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1 Persistent and disinfectant-resistant L. monocytogenes The connection between persistent, disinfectant-resistant Listeria monocytogenes 1
strains from two geographically separate Iberian pork processing plants: evidence 2
from comparative genome analysis 3
4
Running title 5
Persistent and disinfectant-resistant L. monocytogenes 6
7
Sagrario Ortiz,a Victoria López-Alonso,b# Pablo Rodríguez,c Joaquín V. Martínez-8
Suáreza# 9
10
Departamento de Tecnología de Alimentos, Instituto Nacional de Investigación y 11
Tecnología Agraria y Alimentaria (INIA), Madrid, Spaina; Unidad de Biología 12
Computacional, UFIEC, Instituto de Salud Carlos III, Majadahonda, Madrid, Spainb, 13
Embutidos Fermín, S.L., La Alberca, Salamanca, Spainc. 14
15
# Address correspondence to Joaquín V. Martínez-Suárez, [email protected], and 16
Victoria López-Alonso, [email protected] 17
18
AEM Accepted Manuscript Posted Online 23 October 2015Appl. Environ. Microbiol. doi:10.1128/AEM.02824-15Copyright © 2015, American Society for Microbiology. All Rights Reserved.
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2 Persistent and disinfectant-resistant L. monocytogenes The aim of this study was to investigate the basis of putative persistence of Listeria 19
monocytogenes in a new industrial facility dedicated to the processing of ready-to-eat 20
(RTE) Iberian pork products. Quaternary ammonium compounds, which include 21
benzalkonium chloride (BAC), were repeatedly used as surface disinfectants in the 22
processing plant. Clean and disinfected surfaces were sampled to evaluate if resistance 23
to disinfectants was associated with persistence. Of the 14 isolates obtained from 24
product-contact and non-product-contact surfaces, only five different Pulsed Field Gel 25
Electrophoresis (PFGE) types were identified during the 27-month study period. Two of 26
these PFGE types (S1 and S10-1) were previously identified as persistent and BAC-27
resistant (BACR) strains in a geographically separate slaughterhouse belonging to the 28
same company. The remaining three PFGE types which were first identified in this 29
study were also BACR. Whole-genome sequencing and in silico multilocus sequence 30
typing (MLST) analysis of five BACR isolates from the different PFGE types identified 31
in this study showed that the S1 PFGE type belongs to MLST type ST31, a low-32
virulence type characterized by mutations in the inlA and prfA genes. The remaining 33
four PFGE types were found to belong to MLST type ST121, a persistent type that has 34
been isolated in several countries. The ST121 strains contained the BAC resistance 35
transposon Tn6188. The disinfection-resistant L. monocytogenes population in this RTE 36
pork product plant comprised two distinct genotypes with different multidrug resistance 37
phenotypes. This work offers insight into the L. monocytogenes subtypes associated 38
with persistence in food-processing environments. 39
40
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3 Persistent and disinfectant-resistant L. monocytogenes Listeria monocytogenes is a Gram-positive bacterium of the phylum Firmicutes. Often 41
found in raw foods, the bacteria can cause the life-threatening disease listeriosis. Most 42
cases of human listeriosis appear to be caused by ready-to-eat (RTE) foods, and the risk 43
of illness increases with the number of cells ingested and with RTE foods that support 44
growth of L. monocytogenes (1). Processed foods can be contaminated by contact with 45
equipment, the handling of raw products by staff, or from post-processing 46
environmental niches in which L. monocytogenes can survive despite routine thorough 47
disinfection procedures (2, 3). Mechanisms that facilitate the survival of L. 48
monocytogenes in food processing environments include biofilm formation (4, 5), 49
acquisition of antimicrobial resistance (6-10), and stress resistance mechanisms (11, 50
12). 51
L. monocytogenes is capable of colonizing food production plants with certain 52
subtypes that are found only in specific sections of the plants (13, 14). Furthermore, 53
some of these subtypes may persist in food processing environments for years (15-17). 54
Persistent strains have been identified as major post-processing contaminants of RTE 55
foods, and in many cases, listeriosis outbreaks have been associated with cases of 56
persistent environmental contamination of processing plants (18-21). To detect and 57
eliminate persistent strains of L. monocytogenes in the food processing environment, 58
environmental sampling is recommended (3). The tendency of L. monocytogenes to 59
persist in food processing plants after disinfection can lead to the selection and 60
dissemination of clones with decreased susceptibility to disinfectants. However, 61
persistent strains of L. monocytogenes showing reduced susceptibility to disinfectants 62
have only been characterized in a few cases (6-10). Therefore, it is necessary to 63
elucidate further the ecological and genetic characteristics of persistent strains of L. 64
monocytogenes with reduced susceptibility to disinfectants. 65
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4 Persistent and disinfectant-resistant L. monocytogenes
Among the large number of microbicides used in the food industry, those most 66
frequently incorporated into disinfectants used on large external or open surfaces are 67
quaternary ammonium compounds (QACs), which include benzalkonium chloride 68
(BAC). The development of stable antimicrobial resistance is a drawback of QACs that 69
is not shared by universal disinfectants such as chlorine, alcohol, and peracetic acid (7, 70
22). For the QACs, a number of genetic markers identified in L. monocytogenes are 71
known to confer stable and low-level resistance to BAC; these markers include the 72
bcrABC resistance cassette (23), the qacH gene of the Tn6188 transposon (9), and 73
various qac determinants originally found in staphylococci (24). The efflux systems 74
