagulase-negative and coagulase-positive Staphylococcusisolates from black pudding in Southern Brazilrdquo Revista daSociedade Brasileira deMedicina Tropical vol 45 no 5 pp 579ndash585 2012

[39] H I Suzuki T Lefebure P P Bitar and M J StanhopeldquoComparative genomic analysis of the genus Staphylococcusincluding Staphylococcus aureus and its newly described sister

BioMed Research International 17

species Staphylococcus simiaerdquo BMC Genomics vol 13 article38 2012

[40] R P Lamers G Muthukrishnan T A Castoe S Tafur A MCole and C L Parkinson ldquoPhylogenetic relationships amongStaphylococcus species and refinement of cluster groups basedon multilocus datardquo BMC Evolutionary Biology vol 12 article171 2012

[41] I V Pinchuk E J Beswick and V E Reyes ldquoStaphylococcalenterotoxinsrdquo Toxins vol 2 no 8 pp 2177ndash2197 2010

[42] S R Monday and G A Bohach ldquoGenes encoding staphylo-coccal enterotoxins G and I are linked and separated by DNArelated to other staphylococcal enterotoxinsrdquo Journal of NaturalToxins vol 10 no 1 pp 1ndash8 2001

[43] D Y Thomas S Jarraud B Lemercier et al ldquoStaphylococcalenterotoxin-like toxins U2 and V two new staphylococcalsuperantigens arising from recombination within the entero-toxin gene clusterrdquo Infection and Immunity vol 74 pp 4724ndash4734 2006

[44] N Indrawattana O Sungkhachat N Sookrung et al ldquoStaphylo-coccus aureus clinical isolates antibiotic susceptibility molecu-lar characteristics and ability to form biofilmrdquo BioMed ResearchInternational vol 2013 Article ID 314654 11 pages 2013

[45] V Pereira C Lopes A Castro J Silva P Gibbs and P TeixeiraldquoCharacterization for enterotoxin production virulence factorsand antibiotic susceptibility of Staphylococcus aureus isolatesfrom various foods in Portugalrdquo Food Microbiology vol 26 no3 pp 278ndash282 2009

[46] I Anacarso C Condo C Sabia and et al ldquoAntimicrobialresistance and other related virulence factors in staphylococcusspp isolated from food environmental and humans in ItalyrdquoUniversal Journal Microbiology Research vol 1 no 1 pp 1ndash92013

[47] B Martın M Garriga M Hugas S Bover-Cid M T Veciana-Nogues and T Aymerich ldquoMolecular technological and safetycharacterization of Gram-positive catalase-positive cocci fromslightly fermented sausagesrdquo International Journal of FoodMicrobiology vol 107 no 2 pp 148ndash158 2006

[48] NM Broekema T Van Tam T AMonson S AMarshall andD M Warshauer ldquoComparison of cefoxitin and oxacillin diskdiffusionmethods for detection ofmecA-mediated resistance inStaphylococcus aureus in a large-scale studyrdquo Journal of ClinicalMicrobiology vol 47 no 1 pp 217ndash219 2009

[49] E Cercenado F Garcıa-Garrote and E Bouza ldquoIn vitro activityof linezolid against multiply resistant Gram-positive clinicalisolatesrdquo Journal of Antimicrobial Chemotherapy vol 47 no 1pp 77ndash81 2001

[50] A Aydin K Muratoglu M Sudagidan K Bostan B Okukluand SHarsa ldquoPrevalence and antibiotic resistance of foodborneStaphylococcus aureus isolates in Turkeyrdquo Foodborne Pathogensand Disease vol 8 no 1 pp 63ndash69 2011

[51] D Sergelidis A Abrahim T Papadopoulos et al ldquoIsolation ofmethicillin-resistant Staphylococcus spp from ready-to-eat fishproductsrdquo Letters in Applied Microbiology vol 59 no 5 pp500ndash506 2014

[52] I PhillipsM Casewell T Cox et al ldquoDoes the use of antibioticsin food animals pose a risk to human health A critical reviewof published datardquo Journal of Antimicrobial Chemotherapy vol53 no 1 pp 28ndash52 2004

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Page 14: Research Article Safety Evaluation of the Coagulase ...downloads.hindawi.com/journals/bmri/2015/483548.pdf · Research Article Safety Evaluation of the Coagulase-Negative Staphylococci

14 BioMed Research International

Table 3 Multiple resistance to antimicrobial as found in CNS strains from salami

Salami origin CNS strains Antimicrobial agent resistance Multiple antimicrobialresistance (MAR) indexlowast

Commercial Staphylococcus spp KF1354451 CIP CLO CPM GEN NEO OXA PENSXT TET and VAN 066

Commercial S carnosus KJ8620021 CIP GEN LZD NEO OXA SXT andTET 046

Commercial S saprophyticus AB6977171 CFO CLO ERI PEN OXA TET andVAN 046

Commercial S saprophyticus EU4309921 CFO CLO ERI GEN LZD NEO OXAPEN SXT TET and VAN 080

Commercial S saprophyticus JX4901221 CFO CLO ERI GEN NEO PEN OXASXT TET and VAN 066

Commercial S saprophyticus KJ0046231 CFO OXA and PEN 020

Commercial S sciuri JX9664361 CFO CLO GEN NEO OXA PEN andTET 046

Commercial S xylosus CP0072081 OXA LZD PEN and VAN 026

Commercial S xylosus CP0087241 CFO CLO ERI GEN NEO OXA PENTET and VAN 060

Artisanal S epidermidis KF6005891 CFO CLI CLO CPM ERI GEN LZDNEO OXA PEN RIF SXT and TET 093

Artisanal S hominis JX5199881 CIP CPM CPO ERI GEN LZD NEOOXA PEN RIF SXT and VAN 080

Artisanal S saprophyticusHF9372521 CFO OXA PEN and SXT 026Artisanal S saprophyticus KJ6991511 OXA PEN and VAN 020

Artisanal S succinus KC3298241 CFO CLO ERI GEN OXA PEN RIFSXT TET and VAN 066

Artisanal S xylosus KF1980801 CFO CLO ERI GEN NEO OXA PENTET and VAN 060

lowastTheMAR index of an isolate is defined as 119886119887 where 119886 represents the number of antimicrobials to which the isolate was resistant and 119887 represents the numberof antimicrobials to which the isolate was subjectedS aureus strains ATCC WB81 (sea) ATCC 13563 (sed) and ATCC 27664 (see) showing a MAR index of 05 and S aureus strains ATCC14458 (seb) andATCCWB72 (sec) and S xylosus ATCC 29971 showing a MAR index of 03 were used as reference strainsCPM cefepime CFO cefoxitin CLO chloramphenicol CIP ciprofloxacin CLI clindamycin ERI erythromycin GEN gentamycin NEO neomycin LZDlinezolid RIF rifampicin TET tetracycline OXA oxacillin PEN penicillin SXT sulfamethoprim and VAN vancomycin

Table 4 Minimal inhibitory concentration (MIC) of compounds used in antimicrobial therapy against staphylococci infections

Salami origin GenBank accession number and similarity () MIC mgmLminus1

Methicillin Ampicillin Vancomycin Linezolid

Commercial

S carnosus KJ8620021 006 003 mdash mdashS xylosus CP0087241 006 003 006 mdash

S saprophyticus JX4901221 006 003 05 mdashS saprophyticus AB6977171 mdash mdash 05 mdashS saprophyticus EU4309921 mdash mdash 025 0125

