Staphylococcus aureus - Hindawi

Research ArticleDetection of Pathogenic Escherichia coli andStaphylococcus aureus from Cattle and Pigs Slaughtered inAbattoirs in Vhembe District South Africa

Nicoline F Tanih1 Eunice Sekwadi1 Roland N Ndip23 and Pascal O Bessong1

1HIVAIDS amp Global Health Research Programme Department of Microbiology University of VendaThohoyandou 0950 South Africa2Department of Microbiology and Parasitology Faculty of Science University of Buea Buea Cameroon3Department of Biochemistry and Microbiology Faculty of Science and Agriculture University of Fort HareAlice 5700 South Africa

Correspondence should be addressed to Nicoline F Tanih nicofrilineyahoocom

Received 11 December 2014 Accepted 26 January 2015

Academic Editor Hassan Zaraket

Copyright copy 2015 Nicoline F Tanih et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Pathogenic food-borne bacteria have been associated with severe morbidity and mortality in humans and animals This studywas aimed at determining the prevalence of Staphylococcus aureus Salmonella spp and Escherichia coli present in cattle and pigsslaughtered in selected abattoirs in Vhembe District and at determining the susceptibility of the isolates to antibiotics A totalof 176 swab samples (28 cattle and 16 pigs) of the rump flank brisket and neck of the animals were analyzed using standardmicrobiological methods E coli isolates were genotyped to detect pathogenic strains Of the 176 samples 104 (675) were positivefor E coli and 50 (325) for S aureus There was no statistically significant difference (119875 gt 005) in the isolation rate fromthe different animal parts or abattoirs Overall 14104 (1346) of the E coli isolates were pathogenic strains which includedenteropathogenic E coli (EPEC) (bfpA) 19 enterotoxigenic E coli (ETEC) (LT) 38 and enteroaggregative E coli (EAEC) (aaiC)76 E coli isolates were resistant (100) to vancomycin and bacitracin S aureus (100) were resistant to oxacillin and nalidixicacidThe presence of resistant strains of these bacteria in food of animal origin could serve as important vehicles transmitting thesebacteria to humans This finding is of epidemiological significance

1 Introduction

Food-borne pathogens have been extensively incriminatedworldwide as common causes of bacterial infections inhumans with food animals serving as important reservoirs[1ndash3] In industrialized countries microbiological food-borneillnesses were reported in up to 30 of the population [4]The majority of morbidity and mortality related to food-borne infections are caused by bacterial agents [5ndash7] Foodpoisoning is commonly manifested as diarrheal diseaseswhich are often triggered either by toxin production by themicrobe or by the hostrsquos reaction to the infection [5 7 8]

A number of pathogenic bacteria have been associatedwith food of animal origin these include amongst others

Salmonella spp Campylobacter spp Staphylococcus aureusListeria monocytogenes Clostridium perfringens Clostridiumbotulinum E coli 0157H7 and enterohemorrhagic E coli(EHEC) [1 3 9 10] Staphylococci are normal inhabitants ofthe skin andmucousmembranes of animals and humans andstrains with pathogenic potential are known to cause diseaseswhich range from simple abscesses and mastitis to the moresevere toxic shock syndrome [11ndash13] Mastitis in cattle hasequally been associated with a number of microorganismsincluding Escherichia coli and S aureus [13 14] Salmonellaenterica is a significant cause of morbidity and mortalityin humans and animals with contaminated food of animalorigin particularly meat products from cattle and pigs beingan important source of human infections [1 2 15]Escherichia

Hindawi Publishing Corporatione Scientific World JournalVolume 2015 Article ID 195972 8 pageshttpdxdoiorg1011552015195972

2 The Scientific World Journal

coli occur as normal flora in the gastrointestinal tract ofhumans and animalsHowever pathogenicE coli strains havebeen reported to cause life threatening infections in humansworldwide [15 16]

Antibiotic resistance remains amajor challenge in humanand animal health Resistance is increasingly being rec-ognized in pathogens isolated from food [1 17ndash19] Foodcontamination with antibiotic-resistant bacteria can there-fore be a major threat to public health as the antibioticresistance determinants can be transferred to other bacteriaof human clinical significance [20 21] Furthermore transferof these resistant bacteria to humans has significant publichealth implications by increasing the number of food-borneillnesses and the potential for treatment failure [21]

Food of animal origin could be contaminated fromthe farm a situation which may be further compoundedif the food is not properly handled during slaughteringand processing giving way for pathogens to multiply [22]The conditions under which these foods are handled raisequestions regarding their microbiological quality Studiesconducted in different countries to investigate the microbio-logical quality of food of animal origin reported the presenceof potential human pathogens [1 2] In South Africa a largeproportion of the population relies on beef and pork astheir source of protein which could expose them to infectionif contaminated [3 16] Even though other studies havereported the health risk associated with consumption of suchproducts there is a paucity of studies on the microbiologicalquality of cattle and pig slaughtered in abattoirs in theVhembeDistrict of Limpopo ProvinceThe present studywascarried out to assess the microbiological quality of beef andpork slaughtered in this area in a bid to throw more light onthe inherent risk associated with such foods

2 Materials and Methods

21 Study Site Description The study sites included Vyge-boomdrift pig abattoir (A1) C-net (A2) Mukwevho (A3)and Shayandima (A4) all of which are found in the VhembeDistrict Limpopo Province South Africa C-net Mukw-evho and Shayandima are cattle abattoirs where peopleoccasionally bring their personal cattle for slaughtering foroccasions such as funerals weddings or family gatheringsVygeboomdrift abattoir is a commercial abattoir where pigsare slaughtered