encoded by all of these genes belong to the small multidrug resistance (SMR) protein 75
family (25). Other efflux pumps, such as MdrL (multidrug resistance Listeria) and Lde 76
(Listeria drug efflux) (26, 27), which belong to the major facilitator superfamily (MFS) 77
(25), are also associated with L. monocytogenes adaptation and resistance to BAC (26, 78
27). 79
Currently, microbial whole genome sequencing is being implemented in public 80
health surveillance. It can be used to characterize foodborne pathogen isolates providing 81
new insights into foodborne pathogen biology and transmission (28, 29). Regarding L. 82
monocytogenes, subtyping by high discriminatory Pulsed Field Gel Electrophoresis 83
(PFGE) has been critical in detecting and investigating outbreaks of foodborne 84
listeriosis (30-32). Genome sequencing will likely replace PFGE, allowing in-depth 85
characterization of L. monocytogenes in food safety laboratories (29, 31). The genome 86
sequencing of several strains of L. monocytogenes has revealed important factors that 87
affect the persistence (20, 33) and disinfectant resistance (9, 34) of the species. 88
Comparison of the genome sequences of disinfection-resistant L. monocytogenes strains 89
may potentially reveal novel genes related to resistance phenotypes (35). 90
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5 Persistent and disinfectant-resistant L. monocytogenes
The aim of this study was to determine whether the BAC resistance associated with 91
L. monocytogenes persistence in one meat processing plant (10) was also associated 92
with persistence in a newly built plant belonging to the same company. Phenotypic 93
differences between the different resistant PFGE types (36) motivated a genomic 94
analysis to determine the actual number of resistant genotypes and to investigate the 95
molecular attributes that can play important roles in the environmental persistence of 96
individual subtypes. 97
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6 Persistent and disinfectant-resistant L. monocytogenes MATERIALS AND METHODS 99
Study design. This study was designed to investigate the presence of persistent and 100
disinfectant-resistant strains of L. monocytogenes in a newly built RTE pork processing 101
plant (Plant B) and to determine whether persistent L. monocytogenes PFGE types could 102
have originally entered the plant through raw products or otherwise. The new plant 103
(Plant B) received fresh and cured products from a slaughter and processing plant [Plant 104
A, described in (15)] located 22 km away. Both plants belong to the same Iberian pork 105
product supplier located in the province of Salamanca (Spain). 106
Disinfection procedures. Plant B was built with easily accessible open processing 107
lines and adequate materials for structures and equipment to be cleaned. For all rooms 108
and all equipment, specific cleaning and disinfection plans were established. 109
Disinfectant concentrations and contact times, as well as sanitation frequencies, were 110
adjusted according to microbiological testing results and following the instructions 111
provided by the disinfectant manufacturer. Tipically, sanitizers were used daily for a 30-112
minute contact time and were preceded by detergent-aided cleaning. Adequate rinsing 113
with water and drying both before and after disinfection was carried out. Furthermore, 114
the regular monitoring of protein residues and biofilms after sanitation was also 115
undertaken. 116
Two different surface disinfectants with broad spectrum biocidal action were rotated 117
weekly: a synergistic combination of QACs, and a disinfectant containing high 118
performance tertiary amines. In addition, a hydroalcoholic solution of QACs was 119
employed for immediate surface disinfection throughout the day at points where contact 120
with food is constant, e.g., for hand tools and cutting tables. 121
Sample collection. As part of the product quality assurance system, Plant B 122
employees regularly collect samples from the products and environment to test for L. 123
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7 Persistent and disinfectant-resistant L. monocytogenes monocytogenes. Environmental samples were obtained from product-contact and non-124
product-contact surfaces at different times. The samples included in the current study 125
were only those taken prior to start up (“before production”) to verify cleaning and 126
sanitation, and they were expected to be negative. If positive, these samples allow 127
identification of where corrections should be made, and could increase the probability 128
of detecting disinfection-resistant strains. The sampling of clean surfaces was conducted 129
with sponges (37) pre-moistened with neutralizing buffer (Whirlpak; Nasco VWR 130
International, Madrid, Spain) (15). 131
Sampling sites and frequencies changed based on the results acquired over time. At a 132
minimum, five sites were sampled monthly. The sampling commenced in high-risk 133
(post-processing) areas and then moved through to the low-risk areas, following an 134
appropriate sampling plan. Twenty-seven sample collections were conducted in the 135
RTE product plant over a 27-month period, including over 135 samples equally 136
distributed in the different production lines and areas in which both raw and RTE 137
products were processed. 138
The sampling of selected environmental surfaces during production and the regular 139
sampling of food products was also conducted according to the US Food Safety and 140
Inspection Service (FSIS) methodology for L. monocytogenes (37). 141
Isolation and identification of L. monocytogenes. The same procedure (37) was 142
followed for the isolation and identification of L. monocytogenes. Both primary and 143
secondary enrichments were streaked (0.1 ml) onto both modified Oxford medium and 144
CHROMagarTM Listeria. Simultaneous detection and enumeration of viable Listeria 145
spp. in RTE products was carried out by preparing the enrichment homogenate (37) and 146