S succinus KC3298241 006 025 003 mdash

Artisanal

S hominis JX5199881 006 05 mdash mdashS saprophyticus HF9372521 003 mdash mdash mdash

S xylosus KF1980801 003 05 mdashS saprophyticus KJ6991511 05 025 003 mdashS saprophyticus KJ0046231 05 003 003 mdash

S xylosus CP0072081 006 mdash mdash 025Strains S epidermidis KF6005891 and Staphylococcus spp KF1354451 were not susceptible to the antimicrobial concentrations tested in the present study

BioMed Research International 15

of 003mgmLminus1 two strains S succinus KC3298241 and Ssaprophyticus KJ6991511 showed an MIC of 025mgmLminus1and S hominis JX5199881 showed an MIC of 05mgmLminus1(Table 4) S saprophyticus KJ0046231 also showed an MICof 003mgmLminus1 but does not harbor enterotoxin genes It isimportant to note that most of the CNS strains that showedresistance to ampicillin were also resistant to methicillin ashighlighted by the MIC tests

Five strains S saprophyticus HF9372521 S saprophyti-cus KJ6991511 S saprophyticus JX4901221 S saprophyticusKJ0046231 and S succinus KC3298241 showed an MICfor vancomycin of 003mgmLminus1 one strain S xylosusCP0087241 showed an MIC of 006mgmLminus1 one strain Ssaprophyticus EU4309921 showed an MIC of 025mgmLminus1and two strains S xylosus KF1980801 and S saprophyticusAB6977171 showed an MIC of 05mgmLminus1 (Table 4)

Additionally five CNS strains S xylosus CP0087241S saprophyticus JX 4901221 S succinus KC3298241 Ssaprophyticus KJ6991511 and S saprophyticus KJ0046231showed resistance tomethicillin as demonstrated by theMICdeterminations (Table 4)

Previous studies have demonstrated that linezolid isactive against Gram-positive bacteria including methicillin-resistant staphylococci [47] In the present study 05 strainsshowed linezolid resistance in the disc diffusion test but itwas impossible to establish MIC values for S epidermidisKF6005891 Staphylococcus hominis JX5199881 and Staphy-lococcus carnosus KJ8620021The remaining strains S sapro-phyticus EU4309921 and S xylosus CP0072081 presentedan MIC for linezolid of 0125mgmLminus1 and 025mgmLminus1respectively

The MIC of 003mgmLminus1 for penicillin is in accordancewith the previous values estimated for resistant S aureusstrains found in several food matrices such as meat dairyproducts and ready-to-eat food [48]

There is still a lack of information on the antimicro-bial resistance of staphylococci strains from food matricesalthough they are the most worrisome vehicles of dissemi-nation of antibiotic-resistant pathogens [49]

The high resistance found for salami staphylococci strainscan be ascribed to their inappropriate use as growth promot-ers of antimicrobial agents like oxacillin vancomycin chlo-ramphenicol neomycin and erythromycin which are com-monly used in veterinary medicine to treat infections [50]Antimicrobial therapy of infections staphylococci is basedon results of susceptibility tests in vitro [51] Methicillin-resistant coagulase-negative Staphylococcus spp found inready-to-eat products such as meats fish and dairy productsalso offer risks and although they are not classical foodpoisoning bacteria their presence in food offers significantrisks to public health due to the possible spread of antibioticresistance [52]

The standardization of salami quality should includediagnostic methods to screen and quantify the presence ofclassical and newly described enterotoxins directly in thefood matrices as a routine procedure to be conducted by themeat product industry in Brazil

The safety of salami consumption could also be enhancedby the inclusion of amicrobial barrier such as the inclusion ofprobiotic strains producing natural antibiotics or competitiveflora or even the addition of natural bioagents against spoilageor pathogenic microorganisms

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors acknowledge the financial support fromFundacao Carlos Chagas Filho de Amparo a Pesquisa doEstado do Rio de Janeiro (FAPERJ Rio de Janeiro Brazil)Conselho Nacional de Desenvolvimento Cientıfico eTecnologico (CNPq Brasılia Brazil) and Coordenacao deAperfeicoamento de Pessoal de Nıvel Superior (CAPESBrasılia Brazil)

References

[1] ICMSF International Comission on Microbiological Specifica-tions for FoodsMicroorganisms in FoodsTheir Significance andMethods of Enumerations University of Toronto Press TorontoCanada 3rd edition 1983

[2] J F Veras L S Carmo L C Tong et al ldquoA study of theenterotoxigenicity of coagulase-negative and coagulase-positivestaphylococcal isolates from food poisoning outbreaks inMinasGerais Brazilrdquo International Journal of Infectious Diseases vol12 no 4 pp 410ndash415 2008

[3] K Omoe D-L Hu H Takahashi-Omoe A Nakane and KShinagawa ldquoComprehensive analysis of classical and newlydescribed staphylococcal superantigenic toxin genes in Staphy-lococcus aureus isolatesrdquo FEMS Microbiology Letters vol 246no 2 pp 191ndash198 2005

[4] J Kadariya T C Smith and D Thapaliya ldquoStaphylococcusaureus and staphylococcal food-borne disease an ongoing chal-lenge in public healthrdquoBioMedResearch International vol 2014Article ID 827965 9 pages 2014

[5] KOmoeD-LHuH KOno et al ldquoEmetic potentials of newlyidentified staphylococcal enterotoxin-like toxinsrdquo Infection andImmunity vol 81 no 10 pp 3627ndash3631 2013

[6] J K McCormick J M Yarwood and P M SchlievertldquoToxic shock syndrome and bacterial superantigens an updaterdquoAnnual Review of Microbiology vol 55 pp 77ndash104 2001

[7] T Baba F Takeuchi M Kuroda et al ldquoGenome and virulencedeterminants of high virulence community-acquired MRSArdquoThe Lancet vol 359 no 9320 pp 1819ndash1827 2002

[8] Associacao Brasileira de Industria Produtora e Exportadorada Carne Suınas (ABIPECS) ldquoStatistics slaughter ranking2000ndash2014rdquo 2014 httpabpa-brcombrsetoressuinoculturapublicacoesrelatorios-anuais

[9] N Zdolec M Hadziosmanovic L Kozacinski et al ldquoMicrobialand physicochemical succession in fermented sausages pro-duced with bacteriocinogenic culture of Lactobacillus sakei andsemi-purified bacteriocin mesenterocin Yrdquo Meat Science vol80 no 2 pp 480ndash487 2008

16 BioMed Research International

[10] F Ravyts L D Vuyst and F Leroy ldquoBacterial diversity andfunctionalities in food fermentationsrdquo Engineering in Life Sci-ences vol 12 no 4 pp 356ndash367 2012

[11] TOhlsson andN BengtssonTheHurdle ConceptMinimal Pro-cessing Technologies in the Food IndustryWoodhead Publishing2002

[12] S Bernardi B B Golineli and C J Contreras-CastilloldquoRevisao aspectos da aplicacao de culturas starter na producaode embutidos carneos fermentadosrdquo Brazilian Journal of FoodTechnology vol 13 no 2 pp 133ndash140 2010

[13] F Irlinger ldquoSafety assessment of dairy microorganismscoagulase-negative staphylococcirdquo International Journal of FoodMicrobiology vol 126 no 3 pp 302ndash310 2008

