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Small Produce Farm Environments Can Harbor Diverse Listeria monocytogenes and Listeria spp. Populations.

ABSTRACT:

Abstract

A comprehensive understanding of foodborne pathogen diversity in preharvest environments is necessary to effectively track pathogens on farms and identify sources of produce contamination. As such, this study aimed to characterize Listeria diversity in wildlife feces and agricultural water collected from a New York state produce farm over a growing season. Water samples were collected from a pond (n = 80) and a stream (n = 52). Fecal samples (n = 77) were opportunistically collected from areas <5 m from the water sources; all samples were collected from a <0.5-km2 area. Overall, 86 (41%) and 50 (24%) of 209 samples were positive for Listeria monocytogenes and Listeria spp. (excluding L. monocytogenes), respectively. For each positive sample, one L. monocytogenes or Listeria spp. isolate was speciated by sequencing the sigB gene, thereby allowing for additional characterization based on the sigB allelic type. The 86 L. monocytogenes and 50 Listeria spp. isolates represented 8 and 23 different allelic types, respectively. A subset of L. monocytogenes isolates (n = 44) from pond water and pond-adjacent feces (representing an ∼5,000-m2 area) were further characterized by pulsed-field gel electrophoresis (PFGE); these 44 isolates represented 22 PFGE types, which is indicative of considerable diversity at a small spatial scale. Ten PFGE types were isolated more than once, suggesting persistence or reintroduction of PFGE types in this area. Given the small spatial scale, the prevalence of L. monocytogenes and Listeria spp., as well as the considerable diversity among isolates, suggests traceback investigations may be challenging. For example, traceback of finished product or processing facility contamination with specific subtypes to preharvest sources may require collection of large sample sets and characterization of a considerable number of isolates. Our data also support the adage "absence of evidence does not equal evidence of absence" as applies to L. monocytogenes traceback efforts at the preharvest level.

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SUBMITTER: Belias A

PROVIDER: S-EPMC8000000 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:For decades, quaternary ammonium compounds (QAC)-based sanitizers have been broadly used in food processing environments to control foodborne pathogens such as Listeria monocytogenes. Still, there is a lack of consensus on the likelihood and implication of reduced Listeria susceptibility to benzalkonium chloride (BC) that may emerge due to sublethal exposure to the sanitizers in food processing environments. With a focus on fresh produce processing, we attempted to fill multiple data and evidence gaps surrounding the debate. We determined a strong correlation between tolerance phenotypes and known genetic determinants of BC tolerance with an extensive set of fresh produce isolates. We assessed BC selection on L. monocytogenes through a large-scale and source-structured genomic survey of 25,083 publicly available L. monocytogenes genomes from diverse sources in the United States. With the consideration of processing environment constraints, we monitored the temporal onset and duration of adaptive BC tolerance in both tolerant and sensitive isolates. Finally, we examined residual BC concentrations throughout a fresh produce processing facility at different time points during daily operation. While genomic evidence supports elevated BC selection and the recommendation for sanitizer rotation in the general context of food processing environments, it also suggests a marked variation in the occurrence and potential impact of the selection among different commodities and sectors. For the processing of fresh fruits and vegetables, we conclude that properly sanitized and cleaned facilities are less affected by BC selection and unlikely to provide conditions that are conducive for the emergence of adaptive BC tolerance in L. monocytogenes. IMPORTANCE Our study demonstrates an integrative approach to improve food safety assessment and control strategies in food processing environments through the collective leveraging of genomic surveys, laboratory assays, and processing facility sampling. In the example of assessing reduced Listeria susceptibility to a widely used sanitizer, this approach yielded multifaceted evidence that incorporates population genetic signals, experimental findings, and real-world constraints to help address a lasting debate of policy and practical importance.

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.

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Small Produce Farm Environments Can Harbor Diverse Listeria monocytogenes and Listeria spp. Populations.

Abstract

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