immediately spreading 0.1 ml on CHROMagarTM Listeria (38). All media were 147
obtained from Biolife (Milan, Italy), except dehydrated CHROMagarTM Listeria 148
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8 Persistent and disinfectant-resistant L. monocytogenes (CHROMagar, Paris, France). Confirmatory identification of pathogenic Listeria spp. 149
grown on chromogenic agar as L. monocytogenes consisted of biochemical tests 150
(Listeria API® system, bioMérieux, Marcy-I’Etoile, France). 151
Molecular subtyping and virulence gene sequencing. The confirmed isolates were 152
subtyped by: (i) multiplex-PCR serogrouping (39), an assay that differentiates isolates 153
into PCR serogroups (IIa, IIb, IIc, and IVb) (40), each of which represents more than 154
one serotype (1/2a [or 3a], 1/2b [or 3b], 1/2c [or 3c], and 4b [or 4d and 4e], 155
respectively); (ii) PFGE, using restriction enzymes AscI and ApaI (New England 156
Biolabs, Beverly, MA, US) for cleaving the DNA (41); and (iii) sequencing of the entire 157
inlA gene, as described previously (42). 158
Composite PFGE types were obtained by combining AscI and ApaI profiles and were 159
designated with an ‘‘S’’ followed by a number identifying both the AscI and the ApaI 160
profiles; when more than one ApaI profile corresponded to one AscI profile, the first 161
number identifying the AscI profile was followed by a hyphen and a second number 162
identifying the different ApaI profiles (15). 163
Initially, the PFGE analysis yielded 29 different PFGE types of four PCR serogroups 164
in the characterized isolates from Plant A (15). In the present study, PFGE types of PCR 165
serogroup IIa from Plant A were reanalyzed together with PFGE types from Plant B. 166
This new analysis of the PFGE results was performed in accordance with the optimized 167
PulseNet standardized protocol (41), and using the BioNumerics software (Version 4.5, 168
Applied Maths, Kortrijk, Belgium). The similarity clustering was performed according 169
to the BioNumerics PulseNet manual 170
(http://www.pulsenetinternational.org/protocols/Pages/bionumerics.aspx). 171
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9 Persistent and disinfectant-resistant L. monocytogenes
Antimicrobial susceptibility testing and PCR assays to determine the presence 172
of BAC resistance determinants. The susceptibility of the isolates to BAC, 173
cetyltrimethylammonium bromide (CTAB), chloramphenicol, chlorhexidine, 174
ciprofloxacin, erythromycin, ethidium bromide (EB) and gentamicin was analyzed 175
using the agar dilution method (43, 44). Mueller-Hinton agar plates (Biolife) with 176
different concentrations of antimicrobials (from 0.07 to 40 mg/liter, except in the case 177
of EB [10-320 mg/liter] and gentamicin [0.07-1.25 mg/liter]) were inoculated with 104 178
CFU per spot and incubated for 24 h at 37°C. The minimum inhibitory concentration 179
(MIC) was defined as the lowest concentration of compound that inhibited growth. 180
MICs were determined in at least two separate assays, and each strain was assayed in 181
duplicate. To determine efflux pump activity, the inhibitor reserpine (at a final 182
concentration of 10 mg/liter) was added to each Mueller-Hinton agar plate containing 183
BAC (26, 27). All compounds were purchased from Sigma-Aldrich (Saint Louis, MO, 184
US), and stock solutions were prepared and stored according to the manufacturer's 185
recommendations. 186
A standard reference strain (EGD-e, ATCC BAA-679, http://www.lgcstandards-187
atcc.org/) (45) was included as a control, where necessary. L. monocytogenes strains 188
4423 and CDL 69 (9) were used as controls for the BAC resistance genetic determinants 189
qacH and bcrABC, respectively. PCR was used to determine the presence of such 190
determinants in all strains, as previously described (9, 36). BAC 191
resistance/susceptibility phenotypes were expressed as BACR in the case of resistance 192
(MIC of 10-20 mg/liter) and BACS in the case of susceptibility (MIC of 1.25-2.5 193
mg/liter). 194
Genome sequencing. Three BACR isolates corresponding to PFGE types S2-2, S2-3, 195
and S10-3 obtained in 2010 from Plant B were sequenced by whole-genome sequencing 196
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10 Persistent and disinfectant-resistant L. monocytogenes (WGS) (46). Two additional BACR isolates corresponding to PFGE types S1 and S10-1 197
from a previous study (10) were also subjected to WGS for comparative purposes (46). 198
The five isolates were grown in TSYEB at 37°C, and genomic DNA was extracted 199
using a bacterial genomic DNA purification kit (Wizard, Promega, Madison, WI, US) 200
according to the manufacturer’s protocol. The library was prepared from the extracted 201
genomic DNA using TruSeq technology (Illumina, San Diego, CA, US) and a 2x250-202
nucleotide paired-end sequencing run was performed in a MiSeq platform (Illumina) 203
(46). 204
Assembly, annotation, and analysis of genomes. Prior to assembly, the sequences 205
were filtered to remove reads containing one or more ambiguous base calls. The S1, 206
S10-1, S2-2, S2-3, and S10-3 sequences were assembled separately using the de novo 207
assembler Spades 3.1.1 software (http://bioinf.spbau.ru/en/spades) (47). The genomes 208
were annotated automatically using the RAST (Rapid Annotation using Subsystem 209
Technology) (http://rast.nmpdr.org/) and PGAAP (Prokaryotic Genomes Automatic 210
Annotation Pipeline) (http://www.ncbi.nlm.nih.gov/genome/annotation_prok/) servers. 211
Genome comparisons were assessed using the MAUVE Genome Alignment software 212
(http://asap.ahabs.wisc.edu/mauve/) (48) and BLAST via the NCBI website 213
(http://www.ncbi.nlm.nih.gov/). The genome of L. monocytogenes EGD-e (GenBank 214
accession number NC_003210.1) was downloaded from the NCBI website and used as 215
the reference genome. The similarity between the genomes of the L. monocytogenes 216
strains was also assessed by pairwise genome comparison. For each gene in one 217
genome, a BLAST-Like Alignment (BLAT) was performed against the second genome. 218