[14] B Ghebremedhin F Layer W Konig and B Konig ldquoGeneticclassification and distinguishing of Staphylococcus species basedon different partial gap 16S rRNA hsp60 rpoB sodA and tufgene sequencesrdquo Journal of Clinical Microbiology vol 46 no 3pp 1019ndash1025 2008

[15] I Babic K Markov D Kovacevic et al ldquoIdentification andcharacterization of potential autochthonous starter culturesfrom aCroatian lsquobrandrsquo product lsquoslavonski kulenrsquordquoMeat Sciencevol 88 no 3 pp 517ndash524 2011

[16] MMehrotra GWang andWM Johnson ldquoMultiplex PCR forthe detection of genes for Staphylococcus aureus enterotoxinsexfoliative toxins toxic shock syndrome toxin and methicillinresistancerdquo Journal of Clinical Microbiology vol 38 no 3 pp1032ndash1041 2000

[17] WM Johnson SD Tyler E P Ewan F EAshtonDR Pollardand K R Rozee ldquoDetection of genes for enterotoxins exfolia-tive toxins and toxic shock syndrome toxin 1 in Staphylococcusaureus by the polymerase chain reactionrdquo Journal of ClinicalMicrobiology vol 29 no 3 pp 426ndash430 1991

[18] J Mclauchlin G L Narayanan VMithani andG OrsquoNeill ldquoThedetection of enterotoxins and toxic shock syndrome toxin genesin Staphylococcus aureus by polymerase chain reactionrdquo Journalof Food Protection vol 63 no 4 pp 479ndash488 2000

[19] J P Rosec and O Gigaud ldquoStaphylococcal enterotoxin genes ofclassical and new types detected by PCR in Francerdquo Interna-tional Journal of Food Microbiology vol 77 no 1-2 pp 61ndash702002

[20] K Omoe D-L Hu H Takahashi-Omoe A Nakane andK Shinagawa ldquoIdentification and characterization of a newstaphylococcal enterotoxin-related putative toxin encoded bytwo kinds of plasmidsrdquo Infection and Immunity vol 71 no 10pp 6088ndash6094 2003

[21] N Sergeev D Volokhov V Chizhikov and A Rasooly ldquoSimul-taneous analysis of multiple staphylococcal enterotoxin genesby an oligonucleotide microarray assayrdquo Journal of ClinicalMicrobiology vol 42 no 5 pp 2134ndash2143 2004

[22] S Holtfreter D Grumann M Schmudde et al ldquoClonal distri-bution of superantigen genes in clinical Staphylococcus aureusisolatesrdquo Journal of Clinical Microbiology vol 45 no 8 pp2669ndash2680 2007

[23] W J Mason J S Blevins K Beenken NWibowo N Ojha andM S Smeltzer ldquoMultiplex PCR protocol for the diagnosis ofstaphylococcal infectionrdquo Journal of Clinical Microbiology vol39 no 9 pp 3332ndash3338 2001

[24] E Rahimi and H G Safai ldquoDetection of classical enterotoxinsof Staphylococcus aureus strains isolated frombovine subclinicalmastitis in Isfahan Iranrdquo Veterinary Microbiology vol 141 no3-4 pp 393ndash394 2010

[25] M A Larkin G Blackshields N P Brown et al ldquoClustalW andClustal X version 20rdquo Bioinformatics vol 23 no 21 pp 2947ndash2948 2007

[26] P H A Sneath and R R Sokal Unweighted Pair Group Methodwith Arithmetic Mean Numerical Taxonomy W H FreemanSan Francisco Calif USA 1973

[27] NCCLS ldquoPerformance standards for antimicrobial susceptibil-ity testing 14th informational supplementrdquo Tech Rep M 100-S14 NCCLS 2004

[28] S Corbiere Morot-Bizot S Leroy and R Talon ldquoMonitoringof staphylococcal starters in two French processing plantsmanufacturing dry fermented sausagesrdquo Journal of AppliedMicrobiology vol 102 no 1 pp 238ndash244 2007

[29] R Talon S Leroy I Lebert et al ldquoSafety improvement andpreservation of typical sensory qualities of traditional dryfermented sausages using autochthonous starter culturesrdquo Inter-national Journal of Food Microbiology vol 126 no 1-2 pp 227ndash234 2008

[30] G Mauriello A Casaburi G Blaiotta and F Villani ldquoIsolationand technological properties of coagulase negative staphylo-cocci from fermented sausages of Southern ItalyrdquoMeat Sciencevol 67 no 1 pp 149ndash158 2004

[31] M Janssens N Myter L De Vuyst and F Leroy ldquoSpeciesdiversity and metabolic impact of the microbiota are low inspontaneously acidified Belgian sausages with an added starterculture of Staphylococcus carnosusrdquo Food Microbiology vol 29no 2 pp 167ndash177 2012

[32] G Blaiotta D Ercolini G Mauriello G Salzano and F VillanildquoRapid and reliable identification of Staphylococcus equorum bya species-specific PCR assay targeting the sodA generdquo Systematicand Applied Microbiology vol 27 no 6 pp 696ndash702 2004

[33] L Kozacinski E Drosinos F Caklovica L Cocolin J Gasparik-Reichardt and S Veskovic ldquoInvestigation of microbial associa-tion of traditionally fermented sausagesrdquo Food Technology andBiotechnology vol 46 no 1 pp 93ndash106 2008

[34] S Leroy P Giammarinaro J-P Chacornac I Lebert and RTalon ldquoBiodiversity of indigenous staphylococci of naturallyfermented dry sausages and manufacturing environments ofsmall-scale processing unitsrdquo Food Microbiology vol 27 no 2pp 294ndash301 2010

[35] E Coton M-H Desmonts S Leroy et al ldquoBiodiversity ofCoagulase-Negative Staphylococci in French cheeses dry fer-mented sausages processing environments and clinical sam-plesrdquo International Journal of Food Microbiology vol 137 no 2-3 pp 221ndash229 2010

[36] S Hosseinzadeh and H D Saei ldquoStaphylococcal species associ-ated with bovinemastitis in the NorthWest of Iran emerging ofcoagulase-negative staphylococcirdquo International Journal of Vet-erinary Science and Medicine vol 2 no 1 pp 27ndash34 2014

[37] A Piette and G Verschraegen ldquoRole of coagulase-negativestaphylococci in human diseaserdquo Veterinary Microbiology vol134 no 1-2 pp 45ndash54 2009

[38] T M Moura F S Campos P A DrsquoAzevedo et al ldquoPrevalenceof enterotoxin-encoding genes and antimicrobial resistancein coagulase-negative and coagulase-positive Staphylococcusisolates from black pudding in Southern Brazilrdquo Revista daSociedade Brasileira deMedicina Tropical vol 45 no 5 pp 579ndash585 2012

[39] H I Suzuki T Lefebure P P Bitar and M J StanhopeldquoComparative genomic analysis of the genus Staphylococcusincluding Staphylococcus aureus and its newly described sister

BioMed Research International 17

species Staphylococcus simiaerdquo BMC Genomics vol 13 article38 2012

[40] R P Lamers G Muthukrishnan T A Castoe S Tafur A MCole and C L Parkinson ldquoPhylogenetic relationships amongStaphylococcus species and refinement of cluster groups basedon multilocus datardquo BMC Evolutionary Biology vol 12 article171 2012