22 Sample Collection Samples were collected according tothe method of Pearce and Bolton [24] A total of 176 swabbedsamples were collected from 28 cattle (8 8 and 12 cattlefrom abattoirs 2 3 and 4 resp) and 16 pigs from abattoir 1Four parts of each animal which included the neck brisketrump and flank were sampled with sterile swab rinsing kit(containing 10mL isotonic buffer rinse solution) Sampleswabbing of the carcasses was performed after the removal ofthe gastrointestinal tract The tip of the swab was moistenedwith rinse solution before swabbing the carcass The area forswabbing was selected by using 100 cm2 sterile disposableplastic template (Analytical Diagnostics USA)The swabbing

was done 10x horizontally and 10x vertically at each site Thetotal areas sampled were 400 cm2 of each carcass Sampleswere transported on ice and processed within 2 hours

23 Microbial Analysis

231 Isolation and Identification of Bacterial PathogensSalmonella spp were identified using 1mL meat rinsedsolution mixed with 9mL buffered peptone water (Oxoid)and incubated at 37∘C for 24 hours After incubation1mL of preenrichment broth was transferred into 9mL ofRappaport-Vassiliadis (RV) soya peptone broth (Oxoid) andBrilliant green agar (Oxoid) plates and incubated at 37∘C for24 hours

For identification of S aureus the swab from swab rinsingsolution was spread-plated on mannitol salt agar (Oxoid)plates and incubated at 37∘C for 24 hours Staphylococcicoagulase positive colonies were confirmed using StaphylaseTest Kit (Oxoid) Yellow colonies which were Gram positivecocci in cluster oxidase negative and coagulase and catalasepositive and which produced clots were recorded as S aureusandmaintained at minus20∘C in 20 glycerol brain heart infusionbroth for further studies S aureus ATCC 25923 was used asa positive control

E coli was identified by pipetting 1mL of rinsed solutionin three test tubes each containing 9mL Brilliant greenbroth (Oxoid) fitted with Durham tubes and incubated at45∘C for 48 hours Tubes with gas bubbles in the Durhamtubes were streaked on MacConkey agar (Oxoid) plate andincubated at 37∘C for 24 hours Suspected colonieswere testedfor indole production Pink colonies on MacConkey mediawhichwere indole positive were considered positive forE coliandmaintained at minus20∘C in 20 glycerol brain heart infusionbroth for further confirmation and characterization by PCRand antibiotic susceptibility testing E coli ATCC 25922 wasused as a positive control

Presumptive bacteria identification was based on colonypigmentation and Gram staining characteristics Pure cul-tures were obtained by streaking a portion of an isolatedcolony on nutrient agar and incubated aerobically at 37∘Cfor 24 h All preliminary results were confirmed using theMicroscan-Autoscan equipment (Siemens Germany) follow-ing themanufacturerrsquos instruction Briefly an inoculumof thebacterium was prepared in saline and transferred onto 96-well plates in an automated pattern Reagents to supplementvarious reactions to aid identification of both Gram negativeand positive bacteria were added to some of the wells andincubated at 37∘C for 24 hours Plates were read and resultsinterpreted

24 Molecular Identification of E coli Using mPCR

241 Extraction of DNA E coli cultures were revived bystreaking on nutrient agar (Oxoid) and incubated at 37∘Cfor 24 hours Thereafter 05120583L of Triton X and 500120583L ofsterile distilled water were mixed with one colony in a 15mLEppendorf tube and mixed by vortexing for 5 seconds Themixture was boiled in a water bath at 100∘C for 20 minutes

The Scientific World Journal 3

Table 1 Primer sequences used in multiplex PCR for detection of pathogenic E coli

E coli type Primer sequences Product size in bp ReferencesETEC (LT)

ETEC 508F 51015840-CACACGGAGCTCCTCAGTC-31015840 508 bp [23]ETEC 508R 51015840-CCCCCAGCCTAGCTTAGTTT-31015840

ETEC (ST)ETEC 147F 51015840-GCTAAACCAGTAGAGGTCTTCAAAA-31015840 147 bp [23]ETEC 147R 51015840-CCCGGTACAGAGCAGGATTACAACA-31015840

EHEC (Stx1)EHEC 384F 51015840-CAGTTAATGTGGTGGCGAAGG-31015840 384 bp [23]EHEC 384R 51015840-CACCAGACAATGTAACCGCTG-31015840

EHEC (Stx2)EHEC 584F 51015840-ATCCTATTCCCGGGAGTTACG-31015840 584 bp [23]EHEC 584R 51015840-GCGTCATCGTATACACAGGAGC-31015840

EPEC (eae)EPEC 881F 51015840-CCCGAATTCGGCACAAGCATAAGC-31015840 881 bp [23]EPEC 881R 51015840-CCCGGATCCGTCTCGCCAGTATTCG-31015840

EPEC (bfpA)EPEC 300F 51015840-GGAAGTCAAATTCATGGGGGTAT-31015840 300 bp [23]EPEC 300R 51015840-GGAATCAGACGCAGACTGGTAGT-31015840