A given gene was considered specific to a strain if no sequence in the queried genome 219
was at least 50% identical to the gene over at least 50% of its length. For single 220
nucleotide polymorphisms (SNPs) detection, the raw sequence data of the strains was 221
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11 Persistent and disinfectant-resistant L. monocytogenes mapped to the EGD-e reference strain and to each de novo-assembled genome. The 222
MUMmer sequence alignment software package (http://mummer.sourceforge.net/) (49) 223
was used to detect SNPs. 224
In silico multilocus sequence typing (MLST) analysis was performed on the genome 225
of each strain using MLST 1.7 (www.cbs.dtu.dk/services/MLST). The MLST profiles 226
were uploaded to the L. monocytogenes MLST database of the Pasteur Institute 227
(http://www.pasteur.fr/recherche/genopole/PF8/mlst/Lmono.html) to determine the 228
sequence type (ST). Prophage sequences were identified in each genome using PHAST 229
(PHAge Search Tool) (http://phast.wishartlab.com/). The detection of CRISPRs in the 230
genomes was accomplished with the CRISPRfinder program (http://crispr.u-psud.fr/). 231
Nucleotide sequence accession numbers. The genome sequences can be found at 232
DDBJ/EMBL/GenBank under accession numbers JWHI00000000 (S1), 233
JWHG00000000 (S10-1), JWHJ00000000 (S2-2), JWHK00000000 (S2-3), and 234
JWHH00000000 (S10-3) (46). The inlA sequences of the isolates of the PFGE types S2-235
2, S2-3, and S10-3 (which were first determined herein) were submitted to GenBank 236
under accession numbers KM590945, KM590946, and KM590947, respectively. 237
238
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12 Persistent and disinfectant-resistant L. monocytogenes RESULTS AND DISCUSSION 239
L. monocytogenes was first isolated from an environmental sample obtained before 240
production (i.e., from clean and disinfected surfaces) in an RTE product plant 12 241
months after the launch of operations. The month in which the first positive test result 242
was obtained was month number 13 of the study. In addition to months 1-12, no 243
positive test results were obtained in months 15, 16, 17, 18, 20, 21, and 25 of the total 244
27-month study period. However, the 14 samples that yielded positive results resulted in 245
14 isolates of L. monocytogenes (Table 1). The samples were taken to verify the 246
effectiveness of the disinfection treatment applied and to detect putative disinfection-247
resistant strains. The following sanitized surfaces were found positive for the presence 248
of L. monocytogenes: the drains (two samples), the internal side of tables (five samples), 249
the inside of processing equipment (four samples), and the cleaning equipment (aprons, 250
two samples, and boot washer, one sample). All these surfaces were thoroughly 251
disinfected, including the hard-to-reach ones, such as the internal side of tables. The 252
inside of processing equipment was specially cleaned and sanitized after dismantling. 253
This low-level contamination detected before production may be a reflection of the 254
ecological behavior of L. monocytogenes in post-processing environments, which tend 255
to be aggressively sanitized (3, 17). In these high-risk areas, the expected result of 256
environmental monitoring before production is a complete absence of L. 257
monocytogenes. An effective sampling program, however, can yield occasionally 258
positive results, which help to identify where corrections should be made (50, 51). The 259
strains of L. monocytogenes that remained viable in this environment despite cleaning 260
and disinfection could be disinfectant-resistant strains. 261
Molecular subtypes. Only the S1 PFGE type (10, 15) was detected the first month 262
that yielded a positive result (month 13). Another known PFGE type, S10-1 (10, 15), 263
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13 Persistent and disinfectant-resistant L. monocytogenes was detected six months later (month 19). PFGE types S1 and S10-1 had been 264
previously detected in raw products in the Plant A (10, 15). As Plant B received fresh 265
and cured products from Plant A (see Study design), the result suggests a possible origin 266
of certain L. monocytogenes PFGE types that eventually colonized Plant B (14, 52). 267
Two new PFGE types (S2-2 and S2-3) were detected after month 22. These four PFGE 268
types were highly variable in their detection frequencies (Table 1). This study focused 269
on the detection of L. monocytogenes before production; nevertheless, selected isolates 270
of L. monocytogenes from samples obtained during production were also subjected to 271
molecular subtyping. One isolate obtained during production was assigned to a new 272
PFGE type (S10-3) (Table 2) which motivated its inclusion in the study. 273
The term “persistence” is used to describe long-term survival of L. monocytogenes in 274
the facility (17). L. monocytogenes strains were arbitrarily considered persistent when 275
(i) isolates of the persistent strain from successive sampling rounds were 276
indistinguishable by PFGE typing, and (ii) when isolates of the persistent strain were 277
found repeatedly (three times or more) in the environment over a minimum of three 278
months (15, 53). According to these criteria, the only strains that could be considered 279
putatively persistent in the current study were those from the S1 and S2-2 PFGE types. 280
S2-2 was isolated from both the environment (in months 22, 23, 24, 26, and 27; Table 281
1) and food (in months 21 and 26). These food samples were the only finished food 282
product (whole cured ham) which had tested positive for the presence of L. 283
monocytogenes throughout the entire study. However, the levels of contamination of 284
this product were consistently below 100 CFU/g. The result suggests that the transfer of 285
bacteria could occur between the food processing environment and the food itself, as the 286
same PFGE type was found in both sample types. The company had a plan to handle 287