[41] I V Pinchuk E J Beswick and V E Reyes ldquoStaphylococcalenterotoxinsrdquo Toxins vol 2 no 8 pp 2177ndash2197 2010

[42] S R Monday and G A Bohach ldquoGenes encoding staphylo-coccal enterotoxins G and I are linked and separated by DNArelated to other staphylococcal enterotoxinsrdquo Journal of NaturalToxins vol 10 no 1 pp 1ndash8 2001

[43] D Y Thomas S Jarraud B Lemercier et al ldquoStaphylococcalenterotoxin-like toxins U2 and V two new staphylococcalsuperantigens arising from recombination within the entero-toxin gene clusterrdquo Infection and Immunity vol 74 pp 4724ndash4734 2006

[44] N Indrawattana O Sungkhachat N Sookrung et al ldquoStaphylo-coccus aureus clinical isolates antibiotic susceptibility molecu-lar characteristics and ability to form biofilmrdquo BioMed ResearchInternational vol 2013 Article ID 314654 11 pages 2013

[45] V Pereira C Lopes A Castro J Silva P Gibbs and P TeixeiraldquoCharacterization for enterotoxin production virulence factorsand antibiotic susceptibility of Staphylococcus aureus isolatesfrom various foods in Portugalrdquo Food Microbiology vol 26 no3 pp 278ndash282 2009

[46] I Anacarso C Condo C Sabia and et al ldquoAntimicrobialresistance and other related virulence factors in staphylococcusspp isolated from food environmental and humans in ItalyrdquoUniversal Journal Microbiology Research vol 1 no 1 pp 1ndash92013

[47] B Martın M Garriga M Hugas S Bover-Cid M T Veciana-Nogues and T Aymerich ldquoMolecular technological and safetycharacterization of Gram-positive catalase-positive cocci fromslightly fermented sausagesrdquo International Journal of FoodMicrobiology vol 107 no 2 pp 148ndash158 2006

[48] NM Broekema T Van Tam T AMonson S AMarshall andD M Warshauer ldquoComparison of cefoxitin and oxacillin diskdiffusionmethods for detection ofmecA-mediated resistance inStaphylococcus aureus in a large-scale studyrdquo Journal of ClinicalMicrobiology vol 47 no 1 pp 217ndash219 2009

[49] E Cercenado F Garcıa-Garrote and E Bouza ldquoIn vitro activityof linezolid against multiply resistant Gram-positive clinicalisolatesrdquo Journal of Antimicrobial Chemotherapy vol 47 no 1pp 77ndash81 2001

[50] A Aydin K Muratoglu M Sudagidan K Bostan B Okukluand SHarsa ldquoPrevalence and antibiotic resistance of foodborneStaphylococcus aureus isolates in Turkeyrdquo Foodborne Pathogensand Disease vol 8 no 1 pp 63ndash69 2011

[51] D Sergelidis A Abrahim T Papadopoulos et al ldquoIsolation ofmethicillin-resistant Staphylococcus spp from ready-to-eat fishproductsrdquo Letters in Applied Microbiology vol 59 no 5 pp500ndash506 2014

[52] I PhillipsM Casewell T Cox et al ldquoDoes the use of antibioticsin food animals pose a risk to human health A critical reviewof published datardquo Journal of Antimicrobial Chemotherapy vol53 no 1 pp 28ndash52 2004

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Page 15: Research Article Safety Evaluation of the Coagulase ...downloads.hindawi.com/journals/bmri/2015/483548.pdf · Research Article Safety Evaluation of the Coagulase-Negative Staphylococci

BioMed Research International 15

of 003mgmLminus1 two strains S succinus KC3298241 and Ssaprophyticus KJ6991511 showed an MIC of 025mgmLminus1and S hominis JX5199881 showed an MIC of 05mgmLminus1(Table 4) S saprophyticus KJ0046231 also showed an MICof 003mgmLminus1 but does not harbor enterotoxin genes It isimportant to note that most of the CNS strains that showedresistance to ampicillin were also resistant to methicillin ashighlighted by the MIC tests

Five strains S saprophyticus HF9372521 S saprophyti-cus KJ6991511 S saprophyticus JX4901221 S saprophyticusKJ0046231 and S succinus KC3298241 showed an MICfor vancomycin of 003mgmLminus1 one strain S xylosusCP0087241 showed an MIC of 006mgmLminus1 one strain Ssaprophyticus EU4309921 showed an MIC of 025mgmLminus1and two strains S xylosus KF1980801 and S saprophyticusAB6977171 showed an MIC of 05mgmLminus1 (Table 4)

Additionally five CNS strains S xylosus CP0087241S saprophyticus JX 4901221 S succinus KC3298241 Ssaprophyticus KJ6991511 and S saprophyticus KJ0046231showed resistance tomethicillin as demonstrated by theMICdeterminations (Table 4)

Previous studies have demonstrated that linezolid isactive against Gram-positive bacteria including methicillin-resistant staphylococci [47] In the present study 05 strainsshowed linezolid resistance in the disc diffusion test but itwas impossible to establish MIC values for S epidermidisKF6005891 Staphylococcus hominis JX5199881 and Staphy-lococcus carnosus KJ8620021The remaining strains S sapro-phyticus EU4309921 and S xylosus CP0072081 presentedan MIC for linezolid of 0125mgmLminus1 and 025mgmLminus1respectively

The MIC of 003mgmLminus1 for penicillin is in accordancewith the previous values estimated for resistant S aureusstrains found in several food matrices such as meat dairyproducts and ready-to-eat food [48]

There is still a lack of information on the antimicro-bial resistance of staphylococci strains from food matricesalthough they are the most worrisome vehicles of dissemi-nation of antibiotic-resistant pathogens [49]

The high resistance found for salami staphylococci strainscan be ascribed to their inappropriate use as growth promot-ers of antimicrobial agents like oxacillin vancomycin chlo-ramphenicol neomycin and erythromycin which are com-monly used in veterinary medicine to treat infections [50]Antimicrobial therapy of infections staphylococci is basedon results of susceptibility tests in vitro [51] Methicillin-resistant coagulase-negative Staphylococcus spp found inready-to-eat products such as meats fish and dairy productsalso offer risks and although they are not classical foodpoisoning bacteria their presence in food offers significantrisks to public health due to the possible spread of antibioticresistance [52]

The standardization of salami quality should includediagnostic methods to screen and quantify the presence ofclassical and newly described enterotoxins directly in thefood matrices as a routine procedure to be conducted by themeat product industry in Brazil

The safety of salami consumption could also be enhancedby the inclusion of amicrobial barrier such as the inclusion ofprobiotic strains producing natural antibiotics or competitiveflora or even the addition of natural bioagents against spoilageor pathogenic microorganisms

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors acknowledge the financial support fromFundacao Carlos Chagas Filho de Amparo a Pesquisa doEstado do Rio de Janeiro (FAPERJ Rio de Janeiro Brazil)Conselho Nacional de Desenvolvimento Cientıfico eTecnologico (CNPq Brasılia Brazil) and Coordenacao deAperfeicoamento de Pessoal de Nıvel Superior (CAPESBrasılia Brazil)

References

[1] ICMSF International Comission on Microbiological Specifica-tions for FoodsMicroorganisms in FoodsTheir Significance andMethods of Enumerations University of Toronto Press TorontoCanada 3rd edition 1983

