Application of Environmental Monitoring Programs and Root Cause Analysis to Identify and Implement Interventions to Reduce or Eliminate Listeria Populations in Apple Packinghouses.

Controlling Listeria in produce packinghouses can be challenging due to the large number of potential contamination routes. For example, repeated isolation of the same Listeria subtype in a packinghouse could indicate persistence in the packinghouse or reintroduction of the same Listeria from an upstream source. To improve understanding of Listeria transmission patterns in packinghouses, we performed a longitudinal study in four apple packinghouses, including testing of 1,339 environmental sponges and whole genome sequencing (WGS)-based characterization of 280 isolates. Root cause analysis and subsequent intervention implementation were also performed and assessed for effectiveness. Listeria prevalence among environmental sponges collected from the four packinghouses was 20% (range of 5-31% for individual packinghouses). Sites that showed high Listeria prevalence included drains, forklift tires and forks, forklift stops, and waxing area equipment frames. A total of 240/280 WGS-characterized isolates were represented in 41 clusters, each containing two or more isolates that differed by ≤50 high-quality single nucleotide polymorphisms (hqSNPs); 21 clusters were isolated from one packinghouse over ≥2 samplings (suggesting persistence or possibly reintroduction), while 11 clusters included isolates from >2 packinghouses, suggesting common upstream sources. Some interventions successfully (i) reduced Listeria detection on forklift tires and forks (across packinghouses) and (ii) mitigated packinghouse-specific Listeria issues (e.g., in catch pans). However, interventions that lacked enhanced equipment disassembly when persistence was suspected typically appeared to be unsuccessful. Overall, while our data suggest a combination of intensive environmental sampling with subtyping and root cause analysis can help identify effective interventions, implementation of effective interventions continues to be a challenge in packinghouses.

Utilizing whole genome sequencing to characterize Listeria spp. persistence and transmission patterns in a farmstead dairy processing facility and its associated farm environment.

Farmstead dairy processing facilities may be particularly susceptible to Listeria spp. contamination due to the close physical proximity of their processing environments (PE) to associated dairy farm environments (FE). In this case study, we supported the implementation of interventions focused on improving (i) cleaning and sanitation efficacy, (ii) hygienic zoning, and (iii) sanitary equipment/facility design and maintenance in a farmstead dairy processing facility, and evaluated their impact on Listeria spp. detection in the farmstead's PE over 1 year. Detection of Listeria spp. in the farmstead's PE was numerically reduced from 50% to 7.5% after 1 year of intervention implementation, suggesting that these interventions were effective at improving Listeria spp. control. In addition, environmental samples were also collected from the farmstead's FE to evaluate the risk of the FE as a potential source of Listeria spp. in the PE. Overall, detection of Listeria spp. was higher in samples collected from the FE (75%, 27/36) compared with samples collected from the PE (24%, 29/120). Whole genome sequencing (WGS) performed on select isolates collected from the PE and FE supported the identification of 6 clusters (range of 3 to 15 isolates per cluster) that showed ≤ 50 high quality single nucleotide polymorphism (hqSNP) differences. Of these 6 clusters, 3 (i.e., clusters 2, 4, and 5) contained isolates that were collected from both the PE and FE, suggesting that transmission between these 2 environments was likely. Moreover, all cluster 2 isolates represented a clonal complex (CC) of L. monocytogenes commonly associated with dairy farm environmental reservoirs (i.e., CC666), which may support that the farmstead's FE represented an upstream source of the cluster 2 isolates that were found in the PE. Overall, our data underscore that, while the FE can represent a potential upstream source of Listeria spp. contamination in a farmstead dairy processing facility, implementation of targeted interventions can help effectively minimize Listeria spp. contamination in the PE.

Strain-specific Growth Parameters are Important to Accurately Model Bacterial Growth on Baby Spinach in Simulation Models.

Digital tools to predict produce shelf life have the potential to reduce food waste and improve consumer satisfaction. To address this need, we (i) performed an observational study on the microbial quality of baby spinach, (ii) completed growth experiments of bacteria that are representative of the baby spinach microbiota, and (iii) developed an initial simulation model of bacterial growth on baby spinach. Our observational data showed that the predominant genera found on baby spinach were Pseudomonas, Pantoea and Exiguobacterium. Rifampicin-resistant mutants (rifR mutants) of representative bacterial subtypes were subsequently generated to obtain strain-specific growth parameters on baby spinach. These experiments showed that: (i) it is difficult to select rifR mutants that do not have fitness costs affecting growth (9 of 15 rifR mutants showed substantial differences in growth, compared to their corresponding wild-type strain) and (ii) based on estimates from primary growth models, the mean (geometric) maximum population of rifR mutants on baby spinach (7.6 log10 CFU/g, at 6°C) appears lower than that of the spinach microbiota (9.6 log10 CFU/g, at 6°C), even if rifR mutants did not have substantial growth-related fitness costs. Thus, a simulation model, parameterized with the data obtained here as well as literature data on home refrigeration temperatures, underestimated bacterial growth on baby spinach. The root mean square error of the simulation's output, compared against data from the observational study, was 1.11 log10 CFU/g. Sensitivity analysis was used to identify key parameters (e.g., strain maximum population) that impact the simulation model's output, allowing for prioritization of future data collection to improve the simulation model. Overall, this study provides a roadmap for the development of models to predict bacterial growth on leafy vegetables with strain-specific parameters and suggests that additional data are required to improve these models.