such a contingency, and appropriate measures were taken immediately, including the 288
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14 Persistent and disinfectant-resistant L. monocytogenes withdrawal of all suspect products from production and a more frequent and in-depth 289
disinfection of the relevant processing line. After these serious incidents, extensive 290
sampling of product contact surfaces and final products was conducted; no L. 291
monocytogenes was found in any of these samples. 292
The five different PFGE types found in this study belonged to PCR serogroup IIa. 293
The inlA gene of five isolates of the different PFGE types identified in the current study 294
were fully sequenced. The results showed different inlA sequences in S1 (inlA subtype 295
1) than in S10-1, S2-2, S2-3, and S10-3, which shared identical sequences (inlA subtype 296
2). All had mutations leading to a premature stop codon (PMSC) in inlA (10, 54); 297
however, two different mutations were found: PMSC type 5 in inlA subtype 1, and 298
PMSC type 6 in inlA subtype 2. In addition to PFGE typing, PCR-based serogrouping 299
and inlA gene sequencing allow for improved understanding of the relationship between 300
the strains of L. monocytogenes (42, 55, 56). L. monocytogenes isolates of serotype 301
1/2a, included in PCR serogroup IIa, are recognized for their ability to survive in food-302
processing environments (55, 56). 303
BACR subtypes. BAC susceptibility tests of the 14 L. monocytogenes isolates 304
obtained from sanitized surfaces (Table 1) plus one isolate of PFGE type S10-3 (Table 305
2), demonstrated that four PFGE types (S10-1, S2-2, S2-3, and S10-3) were BACR, 306
whereas PFGE type S1 was either BACS or BACR depending on the particular isolate 307
(Table 3). PFGE type S1 is a prfA mutant (10), and a similar finding involving the 308
coexistence of resistant and susceptible isolates in the same PFGE type was previously 309
described in Plant A with another prfA mutant, PFGE type S6 (10). The results of PCR 310
assays directed at BAC resistance determinants qacH and bcrABC (9, 23) yielded 311
negative results in the BACR isolate of S1. For this BACR isolate of S1, the 312
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15 Persistent and disinfectant-resistant L. monocytogenes overexpression of MFS efflux pumps similar to MdrL (26, 27) may be related to its 313
multidrug resistance phenotype (Table 3). 314
BAC susceptibility testing and PCR assays to determine the presence of BAC 315
resistance determinants revealed that the PFGE type S10-1 that contained transposon 316
Tn6188 (36) and the PFGE types S2-2, S2-3, and S10-3 were similar in terms of the 317
MIC of BAC (20 mg/liter) and the genetic determinant of resistance to BAC (Tn6188). 318
To determine efflux pump activity, changes in the MICs of BAC were observed both 319
in the presence and absence of reserpine (26, 27). The MICs of BAC revealed an effect 320
of reserpine on the MIC of BAC in BACS and BACR S1 isolates (Table 3). Exposure to 321
BAC may result in reduced susceptibility to other antibacterial compounds including 322
certain antibiotics (27). The BACR isolate of S1 also showed increased MICs of 323
different compounds, especially EB, when compared to the BACS isolates of S1 (Table 324
3). The MICs of chloramphenicol and erythromycin were not affected (data not shown). 325
The effect of reserpine and the increased MICs of EB were not observed in the isolate of 326
the PFGE type S10-1 and the remaining Tn6188-harboring PFGE types (Table 3). 327
Tn6188 harbors the qacH gene, which can confer reduced susceptibility to both QACs 328
and EB (57). However, the MIC of EB in the mutants with the qacH gene introduced by 329
genetic complementation compared to those in which it is genetically inactivated only 330
differs by less than two-fold (57). 331
The resistance to antibiotics found in BACR strains of L. monocytogenes is tipically 332
low-level resistance (Table 3). Therefore, BACR L. monocytogenes may not show cross-333
resistance to antibiotics at a clinically relevant level (58). In food production 334
environments, L. monocytogenes efflux pumps also do not confer resistance to 335
disinfectants at the concentrations commonly used. In resistant strains, the MICs of 336
BAC (10-20 mg/liter) (Table 3) were much lower than the concentrations used in 337
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16 Persistent and disinfectant-resistant L. monocytogenes practice (200 mg/liter or greater) (17, 59); thus, QACs are still considered an effective 338
way to control BACR strains of L. monocytogenes (22). Regardless, the observed 339
changes may be of concern, as efflux pumps can presumably reduce the intracellular 340
concentration of antimicrobials to sublethal levels, enabling bacteria to survive longer 341
than predicted based upon the MIC of that particular organism (60). Low-level BAC 342
resistance in these L. monocytogenes strains may contribute to their adaptation and 343
survival (36, 50); hence, the resistant strains may have the potential to persist in food 344
ecosystems. 345
The indiscriminate use of QACs in a wide range of environments has raised concerns 346
about the resulting selection of QAC-resistant bacteria (59). However, scientific 347
evidence of environmental development of resistance to QACs is limited, and the 348
information is often difficult to interpret and compare (61). For example, to date there 349
are no clear criteria to determine whether a given microbe is resistant to biocides (62). 350
For the purpose of this study, a standard clinical protocol for antibiotics (43, 44) was 351
adopted to measure the changes in the MIC of the biocide, since the standardized tests 352
for the evaluation of chemical disinfectants measure the bactericidal activity of the 353
substance or product (61). The changes observed in the MIC of BAC were stable, and 354
thus the use of the term “resistance” was intended to distinguish inherited reduced 355