[2] J F Veras L S Carmo L C Tong et al ldquoA study of theenterotoxigenicity of coagulase-negative and coagulase-positivestaphylococcal isolates from food poisoning outbreaks inMinasGerais Brazilrdquo International Journal of Infectious Diseases vol12 no 4 pp 410ndash415 2008

[3] K Omoe D-L Hu H Takahashi-Omoe A Nakane and KShinagawa ldquoComprehensive analysis of classical and newlydescribed staphylococcal superantigenic toxin genes in Staphy-lococcus aureus isolatesrdquo FEMS Microbiology Letters vol 246no 2 pp 191ndash198 2005

[4] J Kadariya T C Smith and D Thapaliya ldquoStaphylococcusaureus and staphylococcal food-borne disease an ongoing chal-lenge in public healthrdquoBioMedResearch International vol 2014Article ID 827965 9 pages 2014

[5] KOmoeD-LHuH KOno et al ldquoEmetic potentials of newlyidentified staphylococcal enterotoxin-like toxinsrdquo Infection andImmunity vol 81 no 10 pp 3627ndash3631 2013

[6] J K McCormick J M Yarwood and P M SchlievertldquoToxic shock syndrome and bacterial superantigens an updaterdquoAnnual Review of Microbiology vol 55 pp 77ndash104 2001

[7] T Baba F Takeuchi M Kuroda et al ldquoGenome and virulencedeterminants of high virulence community-acquired MRSArdquoThe Lancet vol 359 no 9320 pp 1819ndash1827 2002

[8] Associacao Brasileira de Industria Produtora e Exportadorada Carne Suınas (ABIPECS) ldquoStatistics slaughter ranking2000ndash2014rdquo 2014 httpabpa-brcombrsetoressuinoculturapublicacoesrelatorios-anuais

[9] N Zdolec M Hadziosmanovic L Kozacinski et al ldquoMicrobialand physicochemical succession in fermented sausages pro-duced with bacteriocinogenic culture of Lactobacillus sakei andsemi-purified bacteriocin mesenterocin Yrdquo Meat Science vol80 no 2 pp 480ndash487 2008

16 BioMed Research International

[10] F Ravyts L D Vuyst and F Leroy ldquoBacterial diversity andfunctionalities in food fermentationsrdquo Engineering in Life Sci-ences vol 12 no 4 pp 356ndash367 2012

[11] TOhlsson andN BengtssonTheHurdle ConceptMinimal Pro-cessing Technologies in the Food IndustryWoodhead Publishing2002

[12] S Bernardi B B Golineli and C J Contreras-CastilloldquoRevisao aspectos da aplicacao de culturas starter na producaode embutidos carneos fermentadosrdquo Brazilian Journal of FoodTechnology vol 13 no 2 pp 133ndash140 2010

[13] F Irlinger ldquoSafety assessment of dairy microorganismscoagulase-negative staphylococcirdquo International Journal of FoodMicrobiology vol 126 no 3 pp 302ndash310 2008

[14] B Ghebremedhin F Layer W Konig and B Konig ldquoGeneticclassification and distinguishing of Staphylococcus species basedon different partial gap 16S rRNA hsp60 rpoB sodA and tufgene sequencesrdquo Journal of Clinical Microbiology vol 46 no 3pp 1019ndash1025 2008

[15] I Babic K Markov D Kovacevic et al ldquoIdentification andcharacterization of potential autochthonous starter culturesfrom aCroatian lsquobrandrsquo product lsquoslavonski kulenrsquordquoMeat Sciencevol 88 no 3 pp 517ndash524 2011

[16] MMehrotra GWang andWM Johnson ldquoMultiplex PCR forthe detection of genes for Staphylococcus aureus enterotoxinsexfoliative toxins toxic shock syndrome toxin and methicillinresistancerdquo Journal of Clinical Microbiology vol 38 no 3 pp1032ndash1041 2000

[17] WM Johnson SD Tyler E P Ewan F EAshtonDR Pollardand K R Rozee ldquoDetection of genes for enterotoxins exfolia-tive toxins and toxic shock syndrome toxin 1 in Staphylococcusaureus by the polymerase chain reactionrdquo Journal of ClinicalMicrobiology vol 29 no 3 pp 426ndash430 1991

[18] J Mclauchlin G L Narayanan VMithani andG OrsquoNeill ldquoThedetection of enterotoxins and toxic shock syndrome toxin genesin Staphylococcus aureus by polymerase chain reactionrdquo Journalof Food Protection vol 63 no 4 pp 479ndash488 2000

[19] J P Rosec and O Gigaud ldquoStaphylococcal enterotoxin genes ofclassical and new types detected by PCR in Francerdquo Interna-tional Journal of Food Microbiology vol 77 no 1-2 pp 61ndash702002

[20] K Omoe D-L Hu H Takahashi-Omoe A Nakane andK Shinagawa ldquoIdentification and characterization of a newstaphylococcal enterotoxin-related putative toxin encoded bytwo kinds of plasmidsrdquo Infection and Immunity vol 71 no 10pp 6088ndash6094 2003

[21] N Sergeev D Volokhov V Chizhikov and A Rasooly ldquoSimul-taneous analysis of multiple staphylococcal enterotoxin genesby an oligonucleotide microarray assayrdquo Journal of ClinicalMicrobiology vol 42 no 5 pp 2134ndash2143 2004

[22] S Holtfreter D Grumann M Schmudde et al ldquoClonal distri-bution of superantigen genes in clinical Staphylococcus aureusisolatesrdquo Journal of Clinical Microbiology vol 45 no 8 pp2669ndash2680 2007

[23] W J Mason J S Blevins K Beenken NWibowo N Ojha andM S Smeltzer ldquoMultiplex PCR protocol for the diagnosis ofstaphylococcal infectionrdquo Journal of Clinical Microbiology vol39 no 9 pp 3332ndash3338 2001

[24] E Rahimi and H G Safai ldquoDetection of classical enterotoxinsof Staphylococcus aureus strains isolated frombovine subclinicalmastitis in Isfahan Iranrdquo Veterinary Microbiology vol 141 no3-4 pp 393ndash394 2010

[25] M A Larkin G Blackshields N P Brown et al ldquoClustalW andClustal X version 20rdquo Bioinformatics vol 23 no 21 pp 2947ndash2948 2007

[26] P H A Sneath and R R Sokal Unweighted Pair Group Methodwith Arithmetic Mean Numerical Taxonomy W H FreemanSan Francisco Calif USA 1973

[27] NCCLS ldquoPerformance standards for antimicrobial susceptibil-ity testing 14th informational supplementrdquo Tech Rep M 100-S14 NCCLS 2004

[28] S Corbiere Morot-Bizot S Leroy and R Talon ldquoMonitoringof staphylococcal starters in two French processing plantsmanufacturing dry fermented sausagesrdquo Journal of AppliedMicrobiology vol 102 no 1 pp 238ndash244 2007

[29] R Talon S Leroy I Lebert et al ldquoSafety improvement andpreservation of typical sensory qualities of traditional dryfermented sausages using autochthonous starter culturesrdquo Inter-national Journal of Food Microbiology vol 126 no 1-2 pp 227ndash234 2008

[30] G Mauriello A Casaburi G Blaiotta and F Villani ldquoIsolationand technological properties of coagulase negative staphylo-cocci from fermented sausages of Southern ItalyrdquoMeat Sciencevol 67 no 1 pp 149ndash158 2004