Intensive Environmental Sampling and Whole Genome Sequence-based Characterization of Listeria in Small- and Medium-sized Dairy Facilities Reveal Opportunities for Simplified and Size-appropriate Environmental Monitoring Strategies.

Small- and medium-sized dairy processing facilities (SMDFs) may face unique challenges with respect to controlling Listeria in their processing environments, e.g., due to limited resources. The aim of this study was to implement and evaluate environmental monitoring programs (EMPs) for Listeria control in eight SMDFs in a ∼1-year longitudinal study; this included a comparison of pre-operation (i.e., after cleaning and sanitation and prior to production) and mid-operation (i.e., at least 4 h into production) sampling strategies. Among 2,072 environmental sponge samples collected across all facilities, 272 (13%) were positive for Listeria. Listeria prevalence among pre- and mid-operation samples (15% and 17%, respectively), was not significantly different. Whole genome sequencing (WGS) performed on select isolates to characterize Listeria persistence patterns revealed repeated isolation of closely related Listeria isolates (i.e., ≤20 high-quality single nucleotide polymorphism [hqSNP] differences) in 5/8 facilities over >6 months, suggesting Listeria persistence and/or reintroduction was relatively common among the SMDFs evaluated here. WGS furthermore showed that for 41 sites where samples collected pre- and mid-operation were positive for Listeria, Listeria isolates obtained were highly related (i.e., ≤10 hqSNP differences), suggesting that pre-operation sampling alone may be sufficient and more effective for detecting sites of Listeria persistence. Importantly, our data also showed that only 1/8 of facilities showed a significant decrease in Listeria prevalence over 1 year, indicating continued challenges with Listeria control in at least some SMDFs. We conclude that options for simplified Listeria EMPs (e.g., with a focus on pre-operation sampling, which allows for more rapid identification of likely persistence sites) may be valuable for improved Listeria control in SMDFs.

Phenotypic and genomic characterizations of Klebsiella pneumoniae ssp. pneumoniae and Rahnella inusitata strains reveal no clear association between genetic content and ropy phenotype.

Ropy defect of pasteurized fluid milk is a type of spoilage which manifests itself by an increased viscosity, slimy body, and string-like flow during pouring. This defect has, among other causes, been attributed to the growth, proliferation and exopolysaccharide production by coliform bacteria, which are most commonly introduced in milk as post-pasteurization contaminants. As we identified both Klebsiella pneumoniae ssp. pneumoniae and Rahnella inusitata that were linked to a ropy defect, the goal of this study was to characterize 3 K. pneumoniae ssp. pneumoniae strains and 2 R. inusitata for (1) their ability to grow and cause ropy defect in milk at 6°C and 21°C and to (2) probe the genetic basis for observed ropy phenotype. Although all K. pneumoniae ssp. pneumoniae and R. inusitata strains showed net growth of >4 log10 over 48 h in UHT milk at 21°C, only R. inusitata strains displayed growth during 28-d incubation period at 6°C (>6 log10). Two out of 3 K. pneumoniae ssp. pneumoniae strains were capable of causing the ropy defect in milk at 21°C, as supported by an increase in the viscosity of milk and string-like flow during pouring; these 2 strains were originally isolated from raw milk. Only one R. inusitata strains was able to cause the ropy defect in milk; this strain was able to cause the defect at both 6°C and 21°C, and was originally isolated from a pasteurized milk. These findings suggest that the potential of K. pneumoniae ssp. pneumoniae and R. inusitata to cause ropy defect in milk is a strain-dependent characteristic. Comparative genomics provided no definitive answer on genetic basis for the ropy phenotype. However, for K. pneumoniae ssp. pneumoniae, genes rffG, rffH, rfbD, and rfbC involved in biosynthesis and secretion of enterobacterial common antigen (ECA) could only be found in the 2 strains that produced ropy defect, and for R. inusitata a set of 2 glycosyltransferase- and flippase genes involved in nucleotide sugar biosynthesis and export could only be identified in the ropy strain. Although these results provide some initial information for potential markers for strains that can cause ropy milk, the relationship between genetic content and ropiness in milk remains poorly understood and merits further investigation.

Role of Whole Genome Sequencing in Assessing Resident and Transient Listeria monocytogenes in a Produce Packinghouse.