EIEC (ipaH)EIEC 423F 51015840-TGGAAAAACTCAGTGCCTCT-31015840 423 bp [23]EIEC 423R 51015840-CCAGTCCGTAAATTCATTCT-31015840

EAEC (aatA)EAEC 650F 51015840-CTGGCGAAAGACTGTATCAT-31015840 650 bp [23]EAEC 650R 51015840-CAATGTATAGAAATCCGCTGTT-31015840

EAEC (aaic)EAEC 215F 51015840-ATTGTCCTCAGGCATTTCAC-31015840 215 bp [23]EAEC 215R 51015840-ACGACACCCCTGATAAACAA-31015840

and centrifuged at 10000 rpm for 10 minutes Five microlitresof the supernatant was used as DNA template for polymerasechain reaction

242 Multiplex Polymerase Chain Reaction (mPCR) Mul-tiplex polymerase chain reaction analysis of the targetedgenes of interest was performed using DreamTaq DNApolymerase (Thermo Scientific USA) For the amplificationfive microlitres of DNA was added to 20 120583L of mastermix containing 125 120583L of DreamTaq DNA polymerase (2XDreamTaq Green Buffer dATP dCTP dGTP and dTTP04mM each and 4mM MgCl

2) (Thermo Scientific USA)

05 120583L (02 120583M) of respective oligonucleotide primers and thereaction volume was made up with nuclease free water PCRwas performed in a thermal cycler (Bio-Rad LaboratoriesUSA) The amplification cycles consisted of an initial DNAdenaturation at 95∘C for 15min followed by 35 cycles ofdenaturation at 94∘C for 45 s primer annealing at 55∘C for45 s extension at 68∘C for 2min and a final single elongationat 72∘C for 5min The primers used to amplify the targetedgenes were as previously reported by Nguyen et al [23] andare summarized in Table 1 Negative controls substitutingDNA template with ultrapure water (Sigma-Aldrich UK)were included in all PCR runs DNA extracted from E coli

ATCC 25922 was used as a positive control Amplified DNAwas resolved by 2 agarose gel electrophoresis and visualisedunder UV transillumination

25 Antibiotic Susceptibility Testing Antibiotic susceptibilitytesting was performed by the Kirby-Bauer disc-diffusion testwhich conforms to the recommended standard of theClinicaland Laboratory Standards Institute (CLSI) as previouslydescribed by Nyenje et al [25] Briefly an inoculum ofeach pure bacterial isolate was emulsified in 3mL of sterilenormal saline and the density adjusted to 05 McFarlandstandard A sterile cotton swab was dipped into the standard-ized suspension of bacterial cultures and used to inoculateMueller-Hinton agar (MHA) plates (Biotec England) andthe plates were allowed to dry Antibiotic discs with thefollowing drug contents ampicillin (10 120583g) bacitracin (10 120583g)erythromycin (15 120583g) oxytetracycline (30 120583g) streptomycin(10 120583g) cephalothin (30 120583g) nalidixic acid (30 120583g) gen-tamycin (10 120583g) vancomycin (30120583g) and oxacillin (1 120583g)(Antibiotic Becton Dickson and Company Sparks USA LePont de Claix France) were placed onto MHA plates Theplates were incubated at 37∘C for 24 hoursThe zone diameterwas measured and results were interpreted based on CLSI[26] The reference strains E coli ATCC 25922 and S aureus


Table 2 Bacteria distribution in the different parts of cattle and pig carcasses examined in the various abattoirs

Abattoirs Animal parts S aureus E coli Pathogenic E coli Number of samples from each abattoir Number of animals sampled

1-pork A1

P1 neck 10 16 2 (EAEC)P2 brisket 2 12 64 16P3 flank 8 16P4 rump 0 6

2-beef A2

B1 neck 2 14B2 brisket 8 8 2 (EAEC) 32 8B3 flank 0 4B4 rump 0 4

3-beef A3

B1 neck 2 0B2 brisket 2 6 32 8B3 flank 4 0B4 rump 2 0

4-beef A4

B1 neck 4 8 2 (EAEC) 2 (ETEC)B2 brisket 2 6 2 (EAEC) 2 (EPEC) 48 12B3 flank 2 2 2 (ETEC)B4 rump 2 2

Total 50 104 14

Table 3 Bacteria distribution in cattle and pig carcasses examined in the various abattoirs

Bacterial isolatesAbattoirs

Number () occurrence1-pork A1 2-beef A2 3-beef A3 4-beef A4(119899 = 64) (119899 = 32) (119899 = 32) (119899 = 48)

E coli 50 (325) 30 (195) 6 (389) 18 (117) 104 (675)S aureus 20 (1298) 10 (65) 10 (65) 10 (65) 50 (326)Total 70 (455) 40 (205) 16 (104) 28 (182) 154 (875)

ATCC 25923 were used to verify the quality and accuracy ofthe testing procedure

26 Statistical Analysis Statistical analysis was performedusing SPSS version 22 The chi-square test was used tocompare rate of isolation of the various pathogens in beef andpork and the different animal parts sampled Comparisonswere also done among the abattoirs Differences were consid-ered significant at 119875 lt 005

3 Results

31 Prevalence of Bacteria Pathogens in the Various Ani-mal and Abattoir Types Table 2 depicts the prevalence ofpathogens investigated in the 176 samples examined Overalla high prevalence of 875 (154176) was reported from thesamples examined Bacteria were isolated in all the abattoirtypes themost prevalent bacteriawereE coli 675 (104154)while S aureus was 325 (50154) No Salmonella wasisolated in this study Both E coli and S aureus were moreprevalent in pork with percentages of 481 (50104) and40 (2050) respectively than in cattle across the differentabattoirs The isolation rate of the pathogens from cattle andpigs was however not statistically significant (119875 gt 005)