susceptibility that is mutational in origin, from noninherited or phenotypic reduced 356
susceptibility (61, 62). Last but not least, the selection of L. monocytogenes isolates that 357
were resistant to QACs due to the presence of stable and inherited resistance 358
mechanisms was observed after repeated use of that class of disinfectants (see Materials 359
and Methods, Disinfection Procedures). 360
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17 Persistent and disinfectant-resistant L. monocytogenes
Comparison of the draft genomes of five BACR isolates. Five BACR isolates 361
corresponding to PFGE types S1, S10-1, S2-2, S2-3, and S10-3 (Table 2) were used for 362
WGS (46). The more relevant features of the next-generation sequencing and the de 363
novo assembly of the genomes are summarized in Table 4. For all genomes, the GC 364
ratio (38.0%) and genome size were within the range of values that are typical for L. 365
monocytogenes (45, 63, 64). The genomes of S1 and S10-1 were, however, smaller and 366
with a lower number of CDS regions than those of S2-2, S2-3, and S10-3. 367
The results of the in silico MLST analysis of the five strains indicated that the strain 368
of PFGE type S1 belonged to sequence type (ST) 31 (abcZ-7, bglA-14, cat-10, dapE-19, 369
dat-9, ldh-8, and lhkA-1), whereas the four strains containing Tn6188 (PFGE types S10-370
1, S2-2, S2-3, and S10-3) belonged to ST121 (abcZ-7, bglA-6, cat-8, dapE-8, dat-6, ldh-371
37, and lhkA-1) (Table 4). The MLST database of the Pasteur Institute in Paris (updated 372
on 03/03/2015) includes only 10 ST31 strains and 69 ST121 strains (most are the 1/2a 373
serotype). The ST121 type has been isolated as a persistent type in food and food 374
associated environments in different countries (16, 42, 65, 66). ST121 is among the six 375
L. monocytogenes STs most prevalent worldwide (67) and seems well-adapted to 376
colonize meat associated environments (67, 68). ST121 frequently harbors a truncated 377
InlA, resulting in attenuated virulence (42, 66). Strains of the same ST may share 378
similarity in the genes necessary for survival in food and food processing environments 379
(56, 66). 380
Similarly, MLST type ST31 has a low virulence potential due to mutations in inlA 381
and also prfA (10, 69). Previous studies of low-virulence strains of L. monocytogenes 382
discovered that strains of three of the low-virulence genotypic groups were distributed 383
between three closely related, specific types: ST13, ST31, and ST193 (69); all strains of 384
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18 Persistent and disinfectant-resistant L. monocytogenes the ST31 type have mutations in inlA, and only half of these strains also have the 385
PrfAΔ174-237 deletion (69). 386
The pairwise differences in the predicted gene content between the genomes of each 387
strain are shown in Table 5. Strains of PFGE types S2-2, S2-3, and S10-3 were 388
extremely similar, with inter-strain differences of only 0.26 to 0.96% of their genomes. 389
In contrast, the strain of PFGE type S10-1 had 3.2 to 4.1% less genes than the other 390
three ST121 strains. The highest inter-strain differences were found in the number of 391
genes present in the four ST121 strains and not in strain of PFGE type S1 (ST31 type) 392
(from 5.11 to 8.04% of their genomes). 393
The conservation and variation in gene content between the genomes were visualized 394
by MAUVE. The five newly sequenced genomes were included in the comparison, and 395
that of EGD-e was used as a reference (Fig. 1). The comparison shows that all 396
sequenced genomes were generally highly similar to each other. However, the genomes 397
are draft assemblies with fragmentation of genes onto multiple individual contigs (51-73 398
contigs, Table 4). Therefore, regions that are not included in the MAUVE alignment 399
most likely represent deletions/insertions, genome fragments replaced by a 400
nonhomologous sequence, or regions not sequenced in one or more genomes. SNP 401
analyses were carried out using EGD-e or each one of the five strains sequenced in the 402
current study as the reference strain. The computed SNP count difference between all 403
strains and L. monocytogenes EGD-e (32,612 to 38,413 SNPs) or strain of PFGE type 404
S10-1 is shown in Table 4. The number of SNPs identified in the comparison of the 405
backbone sequence of strain of PFGE type S10-1 and strains of PFGE types S2-2, S2-3, 406
and S10-3 ranged between 73 and 75 SNPs, whereas testing strains of PFGE types S10-407
1 and S1 against one another identified 39,721 SNPs (Table 4). Testing S2-2, S2-3, and 408
S10-3 against each other identified between 4 and 11 SNPs (data not shown). 409
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19 Persistent and disinfectant-resistant L. monocytogenes
An initial annotation of the predicted CDS regions for the five genomes was 410
performed in RAST and accessed with the SEED viewer (70). The total number of CDS 411
regions determined in each functionally related protein family is presented in Fig. S1 in 412
the supplemental material. The major differences between strains of PFGE types S1 and 413
S10-1 with respect to strains of PFGE types S2-2, S2-3, and S10-3 were found in the 414
accessory genome, including the “prophage, phage, transposable elements, and 415
plasmid” subsystem. In spite of these differences found in the four ST121 strains (S10-416
1, S2-2, S2-3, and S10-3), conserved plasmids, transposons, and prophages were also 417
present (Table 4) as reported for most of the previously investigated ST121 strains (34). 418
The differences between the four ST121 strains and strain of PFGE type S1 were found 419
in the “cofactors, vitamins and prosthetic groups”, “cell wall and capsule”, “cell 420
division and cell cycle”, and “amino acids and carbohydrates” subsystems. The number 421
of SNPs (Table 4), the comparison of the gene content (Table 5, Fig. 1) and the 422
predicted CDSs (Fig. S1) of the five genomes further confirmed the differences between 423