Whole genome sequencing (WGS) is a powerful tool that may be used to assist in identifying Listeria contamination sources and movement within environments, and to assess persistence. This study investigated sites in a produce packinghouse where Listeria had been historically isolated; and aimed to characterize dispersal patterns and identify cases of transient and resident Listeria. Environmental swab samples (n = 402) were collected from 67 sites at two time-points on three separate visits. Each sample was tested for Listeria, and Listeria isolates were characterized by partial sigB sequencing to determine species and allelic type (AT). Representative isolates from the three most common L. monocytogenes ATs (n = 79) were further characterized by WGS. Of the 144 Listeria species positive samples (35.8%), L. monocytogenes was the most prevalent species. L. monocytogenes was often coisolated with another species of Listeria. WGS identified cases of sporadic and continued reintroduction of L. monocytogenes from the cold storages into the packinghouse and demonstrated cases of L. monocytogenes persistence over 2 years in cold storages, drains, and on a forklift. Nine distinct clusters were found in this study. Two clusters showed evidence of persistence. Isolates in these two clusters (N = 11, with one historical isolate) were obtained predominantly and over multiple samplings from cold storages, with sporadic movement to sites in the packing area, suggesting residence in cold storages with opportunistic dispersal within the packinghouse. The other seven clusters demonstrated evidence of transient Listeria, as isolation was sporadic over time and space during the packing season. Our data provide important insights into likely L. monocytogenes harborage points and transfer in a packinghouse, which is key to root cause analysis. While results support Listeria spp. as a suitable indicator organism for environmental monitoring surveys, findings were unable to establish a specific species as an index organism for L. monocytogenes. Findings also suggest long-term persistence with substantial SNP diversification, which may assist in identifying potential contamination sources and implementing control measures.

Taxonomy, ecology, and relevance to food safety of the genus Listeria with a particular consideration of new Listeria species described between 2010 and 2022.

Since 2010, the genus Listeria has had the addition of 22 new species that more than tripled the number of species identified until 2010. Sixteen of these 22 new species are distantly related to the type species, Listeria monocytogenes, and several of these present phenotypes that distinguish them from classical Listeria species (L. monocytogenes, Listeria innocua, Listeria ivanovii, Listeria seeligeri, Listeria welshimeri, and Listeria grayi). These 22 newly described species also show that Listeria is more genetically diverse than previously estimated. While future studies and surveys are needed to clarify the distribution of these species, at least some of these species may not be widely spread, while other species may be frequently found spread to human-related settings (e.g., farms and processing facilities), and others may be adapted to specific environmental habitats. Here, we review the taxonomic, phylogenetic, and ecological characteristics of these new Listeria species identified since 2010 and re-iterate the suggestion of re-classification of some species into three new genera: Murraya, Mesolisteria, and Paenilisteria. We also provide a review of current detection issues and the relevance to food safety related to the identification of these new species. For example, several new non-pathogenic species could be misidentified as the pathogen L. monocytogenes, based on methods that do not target L. monocytogenes-specific virulence genes/factors, leading to unnecessary product recalls. Moreover, eight species in the proposed new genus Mesolisteria are not good indicators of environmental conditions that could allow L. monocytogenes to grow since Mesolisteria species are unable to grow at low temperatures.

Salmonella enterica serovar Cerro displays a phylogenetic structure and genomic features consistent with virulence attenuation and adaptation to cattle.

Salmonella enterica subsp. enterica (S.) serovar Cerro is rarely isolated from human clinical cases of salmonellosis but represents the most common serovar isolated from cattle without clinical signs of illness in the United States. In this study, using a large, diverse set of 316 isolates, we utilized genomic methods to further elucidate the evolutionary history of S. Cerro and to identify genomic features associated with its apparent virulence attenuation in humans. Phylogenetic analyses showed that within this polyphyletic serovar, 98.4% of isolates (311/316) represent a monophyletic clade within section Typhi and the remaining 1.6% of isolates (5/316) form a monophyletic clade within subspecies enterica Clade A1. Of the section Typhi S. Cerro isolates, 93.2% of isolates (290/311) clustered into a large clonal clade comprised of predominantly sequence type (ST) 367 cattle and environmental isolates, while the remaining 6.8% of isolates (21/311), primarily from human clinical sources, clustered outside of this clonal clade. A tip-dated phylogeny of S. Cerro ST367 identified two major clades (I and II), one of which overwhelmingly consisted of cattle isolates that share a most recent common ancestor that existed circa 1975. Gene presence/absence and rarefaction curve analyses suggested that the pangenome of section Typhi S. Cerro is open, potentially reflecting the gain/loss of prophage; human isolates contained the most open pangenome, while cattle isolates had the least open pangenome. Hypothetically disrupted coding sequences (HDCs) displayed clade-specific losses of intact speC and sopA virulence genes within the large clonal S. Cerro clade, while loss of intact vgrG, araH, and vapC occurred in all section Typhi S. Cerro isolates. Further phenotypic analysis suggested that the presence of a premature stop codon in speC does not abolish ornithine decarboxylase activity in S. Cerro, likely due to the activity of the second ornithine decarboxylase encoded by speF, which remained intact in all isolates. Overall, our study identifies specific genomic features associated with S. Cerro's infrequent isolation from humans and its apparent adaptation to cattle, which has broader implications for informing our understanding of the evolutionary events facilitating host adaptation in Salmonella.

Whole-Genome Sequencing-Based Characterization of Listeria Isolates from Produce Packinghouses and Fresh-Cut Facilities Suggests Both Persistence and Reintroduction of Fully Virulent L. monocytogenes.