Overall isolation rate of both organisms combined washighest from the neck samples 364 (56154) followed bybrisket 298 (46154) flank 234 (36154) and rump 104(16154) (Table 2)

Abattoirs 1-pork A1 and 2-beef A2 had the highestisolation rates of 4545 (70154) and 1948 (30154)respectively The lowest isolation rate of 91 (14154) wasobtained in abattoir 3-beef A3 (Table 3) The isolation rateof the pathogens between the different abattoirs was notstatistically significant (119875 gt 005)

32 Prevalence of Pathogenic E coli Overall pathogenic Ecoli was detected in 1346 (14104) with 192 of bfpA(EPEC) 384 of LT (ETEC) and 769 of aaiC (EAEC)(Table 4) EIEC (enteroinvasive E coli) was not detected inthis study

33 Antimicrobial Patterns All E coli isolates tested (100)were susceptible to nalidixic acid cephalothin gentamycinand ampicillin 90 were susceptible to streptomycin Ahundred percent resistance was recorded for bacitracinand vancomycin while resistances of 98 92 and 57were reported for oxacillin erythromycin and streptomycinrespectively The rest showed either intermediate or total


Table 4 Prevalence of pathogenic E coli from the various abattoirs

AbattoirsE coli pathotypes and associated genes Total E coli

119873

Total ()pathogenicEPEC ETEC EIEC EAEC EHEC

bfpA eae LT ST ipaH aatA aaiC Stx12Abattoir 1 2 54 2 (1428)Abattoir 2 2 26 2Abattoir 3 6 0 (0)Abattoir 4 2 4 4 18 10Total 2 4 8 104 14 (1346)

Table 5 Antimicrobial susceptibility profile of S aureus and E coli isolated from cattle and pigs carcasses

Antibiotics E coli (119899 = 104) () S aureus (119899 = 50) ()R I S R 1 S

Bacitracin 104 (100) mdash mdash 6 (12) mdash 44 (88)Erythromycin 96 (923) 2 (192) 6 (57) mdash mdash 50 (100)Vancomycin 104 (100) mdash mdash mdash mdash 50 (100)Oxacillin 102 (98) 2 (192) mdash 50 (100) mdash mdashOxytetracycline 100 (96) mdash 4 (38) 22 (44) mdash 28 (56)Nalidixic acid mdash mdash 104 (100) 50 (100) mdash mdashCephalothin mdash mdash 104 (100) mdash mdash 50 (100)Gentamycin mdash mdash 104 (100) mdash mdash 50 (100)Ampicillin mdash mdash 104 (100) mdash mdash 50 (100)Streptomycin 6 (57) 4 (38) 94 (90) mdash mdash 50 (100)

resistance to these antibiotics (Table 5) Of the 50 S aureusisolates 100 susceptibility was recorded for cephalothingentamycin ampicillin streptomycin vancomycin and ery-thromycin while 88 and 56 were recorded against oxyte-tracycline and bacitracin respectively On the other hand100 resistance was noted for nalidixic acid and oxacillin(Table 5)Multidrug resistancewas not common in this studyOnly two isolates were resistant to more than two antibioticsthis included oxacillin nalidixic acid oxytetracycline andbacitracin

4 Discussion

Foods contaminated with enteropathogenic bacteria are animportant factor contributing to the high incidence ofdiarrhea in developing countries [27] Pathogenic E colinontyphoid Salmonella serovars and S aureus remain apotential threat to human health with beef broiler chickensand pork serving as possible sources of these organismsin the environment [2 15 16] The clinical significance ofthese pathogens cannot be overemphasized Pathogenic Ecoli is recognized as an important pathogen in outbreaksof acute diarrhea especially in developing countries [7 2829] This study investigated the prevalence and antibiogramof these pathogens in a bid to provide baseline data forepidemiological surveillance

Overall E coli 104154 (675) was the most detectedpathogen followed by S aureus 50154 (325)These findingscorroborate those of other studies that equally reported a highprevalence of either E coli S aureus or both [19 21 22 30]

Several studies have reported the presence of E coli 0157H7in beef and pork carcasses [5 15 16 31 32] However ourstudy focused on diarrheagenic E coli pathotypes Interest-ingly some E coli pathotypes were detected in this studyRivas Pala and Sevilla [33] in their study also found S aureusin 1690 of meat samples Salmonella was not detected inour study a finding which is similar to that of Movassaghet al [3] who did not also report Salmonella isolates intheir study on beef carcasses The high prevalence of theseorganisms in these animals could result from consumption ofcontaminated feed [2] or grazing plants that may have beencontaminated through fertilization with untreated effluentsor sludgeThere is a high probability that the immediate envi-ronment of these animals was not endemic with SalmonellaHowever our findings are contrary to other studies whichreported the presence of Salmonella in beef and pork [1 2 1532 34 35] Both organisms (E coli and S aureus) combinedwere more isolated from neck samples 364 (56154) Ourresults tie with the finding of Pearce and Bolton [24] whoreported a higher isolation rate of Enterobacteriaceae fromthe neck and shoulder regions of slaughtered animals in theirstudyThe rumpwas the site with the least isolation rate 104(16154) This could be due to the fact that microorganismneeds enough nutrients and oxygen to grow and multiplewhich could be absent in the rump given ismade up ofmostlymuscles Initial contamination of meat is likely to occurduring slaughtering [36] According to studies by Podpecan etal [36] the presence of S aureus in meat commonly indicatescontamination that may be directly introduced by the handsof workers and contaminated equipment


