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Characteristics of Listeria Monocytogenes Strains Persisting in a Meat Processing Facility over a 4-Year Period.

ABSTRACT: Listeria monocytogenes can persist in food production facilities, resulting in serious threats to consumers due to the high mortality associated with listeriosis, especially in the very young, old and pregnant. We subtyped 124 strains of L. monocytogenes isolated from a meat processing facility in Switzerland by serotyping, multi locus sequence typing (MLST) typing and whole genome sequencing. We then analyzed their ability to form biofilms and their resistance to the disinfectants benzalkonium chloride (BC) and peracetic acid (PAA). The genotyping results of the strains showed that several clonal populations of L. monocytogenes belonging to CC9, CC204 and CC121 had persisted in this meat processing facility for at least four years. All of the strains showed biofilm forming capacity comparable to a known high biofilm forming strain. Known efflux pumps for BC were present in CC204, CC9 (brcABC) and CC121 (qacH) strains, while strains from other CC showed very low minimal inhibitory concentrations (MICs) for BC. For PAA, minimal bactericidal concentrations of 1.2?1.6% for 20 min and minimal inhibitory concentrations between 0.1 and 0.2% were observed. These values were close to or above the recommended concentration for use (0.5?1%), suggesting that PAA might be ineffective at controlling L. monocytogenes in this and potentially other meat processing facilities.

SUBMITTER: Stoller A

PROVIDER: S-EPMC6471029 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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Characteristics of Listeria Monocytogenes Strains Persisting in a Meat Processing Facility over a 4-Year Period.

Stoller Andrea A Stevens Marc J A MJA Stephan Roger R Guldimann Claudia C

Pathogens (Basel, Switzerland) 20190307 1

<i>Listeria monocytogenes</i> can persist in food production facilities, resulting in serious threats to consumers due to the high mortality associated with listeriosis, especially in the very young, old and pregnant. We subtyped 124 strains of <i>L. monocytogenes</i> isolated from a meat processing facility in Switzerland by serotyping, multi locus sequence typing (MLST) typing and whole genome sequencing. We then analyzed their ability to form biofilms and their resistance to the disinfectants ...[more]

PMID: 30866522

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Project description:Listeria monocytogenes is frequently found in foods and processing facilities, where it can persist, creating concerns for the food industry. Its ability to survive under a wide range of environmental conditions enhances the potential for cross-contamination of the final food products, leading to possible outbreaks of listeriosis. In this study, whole-genome sequencing (WGS) was applied as a tool to characterize and track 100 L. monocytogenes isolates collected from three food processing environments. These WGS data from environmental and food isolates were analyzed to (i) assess the genomic diversity of L. monocytogenes, (ii) identify possible source(s) of contamination, cross-contamination routes, and persistence, (iii) detect absence/presence of antimicrobial resistance-encoding genes, (iv) assess virulence genotypes, and (v) explore in vivo pathogenicity of selected L. monocytogenes isolates carrying different virulence genotypes. The predominant L. monocytogenes sublineages (SLs) identified were SL101 (21%), SL9 (17%), SL121 (12%), and SL5 (12%). Benzalkonium chloride (BC) tolerance-encoding genes were found in 62% of these isolates, a value that increased to 73% among putative persistent subgroups. The most prevalent gene was emrC followed by bcrABC, qacH-Tn6188, and qacC. The L. monocytogenes major virulence factor inlA was truncated in 31% of the isolates, and only one environmental isolate (L. monocytogenes CFS086) harbored all major virulence factors, including Listeria pathogenicity island 4 (LIPI-4), which has been shown to confer hypervirulence. A zebrafish embryo infection model showed a low (3%) embryo survival rate for all putatively hypervirulent L. monocytogenes isolates assayed. Higher embryo survival rates were observed following infection with unknown virulence potential (20%) and putatively hypovirulent (53 to 83%) L. monocytogenes isolates showing predicted pathogenic phenotypes inferred from virulence genotypes.IMPORTANCE This study extends current understanding of the genetic diversity among L. monocytogenes from various food products and food processing environments. Application of WGS-based strategies facilitated tracking of this pathogen of importance to human health along the production chain while providing insights into the pathogenic potential for some of the L. monocytogenes isolates recovered. These analyses enabled the grouping of selected isolates into three putative virulence categories according to their genotypes along with informing selection for phenotypic assessment of their pathogenicity using the zebrafish embryo infection model. It has also facilitated the identification of those isolates with genes conferring tolerance to commercially used biocides. Findings from this study highlight the potential for the application of WGS as a proactive tool to support food safety controls as applied to L. monocytogenes.