the strain of ST31 and the four ST121 strains. 424
The differences in genes encoding putative phage proteins in the genomes were 425
confirmed with the results of PHAST analysis, showing the differences in number and 426
type of prophages between the strains studied (Table 4). The predicted prophage regions 427
in S10-3, S2-2, and S2-3 were split into different contigs, which were arranged in 428
MAUVE at the ends of the draft genomes (Fig. 1), highlighting the difficulty in 429
assembling these regions. A prophage integrated into the comK gene (major competence 430
transcription factor) was present both in the strain of ST31 and in three of the ST121 431
genomes of this study (S2-2, S2-3, and S10-3). In the strain of ST31 , the prophage 432
insertion was 42.3 kbp. In the four ST121 type genomes, one prophage with highly 433
homologous regions to Listeria phage A118 was inserted downstream of tRNA-Arg-434
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20 Persistent and disinfectant-resistant L. monocytogenes CCG. In S2-2, S2-3 and S10-3, another prophage with homologous regions to Listeria 435
phage LP-101 was inserted downstream of tRNA-Arg-TCT (Table 4) (34). 436
Different studies have demonstrated the importance of bacteriophages in the 437
evolution of closely related strains of L. monocytogenes (20, 30-32). WGS analysis can 438
reveal prophage regions that explain differences between phylogenetically similar 439
populations with different PFGE types (33). In the current study, the difference in PFGE 440
type of the four strains carrying Tn6188 (S10-1, S2-2, S2-3, and S10-3) was associated 441
with increasing genome size. The S10-3 genome contained three prophage sequences 442
and exceeded the genome size of S10-1 by 104 kbp, which only contained one prophage 443
sequence (Table 4). These results suggest that differences in genome size are most 444
likely due to the different prophage sequence content. 445
Phages profoundly influence ecological networks in bacterial communities by 446
serving as reservoirs of genetic diversity (71). Within the Listeria genus, prophages are 447
considered the major source of diversity (63, 64) and can constitute up to 7% of the 448
Listeria coding genes (45). Bacteriophages also play an important role in the acquisition 449
of traits by L. monocytogenes that can increase survival capacity (63, 64). For example, 450
phages inserted into the comK gene might provide fitness advantages in food production 451
environments (20), including rapid niche-specific adaptation, biofilm formation, and 452
persistence (72). In this study, a prophage sequence was present as an insertion into 453
comK in four strains, whereas it was absent in S10-1 (Table 4). The lack of an intact 454
prophage insertion in comK has been reported for most of the ST121 strains (34). 455
Strains without the insertion in comK, such as the strain of the PFGE type S10-1, could 456
evolve to contain a prophage in comK, and it could potentially be involved in the 457
persistence mechanism (72). 458
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21 Persistent and disinfectant-resistant L. monocytogenes
The three strains of inlA subtype 2 corresponding to PFGE types S2-2, S2-3, and 459
S10-3 were different but very closely related, according to the low number of SNPs and 460
variable presence of phage sequences in these strains (Table 4). Both the SNPs, which 461
represent diversification, and phage sequences, which were likely acquired from natural 462
bacterial populations (31), may be related to the stress caused by disinfectants. 463
Exposure to antimicrobials can trigger DNA damage and genomic instability; this 464
instability ultimately leads to mutations, which include both SNPs and genome 465
reorganization due to mobile genetic elements (73). 466
The results of comparative analysis of the five genomes also suggest that the two STs 467
were equipped with different antimicrobial- and stress-resistance genes. The four ST121 468
strains contained Tn6188 (Table 4), which may be related to greater capability of the 469
ST121 strains to survive in the presence of QACs. Five additional ST121 genomes are 470
known that also contain this BAC resistance transposon (34). Additionally, plasmids 471
often carry extra genes that allow bacteria to live in stressful environments. The four 472
ST121 type L. monocytogenes harbored a plasmid (Table 4), that includes cadmium 473
resistance transposon Tn5422 (63, 74) and clpL genes, which most likely provide 474
additional stress response potential for the ST121 strains (34). A Tn5422-like 475
transposon associated with the cadmium resistance gene cadA1 was also found in S1 476
(Table 4). In contrast, the particularly long persistence of ST31 (10, 15) (Table 1) could 477
be related to the presence of a region identical to the so-called ''stress survival islet 1'' 478
(SSI-1) (65). The five-gene SSI-1 islet can contribute to elevated resistance to several 479
types of stress in certain strains of L. monocytogenes (65). 480
Conclusions. This study demonstrates that large genomic differences exist between 481
two groups of disinfectant-resistant L. monocytogenes strains that were isolated from 482
the same food processing environment. The two major types identified were the prfA 483
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22 Persistent and disinfectant-resistant L. monocytogenes mutant S1 (type ST31) and the strains containing Tn6188 (type ST121). Both types 484
were identified as persistent contaminants in the RTE pork product Plant B in samples 485
obtained from clean and disinfected surfaces. The selection of L. monocytogenes 486
isolates with low-level and stable resistance to QACs was observed after repeated use of 487
that class of disinfectants. 488
489
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23 Persistent and disinfectant-resistant L. monocytogenes ACKNOWLEDGMENTS 490
This work was supported by Research Project grants RTA2011-00098-C02 and 491
RTA2014-00045-C03 (INIA FEDER) from the Spanish Ministry of Economy and 492