The rates of microbial contamination of abattoirs meatwith E coli and S aureus in this study ranged from 65to 325 in the different abattoirs The pathogens wereisolated more frequently from 1-pork A1 abattoir though thedifference was not statistically significant (119875 gt 005) Worthyof note is the fact that 1-pork A1 abattoir is a pig abattoirThisfinding may not be far from reality given that pigs are filthycompared to cattle The organisms were also isolated in theother abattoirs in our study The lowest isolation rate of 91(14154) was found in abattoir 3-beef A3 The sanitation levelin this abattoirwas seemingly better than the others and couldexplain this difference

Of significance is the fact that 1346 (14104) E colistrains isolated were positive for pathogenic E coli EAECwas the most detected pathotype with a prevalence of 769(8104) followed by ETEC 384 (4104) and EPEC 192(2104) Enteroinvasive E coli (EIEC) and EHEC were notdetected in this study The extremely high prevalence ofnonpathogenic E coli 923 (96104) in this study maynot be surprising The majority of E coli are harmlesscommensals of the mammalian gastrointestinal tract [37]The presence of EAEC in this study is consistent with theworks of Harrington et al [37] who previously reportedthis organism in association with food-borne diseases AlsoEAEC is increasingly being reported as an emerging diarrhealpathogen worldwide [29] The absence of STEC (EHEC) andEIEC inmeat analysed in this study is interesting consideringthat other studies in different countries have reported E coli0157H7 and other strains of STEC in abattoirmeat especiallybeef [38] Specific pathotypes of E coli have been reportedto be prevalent in different geographical regions hence ourenvironment may be void of these pathotypes or they mayexist in low prevalence

The growing problem of antibiotic resistance has becomea significant public health concern [19] S aureus was 100resistant to oxacillin and nalidixic acid in our study This isin line with studies by Haimanot et al [39] who reportedS aureus resistance of 90 to oxacillin Also Ateba et al[13] reported high sensitivity of S aureus to vancomycinsimilar to our findings and high resistance to ampicillinacross the different farms studied in South Africa contraryto the findings of our study Susceptibility to antibioticschanges with time and geographical location [1] Also differ-ent antibiotic practices may account for such trends Eightpercent (450) of S aureus were resistant to more than 3antibiotics

E coli was susceptible to most of the antibiotics used inthis study Studies by Nontongana et al [40] reported 98E coli resistance to ampicillin contrary to our result with100 susceptibility to ampicillin However their study wasfocused on E coli isolates from water sample Our results arehowever similar to other studies that had previously reportedresistances to one or more of the antibiotics that we recordedresistances to [13 40 41] Resistance of these organisms tothe antibiotics may be due to the frequent use of antibioticsin animal husbandry practices [13 41] as most of theseantibiotics are used both in human and in animal medicinesResistant commensal bacteria of food animals such as pig andcattle may increase resistance of pathogenic bacteria in the

intestinal tract of humans [42] causing disease that may bedifficult to treat [43]

5 Conclusion

Our results indicate that cattle and pigs could serve asreservoirs of S aureus and E coli in the Limpopo Provinceof South Africa These isolates were highly susceptible to anumber of antibiotics which could form the basis for empirictreatment of infections caused by these pathogens in ourenvironment We are led to conclude that the absence of astatistically significant difference between different beef andpork carcasses from the different abattoirs might be dueto the small sample size even though some studies havenot reported any statistical difference between the differentanimal carcasses It may be that a large sample size must bestudied to reveal statistically significant relations between thedifferent animal carcasses

Conflict of Interests

The authors declare no conflict of interests

Acknowledgment

The authors are grateful for the collaboration received fromthe management of the abattoirs where samples were col-lected

References

[1] J-F T K Akoachere N F Tanih L M Ndip and R NNdip ldquoPhenotypic characterization of Salmonella typhimuriumisolates from food-animals and abattoir drains in BueaCameroonrdquo Journal of Health Population and Nutrition vol27 no 5 pp 612ndash618 2009

[2] R H Davies R Dalziel J C Gibbens et al ldquoNational survey forSalmonella in pigs cattle and sheep at slaughter in Great Britain(1999-2000)rdquo Journal of Applied Microbiology vol 96 no 4 pp750ndash760 2004

[3] M H Movassagh M Shakoori and J Zolfaghari ldquoThe preva-lence of Salmonella spp In Bovine carcass at Tabriz slaughter-house Iranrdquo Global Veterinaria vol 5 no 2 pp 146ndash149 2010

[4] World Health Organization Initiative to Estimate the GlobalBurden of FoodborneDiseases Information and PublicationsWorld Health Organization Lyon France 2011 httpwwwwhointfoodsafetyfoodborne diseasefergenindex7html

[5] C N AtebaMMbewe andC C Bezuidenhout ldquoPrevalence ofEscherichia coliO157 strains in cattle pigs and humans in NorthWest province South Africardquo South African Journal of Sciencevol 104 no 1-2 pp 7ndash8 2008