Project description:Listeria monocytogenes is an important foodborne pathogen responsible for the disease listeriosis, and can be found throughout the environment, in many foods and in food processing facilities. The main cause of listeriosis is consumption of food contaminated from sources in food processing environments. Persistence in food processing facilities has previously been shown for the L. monocytogenes sequence type (ST) 8 subtype. In the current study, five ST8 strains were subjected to whole-genome sequencing and compared with five additionally available ST8 genomes, allowing comparison of strains from salmon, poultry and cheese industry, in addition to a human clinical isolate. Genome-wide analysis of single-nucleotide polymorphisms (SNPs) confirmed that almost identical strains were detected in a Danish salmon processing plant in 1996 and in a Norwegian salmon processing plant in 2001 and 2011. Furthermore, we show that L. monocytogenes ST8 was likely to have been transferred between two poultry processing plants as a result of relocation of processing equipment. The SNP data were used to infer the phylogeny of the ST8 strains, separating them into two main genetic groups. Within each group, the plasmid and prophage content was almost entirely conserved, but between groups, these sequences showed strong divergence. The accessory genome of the ST8 strains harbored genetic elements which could be involved in rendering the ST8 strains resilient to incoming mobile genetic elements. These included two restriction-modification loci, one of which was predicted to show phase variable recognition sequence specificity through site-specific domain shuffling. Analysis indicated that the ST8 strains harbor all important known L. monocytogenes virulence factors, and ST8 strains are commonly identified as the causative agents of invasive listeriosis. Therefore, the persistence of this L. monocytogenes subtype in food processing facilities poses a significant concern for food safety.

Project description:The emergence of microorganisms exerting resistance to biocides is a challenge to meat-processing environments. Bacteria can be intrinsically resistant to biocides but resistance can also be acquired by adaptation to their sub-lethal concentrations. Moreover, the presence of biocide resistance determinants, which is closely linked to antibiotic resistance determinants, could lead to co-selection during disinfection practices along the food chain, and select cross-resistant foodborne pathogens. The purpose of this work was to test the resistance of wild strains of Listeria monocytogenes, isolated from pork meat processing plants, toward benzalkonium chloride (BC), used as proxy of quaternary ammonium compounds. Furthermore, the expression of two non-specific efflux pumps genes (lde and mdrL) under biocide exposure was evaluated. L. monocytogenes were isolated from five processing plants located in the Veneto region (northeast of Italy) before and after cleaning and disinfection (C&D) procedures. A total of 45 strains were collected: 36 strains before and nine after the C&D procedures. Collected strains were typed according to MLST and ERIC profiles. Strains sampled in the same site, isolated before, and after the C&D procedures and displaying the same MLST and ERIC profiles were tested for their sensitivity to different concentrations of BC, in a time course assay. The expression of non-specific efflux pumps was evaluated at each time point by qPCR using tufA gene as housekeeping. A differential expression of the two investigated genes was observed: lde was found to be more expressed by the strains isolated before C&D procedures while its expression was dose-dependent in the case of the post C&D procedures strain. On the contrary, the expression of mdrL was inhibited under low biocidal stress (10 ppm BC) and enhanced in the presence of high stress (100 ppm BC). These findings suggests a possible role for C&D procedures to select L. monocytogenes persisters, pointing out the importance of dealing with the identification of risk factors in food plants sanification procedures that might select more tolerant strains.