[31] M Janssens N Myter L De Vuyst and F Leroy ldquoSpeciesdiversity and metabolic impact of the microbiota are low inspontaneously acidified Belgian sausages with an added starterculture of Staphylococcus carnosusrdquo Food Microbiology vol 29no 2 pp 167ndash177 2012

[32] G Blaiotta D Ercolini G Mauriello G Salzano and F VillanildquoRapid and reliable identification of Staphylococcus equorum bya species-specific PCR assay targeting the sodA generdquo Systematicand Applied Microbiology vol 27 no 6 pp 696ndash702 2004

[33] L Kozacinski E Drosinos F Caklovica L Cocolin J Gasparik-Reichardt and S Veskovic ldquoInvestigation of microbial associa-tion of traditionally fermented sausagesrdquo Food Technology andBiotechnology vol 46 no 1 pp 93ndash106 2008

[34] S Leroy P Giammarinaro J-P Chacornac I Lebert and RTalon ldquoBiodiversity of indigenous staphylococci of naturallyfermented dry sausages and manufacturing environments ofsmall-scale processing unitsrdquo Food Microbiology vol 27 no 2pp 294ndash301 2010

[35] E Coton M-H Desmonts S Leroy et al ldquoBiodiversity ofCoagulase-Negative Staphylococci in French cheeses dry fer-mented sausages processing environments and clinical sam-plesrdquo International Journal of Food Microbiology vol 137 no 2-3 pp 221ndash229 2010

[36] S Hosseinzadeh and H D Saei ldquoStaphylococcal species associ-ated with bovinemastitis in the NorthWest of Iran emerging ofcoagulase-negative staphylococcirdquo International Journal of Vet-erinary Science and Medicine vol 2 no 1 pp 27ndash34 2014

[37] A Piette and G Verschraegen ldquoRole of coagulase-negativestaphylococci in human diseaserdquo Veterinary Microbiology vol134 no 1-2 pp 45ndash54 2009

[38] T M Moura F S Campos P A DrsquoAzevedo et al ldquoPrevalenceof enterotoxin-encoding genes and antimicrobial resistancein coagulase-negative and coagulase-positive Staphylococcusisolates from black pudding in Southern Brazilrdquo Revista daSociedade Brasileira deMedicina Tropical vol 45 no 5 pp 579ndash585 2012

[39] H I Suzuki T Lefebure P P Bitar and M J StanhopeldquoComparative genomic analysis of the genus Staphylococcusincluding Staphylococcus aureus and its newly described sister

BioMed Research International 17

species Staphylococcus simiaerdquo BMC Genomics vol 13 article38 2012

[40] R P Lamers G Muthukrishnan T A Castoe S Tafur A MCole and C L Parkinson ldquoPhylogenetic relationships amongStaphylococcus species and refinement of cluster groups basedon multilocus datardquo BMC Evolutionary Biology vol 12 article171 2012

[41] I V Pinchuk E J Beswick and V E Reyes ldquoStaphylococcalenterotoxinsrdquo Toxins vol 2 no 8 pp 2177ndash2197 2010

[42] S R Monday and G A Bohach ldquoGenes encoding staphylo-coccal enterotoxins G and I are linked and separated by DNArelated to other staphylococcal enterotoxinsrdquo Journal of NaturalToxins vol 10 no 1 pp 1ndash8 2001

[43] D Y Thomas S Jarraud B Lemercier et al ldquoStaphylococcalenterotoxin-like toxins U2 and V two new staphylococcalsuperantigens arising from recombination within the entero-toxin gene clusterrdquo Infection and Immunity vol 74 pp 4724ndash4734 2006

[44] N Indrawattana O Sungkhachat N Sookrung et al ldquoStaphylo-coccus aureus clinical isolates antibiotic susceptibility molecu-lar characteristics and ability to form biofilmrdquo BioMed ResearchInternational vol 2013 Article ID 314654 11 pages 2013

[45] V Pereira C Lopes A Castro J Silva P Gibbs and P TeixeiraldquoCharacterization for enterotoxin production virulence factorsand antibiotic susceptibility of Staphylococcus aureus isolatesfrom various foods in Portugalrdquo Food Microbiology vol 26 no3 pp 278ndash282 2009

[46] I Anacarso C Condo C Sabia and et al ldquoAntimicrobialresistance and other related virulence factors in staphylococcusspp isolated from food environmental and humans in ItalyrdquoUniversal Journal Microbiology Research vol 1 no 1 pp 1ndash92013

[47] B Martın M Garriga M Hugas S Bover-Cid M T Veciana-Nogues and T Aymerich ldquoMolecular technological and safetycharacterization of Gram-positive catalase-positive cocci fromslightly fermented sausagesrdquo International Journal of FoodMicrobiology vol 107 no 2 pp 148ndash158 2006

[48] NM Broekema T Van Tam T AMonson S AMarshall andD M Warshauer ldquoComparison of cefoxitin and oxacillin diskdiffusionmethods for detection ofmecA-mediated resistance inStaphylococcus aureus in a large-scale studyrdquo Journal of ClinicalMicrobiology vol 47 no 1 pp 217ndash219 2009

[49] E Cercenado F Garcıa-Garrote and E Bouza ldquoIn vitro activityof linezolid against multiply resistant Gram-positive clinicalisolatesrdquo Journal of Antimicrobial Chemotherapy vol 47 no 1pp 77ndash81 2001

[50] A Aydin K Muratoglu M Sudagidan K Bostan B Okukluand SHarsa ldquoPrevalence and antibiotic resistance of foodborneStaphylococcus aureus isolates in Turkeyrdquo Foodborne Pathogensand Disease vol 8 no 1 pp 63ndash69 2011

[51] D Sergelidis A Abrahim T Papadopoulos et al ldquoIsolation ofmethicillin-resistant Staphylococcus spp from ready-to-eat fishproductsrdquo Letters in Applied Microbiology vol 59 no 5 pp500ndash506 2014

[52] I PhillipsM Casewell T Cox et al ldquoDoes the use of antibioticsin food animals pose a risk to human health A critical reviewof published datardquo Journal of Antimicrobial Chemotherapy vol53 no 1 pp 28ndash52 2004

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Page 16: Research Article Safety Evaluation of the Coagulase ...downloads.hindawi.com/journals/bmri/2015/483548.pdf · Research Article Safety Evaluation of the Coagulase-Negative Staphylococci

16 BioMed Research International

[10] F Ravyts L D Vuyst and F Leroy ldquoBacterial diversity andfunctionalities in food fermentationsrdquo Engineering in Life Sci-ences vol 12 no 4 pp 356ndash367 2012

[11] TOhlsson andN BengtssonTheHurdle ConceptMinimal Pro-cessing Technologies in the Food IndustryWoodhead Publishing2002

[12] S Bernardi B B Golineli and C J Contreras-CastilloldquoRevisao aspectos da aplicacao de culturas starter na producaode embutidos carneos fermentadosrdquo Brazilian Journal of FoodTechnology vol 13 no 2 pp 133ndash140 2010

[13] F Irlinger ldquoSafety assessment of dairy microorganismscoagulase-negative staphylococcirdquo International Journal of FoodMicrobiology vol 126 no 3 pp 302ndash310 2008