[6] J C Buzby andT Roberts ldquoThe economics of enteric infectionshuman foodborne disease costsrdquo Gastroenterology vol 136 no6 pp 1851ndash1862 2009

[7] Centers for Disease Control and Prevention [CDC] Estimatesof Foodborne Illness in the United States 2011 httpwwwcdcgovfoodborneburdenindexhtml

[8] M Teplitski A CWright andG Lorca ldquoBiological approachesfor controlling shellfish-associated pathogensrdquoCurrent Opinionin Biotechnology vol 20 no 2 pp 185ndash190 2009


[9] M E Nyenje C E Odjadjare N F Tanih E Green andR N Ndip ldquoFoodborne pathogens recovered from ready-to-eat foods from roadside cafeterias and retail outlets in aliceeastern cape province SouthAfrica public health implicationsrdquoInternational Journal of Environmental Research and PublicHealth vol 9 no 8 pp 2608ndash2619 2012

[10] O A Olaoye ldquoMeat an overview of its composition biochemi-cal changes and associatedmicrobial agentsrdquo International FoodResearch Journal vol 18 no 3 pp 877ndash885 2011

[11] C S F Easmon and C Adlam Staphylococci and StaphylococcalInfections Volumes 1 and 2 Academic Press London UK 1983

[12] H D Larsen K H Sloth C Elsberg et al ldquoThe dynamics ofStaphylococcus aureus intramammary infection in nine Danishdairy herdsrdquoVeterinaryMicrobiology vol 71 no 1-2 pp 89ndash1012000

[13] C N Ateba M Mbewe M S Moneoang and C C Bezuiden-hout ldquoAntibiotic-resistant Staphylococcus aureus isolated frommilk in theMafikeng Area NorthWest province South AfricardquoSouth African Journal of Science vol 106 no 11-12 pp 1ndash6 2010

[14] I U Khan A A Hassan A Abdulmawjood C Lammler WWolter and M Zschock ldquoIdentification and epidemiologicalcharacterization of Streptococcus uberis isolated from bovinemastitis using conventional and molecular methodsrdquo Journal ofVeterinary Science vol 4 no 3 pp 213ndash224 2003

[15] S Bonardi F Brindani G Pizzin et al ldquoDetection ofSalmonella spp Yersinia enterocolitica and verocytotoxin-producing Escherichia coli O157 in pigs at slaughter in ItalyrdquoInternational Journal of Food Microbiology vol 85 no 1-2 pp101ndash110 2003

[16] C N Ateba and M Mbewe ldquoDetection of Escherichia coliO157H7 virulence genes in isolates from beef pork waterhuman and animal species in the northwest province SouthAfrica public health implicationsrdquo Research in Microbiologyvol 162 no 3 pp 240ndash248 2011

[17] G Arlet T J Barrett P Butaye A Cloeckaert M R Mulveyand D G White ldquoSalmonella resistant to extended-spectrumcephalosporins prevalence and epidemiologyrdquo Microbes andInfection vol 8 no 7 pp 1945ndash1954 2006

[18] M N Skov J S Andersen S Aabo et al ldquoAntimicrobialdrug resistance of Salmonella isolates from meat and humansDenmarkrdquo Emerging Infectious Diseases vol 13 no 4 pp 638ndash641 2007

[19] C S Shekh V V Deshmukh R N Waghamare N MMarkandeya and M S Vaidya ldquoIsolation of pathogenicEscherichia coli from buffalo meat sold in Parbhani city Maha-rashtra IndiardquoVeterinaryWorld vol 6 no 5 pp 277ndash279 2013

[20] T T H Van G Moutafis L T Tran and P J Coloe ldquoAntibioticresistance in food-borne bacterial contaminants in VietnamrdquoApplied and Environmental Microbiology vol 73 no 24 pp7906ndash7911 2007

[21] Y O Adesiji O T Alli M A Adekanle and J B JolayemildquoPrevalence of Arcobacter Escherichia coli Staphylococcusaureus and Salmonella species in retail raw chicken pork beefand goat meat in Osogbo Nigeriardquo Sierra Leone Journal ofBiomedical Research vol 3 no 1 pp 8ndash12 2011

[22] M Ghosh S Wahi M Kumar and A Ganguli ldquoPrevalenceof enterotoxigenic Staphylococcus aureus and Shigella spp insome raw street vended Indian foodsrdquo International Journal ofEnvironmental Health Research vol 17 no 2 pp 151ndash156 2007

[23] T V Nguyen P le Van C le Huy K N Gia and A WeintraubldquoDetection and characterization of diarrheagenic Escherichia

coli from young children in Hanoi Vietnamrdquo Journal of ClinicalMicrobiology vol 43 no 2 pp 755ndash760 2005

[24] R A Pearce and D J Bolton ldquoExcision vs sponge swabbingmdasha comparison of methods for the microbiological sampling ofbeef pork and lamb carcassesrdquo Journal of Applied Microbiologyvol 98 no 4 pp 896ndash900 2005

[25] M E Nyenje N F Tanih E Green and R N Ndip ldquoCurrentstatus of antibiograms of Listeria ivanovii and Enterobactercloacae isolated from ready-to-eat foods in Alice South AfricardquoInternational Journal of Environmental Research and PublicHealth vol 9 no 9 pp 3101ndash3114 2012

[26] Clinical and Laboratory Standards Institute (CLSI) ldquoDiskdiffusion supplemental tablesrdquo Tech Rep M100-S17 Clinicaland Laboratory Standards Institute (CLSI) Wayne Pa USA2007