Project description:BACKGROUND:Listeria monocytogenes Clonal Complexes (CCs) have been epidemiologically associated with foods, especially ready-to-eat (RTE) products for which the most likely source of contamination depends on the occurrence of persisting clones in food-processing environments (FPEs). As the ability of L. monocytogenes to adapt to environmental stressors met in the food chain challenges the efforts to its eradication from FPEs, the threat of persistent strains to the food industry and public health authorities continues to rise. In this study, 94 food and FPEs L. monocytogenes isolates, representing persistent subtypes contaminating three French seafood facilities over 2-6 years, were whole-genome sequenced to characterize their genetic diversity and determine the biomarkers associated with long-term survival in FPEs. RESULTS:Food and FPEs isolates belonged to five CCs, comprising long-term intra- and inter-plant persisting clones. Mobile genetic elements (MGEs) such as plasmids, prophages and transposons were highly conserved within CCs, some of which harboured genes for resistance to chemical compounds and biocides used in the processing plants. Some of these genes were found in a 90.8 kbp plasmid, predicted to be" mobilizable", identical in isolates from CC204 and CC155, and highly similar to an 81.6 kbp plasmid from isolates belonging to CC7. These similarities suggest horizontal transfer between isolates, accompanied by deletion and homologous recombination in isolates from CC7. Prophage profiles characterized persistent clonal strains and several prophage-loci were plant-associated. Notably, a persistent clone from CC101 harboured a novel 31.5 kbp genomic island that we named Listeria genomic island 3 (LGI3), composed by plant-associated loci and chromosomally integrating cadmium-resistance determinants cadA1C. CONCLUSIONS:Genome-wide analysis indicated that inter- and intra-plant persisting clones harbour conserved MGEs, likely acquired in FPEs and maintained by selective pressures. The presence of closely related plasmids in L. monocytogenes CCs supports the hypothesis of horizontal gene transfer conferring enhanced survival to FPE-associated stressors, especially in hard-to-clean harbourage sites. Investigating the MGEs evolutionary and transmission dynamics provides additional resolution to trace-back potentially persistent clones. The biomarkers herein discovered provide new tools for better designing effective strategies for the removal or reduction of resident L. monocytogenes in FPEs to prevent contamination of RTE seafood.

Project description:Listeria monocytogenes is a foodborne pathogen adapted to survive and persist in multiple environments. Following two previous studies on prevalence and virulence of L. monocytogenes ST121 and ST14 repeatedly collected in a the same rabbit-meat processing plant, the research questions of the present study were to: (1) assess persistence of L. monocytogenes isolates from the rabbit-plant; (2) select genes associated to physiological adaptation to the food-processing environment; (3) compare presence/absence/truncation of these genes in newly sequenced and publicly available ST121 and ST14 genomes. A total of 273 draft genomes including ST121 and ST14 newly sequenced and publicly available draft genomes were analyzed. Whole-genome Single Nucleotide Polymorfism (wgSNP) analysis was performed separately on the assemblies of ST121 and ST14 draft genomes. SNPs alignments were used to infer phylogeny. A dataset of L. monocytogenes ecophysiology genes was built based on a comprehensive literature review. The 94 selected genes were screened on the assemblies of all ST121 and ST14 draft genomes. Significant gene enrichments were evaluated by statistical analyses. A persistent ST14 clone, including 23 out of 27 newly sequenced genomes, was circulating in the rabbit-meat plant along with two not persistent clones. A significant enrichment was observed in ST121 genomes concerning stress survival islet 2 (SSI-2) (alkaline and oxidative stress), qacH gene (resistance to benzalkonium chloride), cadA1C gene cassette (resistance to 70 mg/l of cadmium chloride) and a truncated version of actA gene (biofilm formation). Conversely, ST14 draft genomes were enriched with a full-length version of actA gene along with the Listeria Genomic Island 2 (LGI 2) including the ars operon (arsenic resistance) and the cadA4C gene cassette (resistance to 35 mg/l of cadmium chloride). Phenotypic tests confirmed ST121 as a weak biofilm producer in comparison to ST14. In conclusion, ST121 carried the qacH gene and was phenotypically resistant to quaternary ammonium compounds. This property might contribute to the high prevalence of ST121 in food processing plants. ST14 showed greater ability to form biofilms, which might contribute to the occasional colonization and persistence on harborage sites where sanitizing procedures are difficult to display.