[14] B Ghebremedhin F Layer W Konig and B Konig ldquoGeneticclassification and distinguishing of Staphylococcus species basedon different partial gap 16S rRNA hsp60 rpoB sodA and tufgene sequencesrdquo Journal of Clinical Microbiology vol 46 no 3pp 1019ndash1025 2008

[15] I Babic K Markov D Kovacevic et al ldquoIdentification andcharacterization of potential autochthonous starter culturesfrom aCroatian lsquobrandrsquo product lsquoslavonski kulenrsquordquoMeat Sciencevol 88 no 3 pp 517ndash524 2011

[16] MMehrotra GWang andWM Johnson ldquoMultiplex PCR forthe detection of genes for Staphylococcus aureus enterotoxinsexfoliative toxins toxic shock syndrome toxin and methicillinresistancerdquo Journal of Clinical Microbiology vol 38 no 3 pp1032ndash1041 2000

[17] WM Johnson SD Tyler E P Ewan F EAshtonDR Pollardand K R Rozee ldquoDetection of genes for enterotoxins exfolia-tive toxins and toxic shock syndrome toxin 1 in Staphylococcusaureus by the polymerase chain reactionrdquo Journal of ClinicalMicrobiology vol 29 no 3 pp 426ndash430 1991

[18] J Mclauchlin G L Narayanan VMithani andG OrsquoNeill ldquoThedetection of enterotoxins and toxic shock syndrome toxin genesin Staphylococcus aureus by polymerase chain reactionrdquo Journalof Food Protection vol 63 no 4 pp 479ndash488 2000

[19] J P Rosec and O Gigaud ldquoStaphylococcal enterotoxin genes ofclassical and new types detected by PCR in Francerdquo Interna-tional Journal of Food Microbiology vol 77 no 1-2 pp 61ndash702002

[20] K Omoe D-L Hu H Takahashi-Omoe A Nakane andK Shinagawa ldquoIdentification and characterization of a newstaphylococcal enterotoxin-related putative toxin encoded bytwo kinds of plasmidsrdquo Infection and Immunity vol 71 no 10pp 6088ndash6094 2003

[21] N Sergeev D Volokhov V Chizhikov and A Rasooly ldquoSimul-taneous analysis of multiple staphylococcal enterotoxin genesby an oligonucleotide microarray assayrdquo Journal of ClinicalMicrobiology vol 42 no 5 pp 2134ndash2143 2004

[22] S Holtfreter D Grumann M Schmudde et al ldquoClonal distri-bution of superantigen genes in clinical Staphylococcus aureusisolatesrdquo Journal of Clinical Microbiology vol 45 no 8 pp2669ndash2680 2007

[23] W J Mason J S Blevins K Beenken NWibowo N Ojha andM S Smeltzer ldquoMultiplex PCR protocol for the diagnosis ofstaphylococcal infectionrdquo Journal of Clinical Microbiology vol39 no 9 pp 3332ndash3338 2001

[24] E Rahimi and H G Safai ldquoDetection of classical enterotoxinsof Staphylococcus aureus strains isolated frombovine subclinicalmastitis in Isfahan Iranrdquo Veterinary Microbiology vol 141 no3-4 pp 393ndash394 2010

[25] M A Larkin G Blackshields N P Brown et al ldquoClustalW andClustal X version 20rdquo Bioinformatics vol 23 no 21 pp 2947ndash2948 2007

[26] P H A Sneath and R R Sokal Unweighted Pair Group Methodwith Arithmetic Mean Numerical Taxonomy W H FreemanSan Francisco Calif USA 1973

[27] NCCLS ldquoPerformance standards for antimicrobial susceptibil-ity testing 14th informational supplementrdquo Tech Rep M 100-S14 NCCLS 2004

[28] S Corbiere Morot-Bizot S Leroy and R Talon ldquoMonitoringof staphylococcal starters in two French processing plantsmanufacturing dry fermented sausagesrdquo Journal of AppliedMicrobiology vol 102 no 1 pp 238ndash244 2007

[29] R Talon S Leroy I Lebert et al ldquoSafety improvement andpreservation of typical sensory qualities of traditional dryfermented sausages using autochthonous starter culturesrdquo Inter-national Journal of Food Microbiology vol 126 no 1-2 pp 227ndash234 2008

[30] G Mauriello A Casaburi G Blaiotta and F Villani ldquoIsolationand technological properties of coagulase negative staphylo-cocci from fermented sausages of Southern ItalyrdquoMeat Sciencevol 67 no 1 pp 149ndash158 2004

[31] M Janssens N Myter L De Vuyst and F Leroy ldquoSpeciesdiversity and metabolic impact of the microbiota are low inspontaneously acidified Belgian sausages with an added starterculture of Staphylococcus carnosusrdquo Food Microbiology vol 29no 2 pp 167ndash177 2012

[32] G Blaiotta D Ercolini G Mauriello G Salzano and F VillanildquoRapid and reliable identification of Staphylococcus equorum bya species-specific PCR assay targeting the sodA generdquo Systematicand Applied Microbiology vol 27 no 6 pp 696ndash702 2004

[33] L Kozacinski E Drosinos F Caklovica L Cocolin J Gasparik-Reichardt and S Veskovic ldquoInvestigation of microbial associa-tion of traditionally fermented sausagesrdquo Food Technology andBiotechnology vol 46 no 1 pp 93ndash106 2008

[34] S Leroy P Giammarinaro J-P Chacornac I Lebert and RTalon ldquoBiodiversity of indigenous staphylococci of naturallyfermented dry sausages and manufacturing environments ofsmall-scale processing unitsrdquo Food Microbiology vol 27 no 2pp 294ndash301 2010

[35] E Coton M-H Desmonts S Leroy et al ldquoBiodiversity ofCoagulase-Negative Staphylococci in French cheeses dry fer-mented sausages processing environments and clinical sam-plesrdquo International Journal of Food Microbiology vol 137 no 2-3 pp 221ndash229 2010

[36] S Hosseinzadeh and H D Saei ldquoStaphylococcal species associ-ated with bovinemastitis in the NorthWest of Iran emerging ofcoagulase-negative staphylococcirdquo International Journal of Vet-erinary Science and Medicine vol 2 no 1 pp 27ndash34 2014

[37] A Piette and G Verschraegen ldquoRole of coagulase-negativestaphylococci in human diseaserdquo Veterinary Microbiology vol134 no 1-2 pp 45ndash54 2009

[38] T M Moura F S Campos P A DrsquoAzevedo et al ldquoPrevalenceof enterotoxin-encoding genes and antimicrobial resistancein coagulase-negative and coagulase-positive Staphylococcusisolates from black pudding in Southern Brazilrdquo Revista daSociedade Brasileira deMedicina Tropical vol 45 no 5 pp 579ndash585 2012

[39] H I Suzuki T Lefebure P P Bitar and M J StanhopeldquoComparative genomic analysis of the genus Staphylococcusincluding Staphylococcus aureus and its newly described sister

BioMed Research International 17

species Staphylococcus simiaerdquo BMC Genomics vol 13 article38 2012

[40] R P Lamers G Muthukrishnan T A Castoe S Tafur A MCole and C L Parkinson ldquoPhylogenetic relationships amongStaphylococcus species and refinement of cluster groups basedon multilocus datardquo BMC Evolutionary Biology vol 12 article171 2012