The contamination of ready-to-eat produce with Listeria monocytogenes (LM) can often be traced back to environmental sources in processing facilities and packinghouses. To provide an improved understanding of Listeria sources and transmission in produce operations, we performed whole-genome sequencing (WGS) of LM (n = 169) and other Listeria spp. (n = 107) obtained from 13 produce packinghouses and three fresh-cut produce facilities. Overall, a low proportion of LM isolates (9/169) had inlA premature stop codons, and a large proportion (83/169) had either or both of the LIPI-3 or LIPI-4 operons, which have been associated with hypervirulence. The further analysis of the WGS data by operation showed a reisolation (at least 2 months apart) of highly related isolates (<10 hqSNP differences) in 7/16 operations. Two operations had highly related strains reisolated from samples that were collected at least 1 year apart. The identification of isolates collected during preproduction (i.e., following sanitation but before the start of production) that were highly related to isolates collected during production (i.e., after people or products have entered and begun moving through the operation) provided evidence that some strains were able to survive standard sanitation practices. The identification of closely related isolates (<20 hqSNPs differences) in different operations suggests that cross-contamination between facilities or introductions from common suppliers may also contribute to Listeria transmission. Overall, our data suggest that the majority of LM isolates collected from produce operations are fully virulent and that both persistence and reintroduction may lead to the repeat isolation of closely related Listeria in produce operations. IMPORTANCE Listeria monocytogenes is of particular concern to the produce industry due to its frequent presence in natural environments as well as its ability to survive in packinghouses and fresh-cut processing facilities over time. The use of whole-genome sequencing, which provides high discriminatory power for the characterization of Listeria isolates, along with detailed source data (isolation date and sample location) shows that the presence of Listeria in produce operations appears to be due to random and continued reintroduction as well as to the persistence of highly related strains in both packinghouses and fresh-cut facilities. These findings indicate the importance of using high-resolution characterization approaches for root cause analyses of Listeria contamination issues. In cases of repeat isolation of closely related Listeria in a given facility, both persistence and reintroduction need to be considered as possible root causes.

Soil Collected in the Great Smoky Mountains National Park Yielded a Novel Listeria sensu stricto Species, L. swaminathanii.

Soil samples collected in the Great Smoky Mountains National Park yielded a Listeria isolate that could not be classified to the species level. Whole-genome sequence-based average nucleotide identity BLAST and in silico DNA-DNA Hybridization analyses confirmed this isolate to be a novel Listeria sensu stricto species with the highest similarity to L. marthii (ANI = 93.9%, isDDH = 55.9%). Additional whole-genome-based analysis using the Genome Taxonomy Database Toolkit further supported delineation as a novel Listeria sensu stricto species, as this tool failed to assign a species identification. Phenotypic and genotypic characterization results indicate that this species is nonpathogenic. Specifically, the novel Listeria species described here is phenotypically (i) nonhemolytic and (ii) negative for phosphatidylinositol-specific phospholipase C activity; the draft genome lacks all virulence genes found in the Listeria pathogenicity islands 1, 2, 3, and 4 as well as the internalin genes inlA and inlB. While the type strain contains an apparently intact catalase gene (kat), this strain is phenotypically catalase-negative (an unusual characteristic for Listeria sensu stricto species). Additional analyses identified a nonsynonymous mutation in a conserved codon of kat that is likely linked to the catalase-negative phenotype. Rapid species identification systems, including two biochemical and one matrix-assisted laser desorption/ionization, misidentified this novel species as either L. monocytogenes, L. innocua, or L. marthii. We propose the name L. swaminathanii, and the type strain is FSL L7-0020T (=ATCC TSD-239T). IMPORTANCEL. swaminathanii is a novel sensu stricto species that originated from a US National Park and it will be the first Listeria identified to date without official standing in the nomenclature. Validation was impeded by the National Park's requirements for strain access, ultimately deemed too restrictive by the International Committee on Systematics of Prokaryotes. However, lack of valid status should not detract from the significance of adding a novel species to the Listeria sensu stricto clade. Notably, detection of non-monocytogenes sensu stricto species in a food processing environment indicate conditions that could facilitate the presence of the pathogen L. monocytogenes. If isolated, our data show a potential for L. swaminathanii to be misidentified as another sensu stricto, notably L. monocytogenes. Therefore, developers of Listeria spp. detection and identification methods, who historically only include validly published species in their validation studies, should include L. swaminathanii to ensure accurate results.

Development of a Modeling Tool To Assess and Reduce Regulatory and Recall Risks for Cold-Smoked Salmon Due to Listeria monocytogenes Contamination.

ABSTRACT: Although public health risk assessments for Listeria monocytogenes (Lm) have been published for various foods, firm-level decision making on interventions targeting Lm involves considerations of both public health and enterprise risks. Smoked seafood is a ready-to-eat product with a high incidence of Lm contamination and has been associated with several recalls. We used cold-smoked salmon as a model product to develop a decision support tool (the regulatory and recall risk [3R] model) to estimate (i) baseline regulatory and recall (RR) risks (i.e., overall risks of a lot sampled and found positive for Lm, e.g., by food regulatory agencies) due to Lm contamination and (ii) the RR risk reduction that can be achieved through interventions with underlying mechanisms such as reducing the prevalence and/or level of Lm and retarding or preventing Lm growth. Given that a set number of samples (e.g., 10) are tested for a given lot, the RR risk equals the likelihood of detecting Lm in at least one sample. Under the baseline scenario, which assumes a 4% Lm prevalence and no interventions, the median predicted RR risk for a given production lot was 0.333 (95% credible interval: 0.288, 0.384) when 10 25-g samples were tested. Nisin treatments, which reduce both the prevalence and initial level of Lm, reduced RR risks in a concentration-dependent manner to 0.109 (0.074, 0.146) with 5 ppm, 0.049 (0.024, 0.083) with 10 ppm, and 0.017 (0.007, 0.033) with 20 ppm. In general, more effective reduction in RR risks can be achieved by reducing Lm prevalence than by retarding Lm growth; the RR risk was reduced to 0.182 (0.153, 0.213) by a 50% prevalence reduction but to only 0.313 (0.268, 0.367) by bacteriostatic growth inhibitors. Sensitivity analysis indicated that prevalence and initial level of Lm and storage temperature have the greatest impact on predicting RR risks, suggesting that reliable data for these parameters will improve model performance.

