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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

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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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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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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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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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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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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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63. Kuenne C, Billion A, Mraheil MA, Strittmatter A, Daniel R, Goesmann A, 735

Barbuddhe S, Hain T, Chakraborty T. 2013. Reassessment of the Listeria 736

monocytogenes pan-genome reveals dynamic integration hotspots and mobile 737

genetic elements as major components of the accessory genome. BMC Genomics 738

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64. den Bakker HC, Desjardins CA, Griggs AD, Peters JE, Zeng Q, Young SK, 740

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L. monocytogenes strains. Appl Environ Microbiol 77:2169-2173. 747

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67. Schoder D, Strauß A, Szakmary-Brändle K, Stessl B, Schlager S, Wagner M. 752

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passenger luggage. Int J Food Microbiol 209:3-12. 754


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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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