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Mobile Elements Harboring Heavy Metal and Bacitracin Resistance Genes Are Common among Listeria monocytogenes Strains Persisting on Dairy Farms.

ABSTRACT: 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.

SUBMITTER: Castro H

PROVIDER: S-EPMC8386393 | biostudies-literature |

REPOSITORIES: biostudies-literature

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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:The molecular epidemiology of Listeria monocytogenes was investigated in a longitudinal study of three Finnish dairy farms during 2013 to 2016. A total of 186 bulk tank milk (BTM), 224 milk filter sock (MFS), and 1,702 barn environment samples were analyzed, and isolates of L. monocytogenes were genotyped using pulsed-field gel electrophoresis. L. monocytogenes occurred throughout the year in all sample types, and the prevalence in MFS increased significantly during the indoor season. L. monocytogenes was more prevalent in MFS (29%) than in BTM (13%) samples. However, the prevalence of L. monocytogenes varied more between farms in samples of MFS (13 to 48%) than in BTM (10 to 16%). For each farm, the L. monocytogenes genotypes detected were classified by persistence (defined as persistent if isolated from ?3 samples during ?6 months) and predominance (defined as predominant if >5% prevalence on at least one farm visit). The prevalence of sporadic genotypes was 4 to 5% on all three farms. In contrast, the prevalence of persistent predominant genotypes varied between farms by 4% to 16%. The highest prevalence of persistent predominant genotypes was observed on the farm with the poorest production hygiene. Persistent predominant genotypes were most prevalent on feeding surfaces, water troughs, and floors. Genotypes isolated from the milking system or from cow udders had a greater relative risk of occurring in BTM and MFS than genotypes that only occurred elsewhere in the farm, supporting the hypothesis that L. monocytogenes is transmitted to milk from contamination on the udder surface or in the milking equipment.IMPORTANCEListeria monocytogenes is a ubiquitous environmental bacterium and the causative agent of a serious foodborne illness, listeriosis. Dairy products are common vehicles of listeriosis, and dairy cattle farms harbor L. monocytogenes genotypes associated with human listeriosis outbreaks. Indeed, dairy cattle farms act as a reservoir of L. monocytogenes, and the organism is frequently detected in bulk tank milk (BTM) and in the feces of clinically healthy cows. The ecology of L. monocytogenes in the farm environment is complex and poorly understood. Isolates of the same L. monocytogenes genotype can occur in the farm for years, but the factors contributing to the persistence of genotypes on dairy farms are unknown. Knowledge of the persistence patterns and contamination routes of L. monocytogenes on dairy farms can improve management of the contamination pressure in the farm environment and aid in the development of focused control strategies to reduce BTM contamination.

Project description:Listeria monocytogenes (L. monocytogenes) is frequently detected in ruminants, especially dairy cattle, and associated with the sporadic and epidemic outbreak of listeriosis in farms. In this epidemiological study, the prevalence, virulence, antibiotic resistance profiles, and genetic diversity of L. monocytogenes in three Egyptian dairy cattle farms were investigated. The risk factors associated with the fecal shedding of L. monocytogenes were analyzed. The L. monocytogenes strains from the three farms were categorized into distinct genotypes based on sampling site and sample type through enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR). A total of 1896 samples were collected from animals, environments, and milking equipment in the three farms. Results revealed that 137 (7.23%) of these samples were L. monocytogenes positive. The prevalence of L. monocytogenes in the animal samples was high (32.1%), and the main environmental source of prevalent genotypes in the three farms was silage. For all sample types, L. monocytogenes was more prevalent in farm I than in farms II and III. Risk factor analysis showed seasonal variation in production hygiene. For all sample types, L. monocytogenes was significantly more prevalent in winter than in spring and summer. The level of L. monocytogenes fecal shedding was high likely because of increasing age, number of parities, and milk yield in dairy cattle. Two virulence genes, namely, hlyA & prfA, were also detected in 93 strains, whereas only one of these genes was found in 44 residual strains. Conversely, iap was completely absent in all strains. The strains exhibited phenotypic resistance to most of the tested antibiotics, but none of them was resistant to netilmicin or vancomycin. According to sample type, the strains from the animal samples were extremely resistant to amoxicillin (95.2%, 80/84) and cloxacillin (92.9%, 78/84). By comparison, the strains from the environmental samples were highly resistant to cefotaxime (86.95%, 20/23). Furthermore, 25 multi-antibiotic resistance (MAR) patterns were observed in L. monocytogenes strains. All strains had a MAR index of 0.22-0.78 and harbored antibiotic resistance genes, including extended-spectrum β-lactamase (blaCTX-M [92.7%] and blaDHA-1 [66.4%]), quinolones (qnrS [91.2%], qnrA [58.4%], parC [58.4%], and qnrB [51%]), macrolides (erm[B] [76.6%], erm(C) [1.5%], and msr(A) [27%]), trimethoprim (dfrD [65.7%]), and tetracyclines (tet(M) [41.6%], tet(S) [8%], and int-Tn [26.3%]). ERIC-PCR confirmed that the strains were genetically diverse and heterogeneous. A total of 137 isolated L. monocytogenes strains were classified into 22 distinct ERIC-PCR groups (A-V). Among them, ERIC E (10.2%) was the most prevalent group. These results indicated that environment and milking equipment served as reservoirs and potential transmission ways of virulent and multidrug-resistant L. monocytogenes to dairy animals, consequently posing threats to public health. Silage is the main environmental source of prevalent genotypes on all three farms. Therefore, hygienic measures at the farm level should be developed and implemented to reduce L. monocytogenes transmission inside dairy cattle farms.

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Mobile Elements Harboring Heavy Metal and Bacitracin Resistance Genes Are Common among Listeria monocytogenes Strains Persisting on Dairy Farms.

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