[27] F E EI-Rami A R Elias T S Fawwak and M A Alexan-der ldquoIdentification of virulence genes among antibacterial-resistance Escherichia coli isolated from poultryrdquo AdvancedStudies in Biology vol 4 no 8 pp 385ndash396 2012

[28] E Woldemariam B Molla D Alemayehu and A MuckleldquoPrevalence and distribution of Salmonella in apparentlyhealthy slaughtered sheep and goats in Debre Zeit EthiopiardquoSmall Ruminant Research vol 58 no 1 pp 19ndash24 2005

[29] N Boisen F Scheutz D A Rasko et al ldquoGenomic characteriza-tion of enteroaggregativeEscherichia coli from children inMalirdquoJournal of Infectious Diseases vol 205 no 3 pp 431ndash444 2012

[30] L S Manguiat and T J Fang ldquoMicrobiological quality ofchicken- and pork-based street-vended foods from TaichungTaiwan and Laguna Philippinesrdquo Food Microbiology vol 36no 1 pp 57ndash62 2013

[31] S Oslash Breum and J Boel ldquoPrevalence of Escherichia coli O157and verocytotoxin producing E coli (VTEC) on Danish beefcarcassesrdquo International Journal of Food Microbiology vol 141no 1-2 pp 90ndash96 2010

[32] M Zarei N Basiri A Jamnejad and M H Eskandari ldquoPreva-lence of Escherichia coli O157H7 Listeria monocytogenes andSalmonella spp in beef buffalo and lamb using multiplex PCRrdquoJundishapur Journal of Microbiology vol 6 no 8 Article IDe7244 2013

[33] T Rivas Pala and A Sevilla ldquoMicrobial contamination ofcarcasses meat and equipment from an Iberian pork cuttingplantrdquo Journal of Food Protection vol 67 no 8 pp 1624ndash16292004

[34] D J Bolton R A Pearce J J Sheridan I S Blair D AMcDow-ell and D Harrington ldquoWashing and chilling as critical controlpoints in pork slaughter hazard analysis and critical controlpoint (HACCP) systemsrdquo Journal of Applied Microbiology vol92 no 5 pp 893ndash902 2002

[35] J M McEvoy A M Doherty J J Sheridan I S Blair and D AMcDowell ldquoThe prevalence of Salmonella spp in bovine faecalrumen and carcass samples at a commercial abattoirrdquo Journal ofApplied Microbiology vol 94 no 4 pp 693ndash700 2003

[36] B Podpecan A Pengov and S Vadnjal ldquoThe source of contam-ination of ground meat for production of meat products withbacteria Staphylococccus aureusrdquo Slovenian Veterinary Researchvol 44 pp 24ndash30 2007

[37] S M Harrington E G Dudley and J P Nataro ldquoPathogenesisof enteroaggregative Escherichia coli infectionrdquo FEMS Microbi-ology Letters vol 254 no 1 pp 12ndash18 2006

[38] R O Elder J E Keen G R Siragusa G A Barkocy-GallagherM Koohmaraie and W W Laegreid ldquoCorrelation of enterohe-morrhagic Escherichia coli O157 prevalence in feces hides and


carcasses of beef cattle during processingrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 97 no 7 pp 2999ndash3003 2000

[39] T Haimanot A Alemseged and G Beyene ldquoMicrobial floraand food pathogens on mincemeat and their susceptibility tomicrobial agentsrdquo Ethiopian Journal of Health Sciences vol 20no 3 pp 137ndash143 2010

[40] N Nontongana T Sibanda E Ngwenya and A I OkohldquoPrevalence and antibiogram profiling of Escherichia coli patho-types isolated from the Kat River and the Fort Beaufort abstrac-tion waterrdquo International Journal of Environmental Research andPublic Health vol 11 no 8 pp 8213ndash8227 2014

[41] V I Enne C Cassar K Sprigings M J Woodward andP M Bennett ldquoA high prevalence of antimicrobial resistantEscherichia coli isolated from pigs and a low prevalence ofantimicrobial resistant E coli from cattle and sheep in GreatBritain at slaughterrdquo FEMS Microbiology Letters vol 278 no 2pp 193ndash199 2008

[42] A E van den Bogaard and E E Stobberingh ldquoEpidemiologyof resistance to antibiotics links between animals and humansrdquoInternational Journal of Antimicrobial Agents vol 14 no 4 pp327ndash335 2000

[43] J N Ombui A M Kimotho and J G Nduhiu ldquoAntimicrobialresistance patterns and plasmid profiles of staphylococcusaureus isolated from milk and meatrdquo East African MedicalJournal vol 77 no 9 pp 463ndash467 2000

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Microbiology


coli occur as normal flora in the gastrointestinal tract ofhumans and animalsHowever pathogenicE coli strains havebeen reported to cause life threatening infections in humansworldwide [15 16]

Antibiotic resistance remains amajor challenge in humanand animal health Resistance is increasingly being rec-ognized in pathogens isolated from food [1 17ndash19] Foodcontamination with antibiotic-resistant bacteria can there-fore be a major threat to public health as the antibioticresistance determinants can be transferred to other bacteriaof human clinical significance [20 21] Furthermore transferof these resistant bacteria to humans has significant publichealth implications by increasing the number of food-borneillnesses and the potential for treatment failure [21]