The Number and Type of Chaperone-Usher Fimbriae Reflect Phylogenetic Clade Rather than Host Range in Salmonella.

Salmonella is one of the most successful foodborne pathogens worldwide, owing in part to its ability to colonize or infect a wide range of hosts. Salmonella serovars are known to encode a variety of different fimbriae (hairlike organelles that facilitate binding to surfaces); however, the distribution, number, and sequence diversity of fimbriae encoded across different lineages of Salmonella were unknown. We queried whole-genome sequence (WGS) data for 242 Salmonella enterica subsp. enterica (subspecies enterica) isolates from the top 217 serovars associated with isolation from humans and agricultural animals; this effort identified 2,894 chaperone-usher (CU)-type fimbrial usher sequences, representing the most conserved component of CU fimbriae. On average, isolates encoded 12 different CU fimbrial ushers (6 to 18 per genome), although the distribution varied significantly (P = 1.328E-08) by phylogenetic clade, with isolates in section Typhi having significantly fewer fimbrial ushers than isolates in clade A2 (medians = 10 and 12 ushers, respectively). Characterization of fimbriae in additional non-enterica subspecies genomes suggested that 8 fimbrial ushers were classified as being unique to subspecies enterica isolates, suggesting that the majority of fimbriae were most likely acquired prior to the divergence of subspecies enterica. Characterization of mobile elements suggested that plasmids represent an important vehicle facilitating the acquisition of a wide range of fimbrial ushers, particularly for the acquisition of fimbriae from other Gram-negative genera. Overall, our results suggest that differences in the number and type of fimbriae encoded most likely reflect differences in phylogenetic clade rather than differences in host range. IMPORTANCE Fimbriae of the CU assembly pathway represent important organelles that mediate Salmonella's interactions with host tissues and abiotic surfaces. Our analyses provide a comprehensive overview of the diversity of CU fimbriae in Salmonella spp., highlighting that the majority of CU fimbriae are distributed broadly across multiple subspecies and suggesting that acquisition most likely occurred prior to the divergence of subspecies enterica. Our data also suggest that plasmids represent the primary vehicles facilitating the horizontal transfer of diverse CU fimbriae in Salmonella. Finally, the observed high sequence similarity between some ushers suggests that different names may have been assigned to closely related fimbrial ushers that likely should be represented by a single designation. This highlights the need to establish standard criteria for fimbria classification and nomenclature, which will also facilitate future studies seeking to associate virulence factors with adaptation to or differences in the likelihood of causing disease in a given host.

Characterization of Listeria monocytogenes isolated from wildlife in central New York.

BACKGROUND: Listeria monocytogenes (Lm) present in farming soil and food-processing facilities threatens food safety, but little is known about the carriage of Lm by wildlife. OBJECTIVES: We estimated the prevalence of faecal Lm shedding among wildlife admitted to a veterinary medical teaching hospital in central New York and characterized a subset of the Lm isolates. METHODS: Wildlife samples were collected between May 2018 and December 2019. We characterized the Lm isolates by assessing the growth at three temperatures approximating the body temperatures of reptiles (25°C), mammals (37°C), and birds (42°C) and identifying genotypic characteristics related to transmission and virulence. RESULTS: The apparent prevalence of faecal Lm shedding was 5.6% [18/324; 95% confidence interval (CI), 3.3%-8.6%]. Among 13 isolates that represented two lineages and 11 clonal complexes, three and five isolates were grouped into the same SNP clusters with human clinical isolates and environmental isolates, respectively. However, specific SNP difference data showed that Lm from wildlife was generally not closely related (>22 SNP differences) to Lm from human clinical sources and the food-processing environment. While the stress response locus SSI-2 was absent, SSI-1 was found in four isolates. Virulence genes prfA, plcA, hly, mpl, actA, plcB, inlA, inlB, inlC, inlE, inlH, inlJ, and inlK were present, without any premature stop codons, in all isolates. Virulence loci Listeria pathogenicity island 3 (LIPI-3) and LIPI-4, which have been linked to hypervirulence, and inlG were found in four, three, and seven isolates, respectively. CONCLUSIONS: Wildlife represents a potential reservoir for genetically diverse and putatively hypervirulent Lm strains. No statistically significant association between growth parameters and hosts was observed. However, compared to lineage I isolates, lineage II isolates showed significantly (p < 0.05) faster growth at 25°C and significantly slower growth at 42°C, suggesting that wildlife Lm isolates that belong to lineages I and II differ in their ability to grow at 25°C and 42°C.

Development of a Genomics-Based Approach To Identify Putative Hypervirulent Nontyphoidal Salmonella Isolates: Salmonella enterica Serovar Saintpaul as a Model.

While differences in human virulence have been reported across nontyphoidal Salmonella (NTS) serovars and associated subtypes, a rational and scalable approach to identify Salmonella subtypes with differential ability to cause human diseases is not available. Here, we used NTS serovar Saintpaul (S. Saintpaul) as a model to determine if metadata and associated whole-genome sequence (WGS) data in the NCBI Pathogen Detection (PD) database can be used to identify (i) subtypes with differential likelihoods of causing human diseases and (ii) genes and single nucleotide polymorphisms (SNPs) potentially responsible for such differences. S. Saintpaul SNP clusters (n = 211) were assigned different epidemiology types (epi-types) based on statistically significant over- or underrepresentation of human clinical isolates, including human associated (HA; n = 29), non-human associated (NHA; n = 23), and other (n = 159). Comparative genomic analyses identified 384 and 619 genes overrepresented among isolates in 5 HA and 4 NHA SNP clusters most significantly associated with the respective isolation source. These genes included 5 HA-associated virulence genes previously reported to be present on Gifsy-1/Gifsy-2 prophages. Additionally, premature stop codons in 3 and 7 genes were overrepresented among the selected HA and NHA SNP clusters, respectively. Tissue culture experiments with strains representing 4 HA and 3 NHA SNP clusters did not reveal evidence for enhanced invasion or intracellular survival for HA strains. However, the presence of sodCI (encoding a superoxide dismutase), found in 4 HA and 1 NHA SNP clusters, was positively correlated with intracellular survival in macrophage-like cells. Post hoc analyses also suggested a possible difference in intracellular survival among S. Saintpaul lineages. IMPORTANCE Not all Salmonella isolates are equally likely to cause human disease, and Salmonella control strategies may unintentionally focus on serovars and subtypes with high prevalence in source populations but are rarely associated with human clinical illness. We describe a framework leveraging WGS data in the NCBI PD database to identify Salmonella subtypes over- and underrepresented among human clinical cases. While we identified genomic signatures associated with HA/NHA SNP clusters, tissue culture experiments failed to identify consistent phenotypic characteristics indicative of enhanced human virulence of HA strains. Our findings illustrate the challenges of defining hypo- and hypervirulent S. Saintpaul and potential limitations of phenotypic assays when evaluating human virulence, for which in vivo experiments are essential. Identification of sodCI, an HA-associated virulence gene associated with enhanced intracellular survival, however, illustrates the potential of the framework and is consistent with prior work identifying specific genomic features responsible for enhanced or reduced virulence of nontyphoidal Salmonella.

Characterization of Basal Transcriptomes Identifies Potential Metabolic and Virulence-Associated Adaptations Among Diverse Nontyphoidal Salmonella enterica Serovars.

The zoonotic pathogen Salmonella enterica includes >2,600 serovars, which differ in the range of hosts they infect and the severity of disease they cause. To further elucidate the mechanisms behind these differences, we performed transcriptomic comparisons of nontyphoidal Salmonella (NTS) serovars with the model for NTS pathogenesis, S. Typhimurium. Specifically, we used RNA-seq to characterize the understudied NTS serovars S. Javiana and S. Cerro, representing a serovar frequently attributed to human infection via contact with amphibians and reptiles, and a serovar primarily associated with cattle, respectively. Whole-genome sequence (WGS) data were utilized to ensure that strains characterized with RNA-seq were representative of their respective serovars. RNA extracted from representative strains of each serovar grown to late exponential phase in Luria-Bertani (LB) broth showed that transcript abundances of core genes were significantly higher (p<0.001) than those of accessory genes for all three serovars. Inter-serovar comparisons identified that transcript abundances of genes in Salmonella Pathogenicity Island (SPI) 1 were significantly higher in both S. Javiana and S. Typhimurium compared to S. Cerro. Together, our data highlight potential transcriptional mechanisms that may facilitate S. Cerro and S. Javiana survival in and adaptation to their respective hosts and impact their ability to cause disease in others. Furthermore, our analyses demonstrate the utility of omics approaches in advancing our understanding of the diversity of metabolic and virulence mechanisms of different NTS serovars.

Sequence Analyses and Phenotypic Characterization Revealed Multidrug Resistant Gene Insertions in the Genomic Region Encompassing Phase 2 Flagellin Encoding fljAB Genes in Monophasic Variant Salmonella enterica Serovar 4,5,12:i:- Isolates From Various Sources in Thailand.

Salmonella enterica serovar 4,5,12:i:- (S. 4,5,12:i:-), a monophasic variant of Salmonella Typhimurium (STm) lacking the phase 2 flagellin encoding genes fljAB, has become increasingly prevalent worldwide. The increasing trends in multidrug resistant (MDR) S. 4,5,12:i:- prevalence also pose an important global health threat. Though many reports have characterized phenotypic and genotypic drug resistance of this serovar, few studies have characterized antimicrobial resistance of this serovar in Thailand. In this study, 108 S. 4,5,12:i:- isolates from various sources in Thailand and four international S. 4,5,12:i:- isolates were screened using polymerase chain reaction (PCR) to detect the presence of five target regions which are associated with antimicrobial resistant (AMR) genes, in the genomic region that contained fljAB genes in STm. We determined AMR phenotypes of all isolates by Kirby-Bauer disk diffusion method. Whole genome sequencing (WGS) was performed on 53 representative isolates (based on differences in the pulsed filed gel electrophoresis profiles, the sources of isolate, and the PCR and AMR patterns) to characterize the genetic basis of AMR phenotype and to identify the location of AMR determinants. Based on PCR screening, nine PCR profiles showing distinct deletion patterns of the five target regions have been observed. Approximately 76% of isolates (or 85 of 112 isolates), all of which were Thai isolates, contained five target regions inserted between STM2759 and iroB gene. A total of 21 phenotypic AMR patterns were identified with the predominant AmpST resistant phenotype [i.e., 84% (or 94 of 112) tested positive for resistance to ampicillin, streptomycin, and tetracycline], and 89% (or 100 of 112) were found to be MDR (defined here as resistant to at least three classes of tested antimicrobials). Using WGS data, a total of 24 genotypic AMR determinants belonging to seven different antimicrobial groups were found. AMR determinants (i.e., blaTEM - 1 , strB-A, sul2, and tetB, conferring resistance to ampicillin, streptomycin, sulfonamides, and tetracycline, respectively) were found to be inserted in a region typically occupied by the phase 2 flagellin encoding genes in STm. These resistant genes were flanked by a number of insertion sequences (IS), and co-localized with mercury tolerance genes. Our findings identify AMR genes, possibly associated with multiple IS26 copies, in the genetic region between STM2759 and iroB genes replacing phase 2 flagellin encoding fljAB genes in Thai S. 4,5,12:i:- isolates.

Recent Evolution and Genomic Profile of Salmonella enterica Serovar Heidelberg Isolates from Poultry Flocks in Brazil.

Salmonella enterica serovar Heidelberg is isolated from poultry-producing regions around the world. In Brazil, S. Heidelberg has been frequently detected in poultry flocks, slaughterhouses, and chicken meat. The goal of the present study was to assess the population structure, recent temporal evolution, and some important genetic characteristics of S. Heidelberg isolated from Brazilian poultry farms. Phylogenetic analysis of 68 S. Heidelberg genomes sequenced here and additional whole-genome data from NCBI demonstrated that all isolates from the Brazilian poultry production chain clustered into a monophyletic group, here called S. Heidelberg Brazilian poultry lineage (SH-BPL). Bayesian analysis defined the time of the most recent common ancestor (tMRCA) as 2004, and the overall population size (Ne) was constant until 2008, when an ∼10-fold Ne increase was observed until circa 2013. SH-BPL presented at least two plasmids with replicons ColpVC (n = 68; 100%), IncX1 (n = 66; 97%), IncA/C2 (n = 65; 95.5%), ColRNAI (n = 43; 63.2%), IncI1 (n = 32; 47%), ColMG828, Col156, IncHI2A, IncHI2, IncQ1, IncX4, IncY, and TrfA (each with n < 4; <4% each). Antibiotic resistance genes were found, with high frequencies of fosA7 (n = 68; 100%), mdf(A) (n = 68; 100%), tet(34) (n = 68; 100%), sul2 (n = 64; 94.1%), and blaCMY-2 (n = 56; 82.3%), along with an overall multidrug resistance (MDR) profile. Ten Salmonella pathogenicity islands (SPI1 to SPI5, SPI9, and SPI11 to SPI14) and 139 virulence genes were also detected. The SH-BPL profile was like those of other previous S. Heidelberg isolates from poultry around the world in the 1990s. In conclusion, the present study demonstrates the recent introduction (2004) and high level of dissemination of an MDR S. Heidelberg lineage in Brazilian poultry operations. IMPORTANCES. Heidelberg is the most frequent serovar in several broiler farms from the main Brazilian poultry-producing regions. Therefore, avian-source foods (mainly chicken carcasses) commercialized in the country and exported to other continents are contaminated with this foodborne pathogen, generating several national and international economic losses. In addition, isolates of this serovar are usually resistant to antibiotics and can cause human invasive and septicemic infection, representing a public health concern. This study demonstrates the use of whole-genome sequencing (WGS) to obtain epidemiological information for one S. Heidelberg lineage highly spread among Brazilian poultry farms. This information will help to define biosecurity measures to control this important Salmonella serovar in Brazilian and worldwide poultry operations.

Alternative σ Factors Regulate Overlapping as Well as Distinct Stress Response and Metabolic Functions in Listeria monocytogenes under Stationary Phase Stress Condition.

Listeria monocytogenes can regulate and fine-tune gene expression, to adapt to diverse stress conditions encountered during foodborne transmission. To further understand the contributions of alternative sigma (σ) factors to the regulation of L. monocytogenes gene expression, RNA-Seq was performed on L. monocytogenes strain 10403S and five isogenic mutants (four strains bearing in-frame null mutations in three out of four alternative σ factor genes, ΔCHL, ΔBHL, ΔBCL, and ΔBCH, and one strain bearing null mutations in all four genes, ΔBCHL), grown to stationary phase. Our data showed that 184, 35, 34, and 20 genes were positively regulated by σB, σL, σH, and σC (posterior probability > 0.9 and Fold Change (FC) > 5.0), respectively. Moreover, σB-dependent genes showed the highest FC (based on comparisons between the ΔCHL and the ΔBCHL strain), with 44 genes showing an FC > 100; only four σL-dependent, and no σH- or σC-dependent genes showed FC >100. While σB-regulated genes identified in this study are involved in stress-associated functions and metabolic pathways, σL appears to largely regulate genes involved in a few specific metabolic pathways, including positive regulation of operons encoding phosphoenolpyruvate (PEP)-dependent phosphotransferase systems (PTSs). Overall, our data show that (i) σB and σL directly and indirectly regulate genes involved in several energy metabolism-related functions; (ii) alternative σ factors are involved in complex regulatory networks and appear to have epistatic effects in stationary phase cells; and (iii) σB regulates multiple stress response pathways, while σL and σH positively regulate a smaller number of specific pathways.