Project description:Listeria monocytogenes is a foodborne pathogen and a resilient environmental saprophyte. Dairy farms are a reservoir of L. monocytogenes, and strains can persist on farms for years. Here, we sequenced the genomes of 250 L. monocytogenes isolates to investigate the persistence and mobile genetic elements (MGEs) of Listeria strains inhabiting dairy farms. We performed a single-nucleotide polymorphism (SNP)-based phylogenomic analysis to identify 14 monophyletic clades of L. monocytogenes persistent on the farms for ≥6 months. We found that prophages and other mobile genetic elements were, on average, more numerous among isolates in persistent than nonpersistent clades, and we demonstrated that resistance genes against bacitracin, arsenic, and cadmium were significantly more prevalent among isolates in persistent than nonpersistent clades. We identified a diversity of mobile elements among the 250 farm isolates, including three novel plasmids, three novel transposons, and a novel prophage harboring cadmium resistance genes. Several of the mobile elements we identified in Listeria were identical to the mobile elements of enterococci, which is indicative of recent transfer between these genera. Through a genome-wide association study, we discovered that three putative defense systems against invading prophages and plasmids were negatively associated with persistence on farms. Our findings suggest that mobile elements support the persistence of L. monocytogenes on dairy farms and that L. monocytogenes inhabiting the agroecosystem is a potential reservoir of mobile elements that may spread to the food industry. IMPORTANCE Animal-derived raw materials are an important source of L. monocytogenes in the food industry. Knowledge of the factors contributing to the pathogen's transmission and persistence on farms is essential for designing effective strategies against the spread of the pathogen from farm to fork. An increasing body of evidence suggests that mobile genetic elements support the adaptation and persistence of L. monocytogenes in the food industry, as these elements contribute to the dissemination of genes encoding favorable phenotypes, such as resilience against biocides. Understanding of the role of farms as a potential reservoir of these elements is needed for managing the transmission of mobile elements across the food chain. Because L. monocytogenes coinhabits the farm ecosystem with a diversity of other bacterial species, it is important to assess the degree to which genetic elements are exchanged between Listeria and other species, as such exchanges may contribute to the rise of novel resistance phenotypes.

Project description:Listeriosis is a serious infectious disease with one of the highest case fatality rates (ca. 20%) among the diseases manifested from bacterial foodborne pathogens in humans, while dairy products are often implicated as sources of human infection with Listeria monocytogenes. In this study, we characterized phenotypically and genetically by whole-genome sequencing (WGS) 54 L. monocytogenes strains isolated from Myzithra, a traditional Greek soft whey cheese (48 isolates), and swabs collected from surfaces of a cheese processing plant (six isolates) in the Epirus region of Greece. All but one strain of L. monocytogenes belonged to the polymerase chain reaction (PCR) serogroups IIa (16.7%) and IIb (81.5%), corresponding to serotypes 1/2a, 3a and 1/2b, 3b, 7, respectively. The latter was identified as a PCR-serogroup IVb strain (1.8%) of serotypes 4b, 4d, 4e. Bioinformatics analysis revealed the presence of five sequence types (STs) and clonal complexes (CCs); ST1, ST3, ST121, ST 155, ST398 and CC1, CC3, CC121, CC155, CC398 were thus detected in 1.9, 83.3, 11.0, 1.9, and 1.9% of the L. monocytogenes isolates, respectively. Antibiograms of the pathogen against a panel of seven selected antibiotics (erythromycin, tetracycline, benzylpenicillin, trimethoprim-sulfamethoxazole, ampicillin, ciprofloxacin, and meropenem) showed that 50 strains (92.6%), the six surface isolates also included, were intermediately resistant to ciprofloxacin and susceptible to the rest of the six antimicrobial agents tested, whereas strong resistance against the use of a single from three implicated antibiotics was recorded to four strains (7.4%) of the pathogen isolated from Myzithra cheese samples. Thence, the minimum inhibitory concentrations (MICs) were determined for erythromycin (MIC = 0.19 μg/mL), ciprofloxacin (MIC ≥ 0.19 μg/mL), and meropenem (MIC = 0.64 μg/mL), and finally, just one strain was deemed resistant to the latter antibiotic. The phylogenetic positions of the L. monocytogenes strains and their genetic variability were determined through WGS, whilst also stress response and virulence gene analysis for the isolates was conducted. Findings of this work should be useful as they could be utilized for epidemiological investigations of L. monocytogenes in the food processing environment, revealing possible contamination scenarios, and acquired antimicrobial resistance along the food production chain.

Dataset Information

Characteristics of Listeria Monocytogenes Strains Persisting in a Meat Processing Facility over a 4-Year Period.

Publications

Characteristics of Listeria Monocytogenes Strains Persisting in a Meat Processing Facility over a 4-Year Period.

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