[41] I V Pinchuk E J Beswick and V E Reyes ldquoStaphylococcalenterotoxinsrdquo Toxins vol 2 no 8 pp 2177ndash2197 2010

[42] S R Monday and G A Bohach ldquoGenes encoding staphylo-coccal enterotoxins G and I are linked and separated by DNArelated to other staphylococcal enterotoxinsrdquo Journal of NaturalToxins vol 10 no 1 pp 1ndash8 2001

[43] D Y Thomas S Jarraud B Lemercier et al ldquoStaphylococcalenterotoxin-like toxins U2 and V two new staphylococcalsuperantigens arising from recombination within the entero-toxin gene clusterrdquo Infection and Immunity vol 74 pp 4724ndash4734 2006

[44] N Indrawattana O Sungkhachat N Sookrung et al ldquoStaphylo-coccus aureus clinical isolates antibiotic susceptibility molecu-lar characteristics and ability to form biofilmrdquo BioMed ResearchInternational vol 2013 Article ID 314654 11 pages 2013

[45] V Pereira C Lopes A Castro J Silva P Gibbs and P TeixeiraldquoCharacterization for enterotoxin production virulence factorsand antibiotic susceptibility of Staphylococcus aureus isolatesfrom various foods in Portugalrdquo Food Microbiology vol 26 no3 pp 278ndash282 2009

[46] I Anacarso C Condo C Sabia and et al ldquoAntimicrobialresistance and other related virulence factors in staphylococcusspp isolated from food environmental and humans in ItalyrdquoUniversal Journal Microbiology Research vol 1 no 1 pp 1ndash92013

[47] B Martın M Garriga M Hugas S Bover-Cid M T Veciana-Nogues and T Aymerich ldquoMolecular technological and safetycharacterization of Gram-positive catalase-positive cocci fromslightly fermented sausagesrdquo International Journal of FoodMicrobiology vol 107 no 2 pp 148ndash158 2006

[48] NM Broekema T Van Tam T AMonson S AMarshall andD M Warshauer ldquoComparison of cefoxitin and oxacillin diskdiffusionmethods for detection ofmecA-mediated resistance inStaphylococcus aureus in a large-scale studyrdquo Journal of ClinicalMicrobiology vol 47 no 1 pp 217ndash219 2009

[49] E Cercenado F Garcıa-Garrote and E Bouza ldquoIn vitro activityof linezolid against multiply resistant Gram-positive clinicalisolatesrdquo Journal of Antimicrobial Chemotherapy vol 47 no 1pp 77ndash81 2001

[50] A Aydin K Muratoglu M Sudagidan K Bostan B Okukluand SHarsa ldquoPrevalence and antibiotic resistance of foodborneStaphylococcus aureus isolates in Turkeyrdquo Foodborne Pathogensand Disease vol 8 no 1 pp 63ndash69 2011

[51] D Sergelidis A Abrahim T Papadopoulos et al ldquoIsolation ofmethicillin-resistant Staphylococcus spp from ready-to-eat fishproductsrdquo Letters in Applied Microbiology vol 59 no 5 pp500ndash506 2014

[52] I PhillipsM Casewell T Cox et al ldquoDoes the use of antibioticsin food animals pose a risk to human health A critical reviewof published datardquo Journal of Antimicrobial Chemotherapy vol53 no 1 pp 28ndash52 2004

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Page 17: Research Article Safety Evaluation of the Coagulase ...downloads.hindawi.com/journals/bmri/2015/483548.pdf · Research Article Safety Evaluation of the Coagulase-Negative Staphylococci

BioMed Research International 17

species Staphylococcus simiaerdquo BMC Genomics vol 13 article38 2012

[40] R P Lamers G Muthukrishnan T A Castoe S Tafur A MCole and C L Parkinson ldquoPhylogenetic relationships amongStaphylococcus species and refinement of cluster groups basedon multilocus datardquo BMC Evolutionary Biology vol 12 article171 2012

[41] I V Pinchuk E J Beswick and V E Reyes ldquoStaphylococcalenterotoxinsrdquo Toxins vol 2 no 8 pp 2177ndash2197 2010

[42] S R Monday and G A Bohach ldquoGenes encoding staphylo-coccal enterotoxins G and I are linked and separated by DNArelated to other staphylococcal enterotoxinsrdquo Journal of NaturalToxins vol 10 no 1 pp 1ndash8 2001

[43] D Y Thomas S Jarraud B Lemercier et al ldquoStaphylococcalenterotoxin-like toxins U2 and V two new staphylococcalsuperantigens arising from recombination within the entero-toxin gene clusterrdquo Infection and Immunity vol 74 pp 4724ndash4734 2006

[44] N Indrawattana O Sungkhachat N Sookrung et al ldquoStaphylo-coccus aureus clinical isolates antibiotic susceptibility molecu-lar characteristics and ability to form biofilmrdquo BioMed ResearchInternational vol 2013 Article ID 314654 11 pages 2013

[45] V Pereira C Lopes A Castro J Silva P Gibbs and P TeixeiraldquoCharacterization for enterotoxin production virulence factorsand antibiotic susceptibility of Staphylococcus aureus isolatesfrom various foods in Portugalrdquo Food Microbiology vol 26 no3 pp 278ndash282 2009

[46] I Anacarso C Condo C Sabia and et al ldquoAntimicrobialresistance and other related virulence factors in staphylococcusspp isolated from food environmental and humans in ItalyrdquoUniversal Journal Microbiology Research vol 1 no 1 pp 1ndash92013

[47] B Martın M Garriga M Hugas S Bover-Cid M T Veciana-Nogues and T Aymerich ldquoMolecular technological and safetycharacterization of Gram-positive catalase-positive cocci fromslightly fermented sausagesrdquo International Journal of FoodMicrobiology vol 107 no 2 pp 148ndash158 2006

[48] NM Broekema T Van Tam T AMonson S AMarshall andD M Warshauer ldquoComparison of cefoxitin and oxacillin diskdiffusionmethods for detection ofmecA-mediated resistance inStaphylococcus aureus in a large-scale studyrdquo Journal of ClinicalMicrobiology vol 47 no 1 pp 217ndash219 2009

[49] E Cercenado F Garcıa-Garrote and E Bouza ldquoIn vitro activityof linezolid against multiply resistant Gram-positive clinicalisolatesrdquo Journal of Antimicrobial Chemotherapy vol 47 no 1pp 77ndash81 2001

[50] A Aydin K Muratoglu M Sudagidan K Bostan B Okukluand SHarsa ldquoPrevalence and antibiotic resistance of foodborneStaphylococcus aureus isolates in Turkeyrdquo Foodborne Pathogensand Disease vol 8 no 1 pp 63ndash69 2011

[51] D Sergelidis A Abrahim T Papadopoulos et al ldquoIsolation ofmethicillin-resistant Staphylococcus spp from ready-to-eat fishproductsrdquo Letters in Applied Microbiology vol 59 no 5 pp500ndash506 2014

[52] I PhillipsM Casewell T Cox et al ldquoDoes the use of antibioticsin food animals pose a risk to human health A critical reviewof published datardquo Journal of Antimicrobial Chemotherapy vol53 no 1 pp 28ndash52 2004

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Page 18: Research Article Safety Evaluation of the Coagulase ...downloads.hindawi.com/journals/bmri/2015/483548.pdf · Research Article Safety Evaluation of the Coagulase-Negative Staphylococci

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology