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In a recent longitudinal surveillance study in 30 U.S. retail delicatessens, 9.7% of environmental surfaces were positive for Listeria monocytogenes, and we found substantial evidence of persistence. In this study, we aimed to reduce the prevalence and persistence of L. monocytogenes in the retail deli environment by developing and implementing practical and feasible intervention strategies (i.e., sanitation standard operating procedures; SSOPs). These SSOPs were standardized across the 30 delis enrolled in this study. SSOP implementation was verified by systems inherent to each retailer. Each deli also was equipped with ATP monitoring systems to verify effective sanitation. We evaluated intervention strategy efficacy by testing 28 food and nonfood contact surfaces for L. monocytogenes for 6 months in all 30 retail delis. The efficacy of the intervention on the delis compared with preintervention prevalence level was not statistically significant; we found that L. monocytogenes could persist despite implementation of enhanced SSOPs. Systematic and accurate use of ATP monitoring systems varied widely among delis. The findings indicate that intervention strategies in the form of enhanced daily SSOPs were not sufficient to eliminate L. monocytogenes from highly prevalent and persistently contaminated delis and that more aggressive strategies (e.g., deep cleaning or capital investment in redesign or equipment) may be necessary to fully mitigate persistent contamination.

Salmonella enterica serovar Heidelberg is currently the 12th most common serovar of Salmonella enterica causing salmonellosis in the United States and results in twice the average incidence of blood infections caused by nontyphoidal salmonellae. Multiple outbreaks of salmonellosis caused by Salmonella Heidelberg resulted from the same poultry processor, which infected 634 people during 2013 and 2014. The hospitalization and invasive illness rates were 38% and 15%, respectively. We hypothesized that the outbreak strains of Salmonella Heidelberg had enhanced stress tolerance and virulence capabilities. We sourced nine food isolates collected during the outbreak investigation and three reference isolates to assess their tolerance to heat and sanitizers, ability to attach to abiotic surfaces, and invasiveness in vitro. We performed RNA sequencing on three isolates (two outbreak-associated isolates and a reference Salmonella Heidelberg strain) with various levels of heat tolerance to gain insight into the mechanism behind the isolates' enhanced heat tolerance. We also performed genomic analyses to determine the genetic relationships among the outbreak isolates. Ultimately, we determined that (i) six Salmonella Heidelberg isolates associated with the foodborne outbreak had enhanced heat tolerance, (ii) one outbreak isolate with enhanced heat tolerance also had an enhanced biofilm-forming ability under stressful conditions, (iii) exposure to heat stress increased the expression of Salmonella Heidelberg multidrug efflux and virulence genes, and (iv) outbreak-associated isolates were likely transcriptionally primed to better survive processing stresses and, potentially, to cause illness. IMPORTANCE This study provides a deep analysis of the intrinsic stress tolerance and virulence capabilities of Salmonella Heidelberg that may have contributed to the length and severity of a recent salmonellosis outbreak. Additionally, this study provides a comprehensive analysis of the transcriptomic response of S. enterica strains to heat stress conditions and compares baseline stationary-phase gene expression among outbreak-and non-outbreak-associated Salmonella Heidelberg isolates. These data can be used in assay development to screen isolates for stress tolerance and subsequent survival. This study adds to our understanding of the strains associated with the outbreak and informs ongoing regulatory discussions on Salmonella in poultry.

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The objective of this study was to develop and assess the efficacy of an aggressive deep cleaning sanitation standard operating procedure (DC-SSOP) in nine retail delicatessens to reduce persistent Listeria monocytogenes environmental contamination. The DC-SSOP was developed from combined daily SSOPs recommended by the Food Marketing Institute and input from experts in Listeria control from food manufacturing and sanitation. The DC-SSOP was executed by a trained professional cleaning service during a single 12-h shutdown period. A modified protocol from the U.S. Food and Drug Administration Bacteriological Analytical Manual was used to detect L. monocytogenes in samples from 28 food and nonfood contact surfaces that were collected immediately before and after each cleaning and in samples collected monthly for 3 months. The DC-SSOP significantly reduced L. monocytogenes prevalence overall during the 3-month follow-up period and produced variable results for persistent L. monocytogenes isolates. Six delis with historically low to moderate L. monocytogenes prevalence had no significant changes in the number of samples positive for L. monocytogenes after deep cleaning. Deep cleaning in very high prevalence delis (20 to 30% prevalence) reduced L. monocytogenes by 25.6% (P < 0.0001, n = 294) overall during the follow-up period. Among delis with extremely high prevalence (>30%), positive samples from nonfood contact surfaces were reduced by 19.6% (P = 0.0002, n = 294) during the follow-up period. The inability of deep cleaning to completely eliminate persistent L. monocytogenes was likely due to the diverse infrastructures in each deli, which may require more individualized intervention strategies.

Recent research demonstrated that some Listeria monocytogenes plasmids contribute to stress survival. However, only a few studies have analyzed gene expression patterns of L. monocytogenes plasmids. In this study, we identified four previously published stress‐response‐associated transcriptomic data sets which studied plasmid‐harboring L. monocytogenes strains but did not include an analysis of the plasmid transcriptomes. The four transcriptome data sets encompass three distinct plasmids from three different L. monocytogenes strains. Differential gene expression analysis of these plasmids revealed that the number of differentially expressed (DE) L. monocytogenes plasmid genes ranged from 30 to 45 with log 2 fold changes of −2.2 to 6.8, depending on the plasmid. Genes often found to be DE included the cadmium resistance genes cadA and cadC , a gene encoding a putative NADH peroxidase, the putative ultraviolet resistance gene uvrX , and several uncharacterized noncoding RNAs (ncRNAs). Plasmid‐encoded ncRNAs were consistently among the highest expressed genes. In addition, one of the data sets utilized the same experimental conditions for two different strains harboring distinct plasmids. We found that the gene expression patterns of these two L. monocytogenes plasmids were highly divergent despite the identical treatments. These data suggest plasmid‐specific gene expression responses to environmental stimuli and differential plasmid regulation mechanisms between L. monocytogenes strains. Our findings further our understanding of the dynamic expression of L. monocytogenes plasmid‐encoded genes in diverse environmental conditions and highlight the need to expand the study of L. monocytogenes plasmid genes' functions.

The popularity of backyard chickens has been growing steadily over the past 10 years, with Covid-19 stay at home orders in 2020 yielding an added boost in popularity. Concurrently, cases of salmonellosis from live poultry exposure have also risen. Previous research on backyard chicken owners has focused primarily on urban chicken owners, which may have differing knowledge and biosecurity habits from rural backyard chicken owners. The goal of this study was to investigate the prevalence of S. enterica in rural and urban flocks of chickens in the state of Vermont and to determine what attitudes toward and knowledge about S. enterica owners had, as well as what biosecurity practices they used. We conducted two surveys in Vermont between 2019–2022; a pilot study tied to sampling for Salmonella enterica in backyard chicken flocks from 2019–2021 and a statewide study in 2022 to determine the prevalence of backyard chickens in Vermont and obtain representative survey data from backyard chicken owners. We found (i) overall, 19% (8/42) backyard chicken flocks from 2019–2021 had S. enterica, but S. enterica rates varied substantially by year; (ii) backyard chicken owners were wealthier and more educated than the average Vermonter and generally lived in rural areas; (iii) participants in the statewide survey had much lower uptake of good biosecurity habits compared to the pilot survey; (iv) despite increased messaging about backyard chicken-associated salmonellosis and good biosecurity measures over the past several years, uptake of biosecurity measures is inconsistent, and rates of unsafe practices such as kissing or cuddling chickens have increased in Vermont. Overall, the data indicate the need for improved messaging on biosecurity and risks associated with backyard chickens

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