Competitiveness, and by Embutidos Fermín, S.L., La Alberca, Salamanca, Spain. 493
We gratefully acknowledge Pilar López (Instituto Nacional de Investigación y 494
Tecnología Agraria y Alimentaria, Madrid, Spain) for technical assistance, and Dr. 495
Stephan Schmitz-Esser (Institute for Milk Hygiene, Vienna, Austria) for providing 496
strains 4423 and CDL 69. 497
498
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Barbuddhe S, Hain T, Chakraborty T. 2013. Reassessment of the Listeria 736
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64. den Bakker HC, Desjardins CA, Griggs AD, Peters JE, Zeng Q, Young SK, 740
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73. Shapiro RS. 2015. Antimicrobial-induced DNA damage and genomic instability in 778
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74. Lebrun M, Audurier A, Cossart P. 1994. Plasmid-borne cadmium resistance 780
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783
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36 Persistent and disinfectant-resistant L. monocytogenes FIGURE LEGENDS 784
785
FIG 1 Multiple sequence alignment of L. monocytogenes strains of PFGE types S1, 786
S10-1, S10-3, S2-2, and S2-3 with reference L. monocytogenes strain EGD-e (accession 787
number NC_003210.1) using MAUVE aligner version 2.3.1. MAUVE viewer generated 788
the figure. Homologous regions are shown in the same color. The height of the 789
similarity profile within each block corresponds to the average level of conservation in 790
that region of the genomes. Conversely, larger white portions identify areas of low 791
similarity. Areas that are completely white within a block are not aligned and most 792
likely contain sequence elements specific to a particular genome. Inverted regions are 793
depicted as blocks below a genome's centerline. 794
795
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37 Persistent and disinfectant-resistant L. monocytogenes TABLES 796
797
TABLE 1 Incidence of detection of L. monocytogenes before production, and PFGE types 798
Month No. a PFGE type detected No. of isolates 13, 14, 23 S1 4 19 S10-1 1 22, 23, 24, 26, 27 S2-2 8 22 S2-3 1 a Nos. 1-27 represent consecutive months from November 2008 (month No. 1) to January 2011 (month No. 27). L. monocytogenes was not 799
detected during the months not included. 800
801
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38 Persistent and disinfectant-resistant L. monocytogenes TABLE 2 Characteristics of the five PFGE types detected in this study 802
PFGE type PCR serogroup inlA subtype Sample collection Reference
S1 IIa 1 Before production This study and (10) S10-1 IIa 2 Before production This study and (10) S2-2 IIa 2 Before production This study S2-3 IIa 2 Before production This study S10-3 b IIa 2 During production This study 803
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39 Persistent and disinfectant-resistant L. monocytogenes TABLE 3 MICs of different compounds for representative BACS and BACR strains a 804
L. monocytogenes strain MIC (mg/liter) BAC BAC with reserpine CTABa Chlorhexidine EBa Ciprofloxacin Gentamicin
EGD-e 1.25 0.6 5 2.5 20 1.25 0.3 S1 BACS b 2.5 1.25 5 2.5 20 1.25 0.3 S1 BACR c 10 5 10 5 160 5.0 0.6 S10-1 d 20 20 20 2.5 40 1.25 0.3 4423 e 20 20 20 2.5 40 1.25 0.6 CDL 69 f 20 20 10 2.5 40 1.25 0.3 a MICs were determined in at least two separate assays, and each strain was assayed in duplicate. Abbreviations: BAC, benzalkonium chloride; 805
BACR, resistant phenotype; BACS, susceptible phenotype. CTAB, cetyltrimethylammonium bromide. EB, ethidium bromide. 806
b Three isolates. 807
c One isolate. 808
d One isolate. Identical results were obtained with different isolates of Tn6188-harboring PFGE types S2-2, S2-3, and S10-3. 809
e qacH gene control. 810
f bcrABC genetic determinant control. 811
812
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40 Persistent and disinfectant-resistant L. monocytogenes TABLE 4 Relevant features of the L. monocytogenes S1, S10-1, S10-3, S2-2, and S2-3 genomes 813
Feature PFGE type of the isolate used for WGS S1 S10-1 S10-3 S2-2 S2-3
Processing plant A A B B B Year of isolation 2008 2008 2010 2010 2010 No. of contigs 51 70 73 52 71 G+C content (%) 38 38 38 38 38 Total length of all de novo-assembled contigs (bp) a 2,997,617 3,009,749 3,114,032 3,112,163 3,086,604 No. of predicted CDS regions b 3,044 2,972 3,108 3,115 3,069 Sequence type (ST) 31 121 121 121 121 No. of SNPs compared to EGD-e 32,612 38,264 38,393 38,413 38,386 No. of SNPs compared to S10-1 39,721 0 73 75 73 bcrABC - - - - - Tn6188 - + + + + No. of prophage sequences 1 1 3 4 3 comk prophage + - + + + tRNA-Arg-CCG prophage - + + + + tRNA-Arg-TCT prophage - - + + + Plasmid ST121 c - + + + + Tn5422 (cadA1) + + d + d + d + d Islet SSI-1 + - - - - CRISPR-I ( lmo0517/lmo0518) e + + + + + CRISPR-II ( lmo2591/lmo2595) - + + + + a Data were obtained after assembling with Spades 814
b Data predicted using RAST 815
c Described in (34) 816
d Within plasmid 817
e CRISPR, clustered regularly interspaced short palindromic repeats systems 818
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41 Persistent and disinfectant-resistant L. monocytogenes TABLE 5 Pairwise genome comparison obtained using BLAT a 819
PFGE type of ‘strain’ PFGE type of the ‘query strain’
S1 S10-1 S10-3 S2-2 S2-3
S1 0 160 146 147 157 (0%) (5.25%) (4.79%) (4.82%) (5.15%)
S10-1 152 0 14 14 12 (5.11%) (0%) (0.47%) (0.47%) (0.40%)
S10-3 250 126 0 9 24 (8.04%) (4.05%) (0%) (0.28%) (0.77%)
S2-2 251 129 12 0 30 (8.05%) (4.14%) (0.38%) (0%) (0.96%)
S2-3 219 100 8 13 0 (7.13%) (3.2%) (0.26%) (0.42%) (0%)
a The table indicates the number of non-shared protein-coding genes in the genome of the ‘query strain’ (indicated in the row) compared with the 820
genome of the ‘strain’ indicated in the column. The percentage shown is based on the ratio of the non-shared protein-coding genes and the total 821
number of predicted genes in the genome of the ‘query strain’. 822
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