Food of animal origin could be contaminated fromthe farm a situation which may be further compoundedif the food is not properly handled during slaughteringand processing giving way for pathogens to multiply [22]The conditions under which these foods are handled raisequestions regarding their microbiological quality Studiesconducted in different countries to investigate the microbio-logical quality of food of animal origin reported the presenceof potential human pathogens [1 2] In South Africa a largeproportion of the population relies on beef and pork astheir source of protein which could expose them to infectionif contaminated [3 16] Even though other studies havereported the health risk associated with consumption of suchproducts there is a paucity of studies on the microbiologicalquality of cattle and pig slaughtered in abattoirs in theVhembeDistrict of Limpopo ProvinceThe present studywascarried out to assess the microbiological quality of beef andpork slaughtered in this area in a bid to throw more light onthe inherent risk associated with such foods

2 Materials and Methods

21 Study Site Description The study sites included Vyge-boomdrift pig abattoir (A1) C-net (A2) Mukwevho (A3)and Shayandima (A4) all of which are found in the VhembeDistrict Limpopo Province South Africa C-net Mukw-evho and Shayandima are cattle abattoirs where peopleoccasionally bring their personal cattle for slaughtering foroccasions such as funerals weddings or family gatheringsVygeboomdrift abattoir is a commercial abattoir where pigsare slaughtered

22 Sample Collection Samples were collected according tothe method of Pearce and Bolton [24] A total of 176 swabbedsamples were collected from 28 cattle (8 8 and 12 cattlefrom abattoirs 2 3 and 4 resp) and 16 pigs from abattoir 1Four parts of each animal which included the neck brisketrump and flank were sampled with sterile swab rinsing kit(containing 10mL isotonic buffer rinse solution) Sampleswabbing of the carcasses was performed after the removal ofthe gastrointestinal tract The tip of the swab was moistenedwith rinse solution before swabbing the carcass The area forswabbing was selected by using 100 cm2 sterile disposableplastic template (Analytical Diagnostics USA)The swabbing

was done 10x horizontally and 10x vertically at each site Thetotal areas sampled were 400 cm2 of each carcass Sampleswere transported on ice and processed within 2 hours

23 Microbial Analysis

231 Isolation and Identification of Bacterial PathogensSalmonella spp were identified using 1mL meat rinsedsolution mixed with 9mL buffered peptone water (Oxoid)and incubated at 37∘C for 24 hours After incubation1mL of preenrichment broth was transferred into 9mL ofRappaport-Vassiliadis (RV) soya peptone broth (Oxoid) andBrilliant green agar (Oxoid) plates and incubated at 37∘C for24 hours

For identification of S aureus the swab from swab rinsingsolution was spread-plated on mannitol salt agar (Oxoid)plates and incubated at 37∘C for 24 hours Staphylococcicoagulase positive colonies were confirmed using StaphylaseTest Kit (Oxoid) Yellow colonies which were Gram positivecocci in cluster oxidase negative and coagulase and catalasepositive and which produced clots were recorded as S aureusandmaintained at minus20∘C in 20 glycerol brain heart infusionbroth for further studies S aureus ATCC 25923 was used asa positive control

E coli was identified by pipetting 1mL of rinsed solutionin three test tubes each containing 9mL Brilliant greenbroth (Oxoid) fitted with Durham tubes and incubated at45∘C for 48 hours Tubes with gas bubbles in the Durhamtubes were streaked on MacConkey agar (Oxoid) plate andincubated at 37∘C for 24 hours Suspected colonieswere testedfor indole production Pink colonies on MacConkey mediawhichwere indole positive were considered positive forE coliandmaintained at minus20∘C in 20 glycerol brain heart infusionbroth for further confirmation and characterization by PCRand antibiotic susceptibility testing E coli ATCC 25922 wasused as a positive control

Presumptive bacteria identification was based on colonypigmentation and Gram staining characteristics Pure cul-tures were obtained by streaking a portion of an isolatedcolony on nutrient agar and incubated aerobically at 37∘Cfor 24 h All preliminary results were confirmed using theMicroscan-Autoscan equipment (Siemens Germany) follow-ing themanufacturerrsquos instruction Briefly an inoculumof thebacterium was prepared in saline and transferred onto 96-well plates in an automated pattern Reagents to supplementvarious reactions to aid identification of both Gram negativeand positive bacteria were added to some of the wells andincubated at 37∘C for 24 hours Plates were read and resultsinterpreted

24 Molecular Identification of E coli Using mPCR

241 Extraction of DNA E coli cultures were revived bystreaking on nutrient agar (Oxoid) and incubated at 37∘Cfor 24 hours Thereafter 05120583L of Triton X and 500120583L ofsterile distilled water were mixed with one colony in a 15mLEppendorf tube and mixed by vortexing for 5 seconds Themixture was boiled in a water bath at 100∘C for 20 minutes






















1-pork A1


2-beef A2


3-beef A3


4-beef A4


Total 50 104 14







3 Results









119873







4 Discussion










5 Conclusion




Acknowledgment


References























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






















1-pork A1


2-beef A2


3-beef A3


4-beef A4


Total 50 104 14







3 Results









119873







4 Discussion










5 Conclusion




Acknowledgment


References























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




119873







4 Discussion










5 Conclusion




Acknowledgment


References























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology







5 Conclusion




Acknowledgment


References























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Staphylococcus aureus - Hindawi

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Transcript of Staphylococcus aureus - Hindawi