Identification of Closely Related Listeria monocytogenes Isolates with No Apparent Evidence for a Common Source or Location: A Retrospective Whole Genome Sequencing Analysis.

ABSTRACT: Public health and regulatory agencies worldwide sequence all Listeria monocytogenes isolates obtained as part of routine surveillance and outbreak investigations. Many of these entities submit the sequences to the National Center for Biotechnology Information Pathogen Detection (NCBI PD) database, which groups the L. monocytogenes isolates into single nucleotide polymorphism (SNP) clusters based on a pairwise SNP difference threshold of 50 SNPs. Our goal was to assess whether isolates with metadata that suggest different sources or locations could show evidence for close genetic relatedness indicating a recent common ancestor and a possible unknown common source. We compared the whole genome sequencing (WGS) data of 249 L. monocytogenes isolates sequenced here, which have detailed metadata, with WGS data of nonclinical isolates on NCBI PD. The 249 L. monocytogenes isolates originated from natural environments (n = 91) as well as from smoked fish (n = 62), dairy (n = 56), and deli meat (n = 40) operations in the United States. Using a combination of subtyping by core genome multilocus sequence typing and high-quality SNP, we observed five SNP clusters in which study isolates and SNP cluster isolates seemed to be closely related and either (i) shared the same geolocation but showed different source types (one SNP cluster); (ii) shared the same source type but showed different geolocations (two SNP clusters); or (iii) shared neither source type nor geolocation (two SNP clusters). For one of the two clusters under (iii), there was, however, no strong bootstrap support for a common ancestor shared between the study isolates and SNP cluster isolates, indicating the value of in-depth evolutionary analyses when WGS data are used for traceback and epidemiological investigations. Overall, our results demonstrate that some L. monocytogenes subtypes may be associated with specific locations or commodities; these associations can help in investigations involving multi-ingredient foods such as sandwiches. However, at least some L. monocytogenes subtypes can be widespread geographically and can be associated with different sources, which may present a challenge to traceback investigations involving these subtypes.

Pre-growth conditions and strain diversity affect nisin treatment efficacy against Listeria monocytogenes on cold-smoked salmon.

Listeria monocytogenes is a human pathogen that is commonly found in environments associated with cold-smoked salmon. Nisin is a natural antimicrobial that can be used as a food preservative. While nisin is active against a number of Gram-positive bacteria, including L. monocytogenes, environmental stresses encountered in cold-smoked salmon processing facilities might affect L. monocytogenes' nisin susceptibility. The objective of this study was to investigate the effect of seafood-relevant pre-growth conditions and L. monocytogenes strain diversity on nisin treatment efficacy on cold-smoked salmon. Six L. monocytogenes strains representing serotypes most commonly associated with cold-smoked salmon (1/2a, 1/2b, and 4b) were initially pre-grown under a number of seafood-relevant conditions and challenged with nisin in growth media modified to represent the characteristics of cold-smoked salmon. The pre-growth conditions with the lowest mean log reduction due to nisin and the highest strain-to-strain variability were selected for experiments on cold-smoked salmon; these included: (i) 4.65% w.p. NaCl ("NaCl"); (ii) pH = 6.1 ("pH"); (iii) 0.5 µg/ml benzalkonium chloride ("Quat"); and a control ("BHI"). Cold-smoked salmon slices with or without nisin were inoculated with L. monocytogenes pre-grown in one of the conditions above, vacuum-packed, and incubated at 7 °C. L. monocytogenes were enumerated on days 1, 15, and 30. A linear mixed effects model was constructed to investigate the effect of pre-growth condition, day in storage, serotype, source of isolation as well as their interactions on nisin efficacy against L. monocytogenes. Compared to pre-growth in "BHI", significant reduction (P < 0.05) in nisin efficacy was induced by pre-growth in "pH" and "Quat" on both days 15 and 30, and by pre-growth in "NaCl" on day 30, indicating a time-dependent cross-protection effect. Additionally, an effect of L. monocytogenes' serotype on the cross-protection to nisin was observed; pre-growth in "pH" significantly reduced nisin efficacy against serotype 1/2a and 4b strains, but not against 1/2b strains. In conclusion, pre-exposure to mildly acidic environment, high salt content, and sublethal concentrations of quaternary ammonium compounds, is likely to provide cross-protection against a subsequent nisin treatment of L. monocytogenes on cold-smoked salmon. Therefore, challenge studies that use pre-growth in "BHI", as well as more susceptible L. monocytogenes strains, may overestimate the efficacy of nisin as a control strategy for cold-smoked salmon.