Analysis of Antimicrobial Resistance in Bacterial Pathogens Recovered from Food and Human Sources: Insights from 639,087 Bacterial Whole-Genome Sequences in the NCBI Pathogen Detection Database.

Understanding the role of foods in the emergence and spread of antimicrobial resistance necessitates the initial documentation of antibiotic resistance genes within bacterial species found in foods. Here, the NCBI Pathogen Detection database was used to query antimicrobial resistance gene prevalence in foodborne and human clinical bacterial isolates. Of the 1,843,630 sequence entries, 639,087 (34.7%) were assigned to foodborne or human clinical sources with 147,788 (23.14%) from food and 427,614 (76.88%) from humans. The majority of foodborne isolates were either Salmonella (47.88%), Campylobacter (23.03%), Escherichia (11.79%), or Listeria (11.3%), and the remaining 6% belonged to 20 other genera. Most foodborne isolates were from meat/poultry (95,251 or 64.45%), followed by multi-product mixed food sources (29,892 or 20.23%) and fish/seafood (6503 or 4.4%); however, the most prominent isolation source varied depending on the genus/species. Resistance gene carriage also varied depending on isolation source and genus/species. Of note, Klebsiella pneumoniae and Enterobacter spp. carried larger proportions of the quinolone resistance gene qnrS and some clinically relevant beta-lactam resistance genes in comparison to Salmonella and Escherichia coli. The prevalence of mec in S. aureus did not significantly differ between meat/poultry and multi-product sources relative to clinical sources, whereas this resistance was rare in isolates from dairy sources. The proportion of biocide resistance in Bacillus and Escherichia was significantly higher in clinical isolates compared to many foodborne sources but significantly lower in clinical Listeria compared to foodborne Listeria. This work exposes the gaps in current publicly available sequence data repositories, which are largely composed of clinical isolates and are biased towards specific highly abundant pathogenic species. We also highlight the importance of requiring and curating metadata on sequence submission to not only ensure correct information and data interpretation but also foster efficient analysis, sharing, and collaboration. To effectively monitor resistance carriage in food production, additional work on sequencing and characterizing AMR carriage in common commensal foodborne bacteria is critical.

Modeling the limits of detection for antimicrobial resistance genes in agri-food samples: a comparative analysis of bioinformatics tools.

BACKGROUND: Although the spread of antimicrobial resistance (AMR) through food and its production poses a significant concern, there is limited research on the prevalence of AMR bacteria in various agri-food products. Sequencing technologies are increasingly being used to track the spread of AMR genes (ARGs) in bacteria, and metagenomics has the potential to bypass some of the limitations of single isolate characterization by allowing simultaneous analysis of the agri-food product microbiome and associated resistome. However, metagenomics may still be hindered by methodological biases, presence of eukaryotic DNA, and difficulties in detecting low abundance targets within an attainable sequence coverage. The goal of this study was to assess whether limits of detection of ARGs in agri-food metagenomes were influenced by sample type and bioinformatic approaches. RESULTS: We simulated metagenomes containing different proportions of AMR pathogens and analysed them for taxonomic composition and ARGs using several common bioinformatic tools. Kraken2/Bracken estimates of species abundance were closest to expected values. However, analysis by both Kraken2/Bracken indicated presence of organisms not included in the synthetic metagenomes. Metaphlan3/Metaphlan4 analysis of community composition was more specific but with lower sensitivity than the Kraken2/Bracken analysis. Accurate detection of ARGs dropped drastically below 5X isolate genome coverage. However, it was sometimes possible to detect ARGs and closely related alleles at lower coverage levels if using a lower ARG-target coverage cutoff (< 80%). While KMA and CARD-RGI only predicted presence of expected ARG-targets or closely related gene-alleles, SRST2 (which allows read to map to multiple targets) falsely reported presence of distantly related ARGs at all isolate genome coverage levels. The presence of background microbiota in metagenomes influenced the accuracy of ARG detection by KMA, resulting in mcr-1 detection at 0.1X isolate coverage in the lettuce but not in the beef metagenome. CONCLUSIONS: This study demonstrates accurate detection of ARGs in synthetic metagenomes using various bioinformatic methods, provided that reads from the ARG-encoding organism exceed approximately 5X isolate coverage (i.e. 0.4% of a 40 million read metagenome). While lowering thresholds for target gene detection improved sensitivity, this led to the identification of alternative ARG-alleles, potentially confounding the identification of critical ARGs in the resistome. Further advancements in sequencing technologies providing increased coverage depth or extended read lengths may improve ARG detection in agri-food metagenomic samples, enabling use of this approach for tracking clinically important ARGs in agri-food samples.

Contribution of Food to the Human Health Burden of Antimicrobial Resistance.

The impact of foodborne antimicrobial resistance (AMR) on the human health burden of AMR infections is unknown. The aim of this review was to evaluate and summarize the scientific literature investigating all potential sources of human AMR infections related to food. A literature search was conducted in Embase (Ovid) and MEDLINE (Ovid) databases to identify appropriate studies published between 2010 and 2023. The results of the search were reviewed and categorized based on the primary subject matter. Key concepts from each category are described from the perspective of food safety as a public health objective. The search yielded 3457 references, 1921 remained after removal of duplicates, abstracts, editorials, comments, notes, retractions, and errata. No properly designed source attribution studies were identified, but 383 journal articles were considered relevant and were classified into eight subcategories and discussed in the context of four streams of evidence: prevalence data, epidemiological studies, outbreak investigations and human health impact estimates. There was sufficient evidence to conclude that AMR genes, whether present in pathogenic or nonpathogenic bacteria, constitute a foodborne hazard. The level of consumer risk owing to this hazard cannot be accurately estimated based on the data summarized here. Key gaps in the literature are noted.

Targeted metagenomics using bait-capture to detect antibiotic resistance genes in retail meat and seafood.

Metagenomics analysis of foods has the potential to provide comprehensive data on the presence and prevalence of antimicrobial resistance (AMR) genes in the microbiome of foods. However, AMR genes are generally present in low abundance compared to other bacterial genes in the food microbiome and consequently require multiple rounds of in-depth sequencing for detection. Here, a metagenomics approach, using bait-capture probes targeting antimicrobial resistance and plasmid genes, is used to characterize the resistome and plasmidome of retail beef, chicken, oyster, shrimp, and veal enrichment cultures (n = 15). Compared to total shotgun metagenomics, bait-capture required approximately 40-fold fewer sequence reads to detect twice the number of AMR gene classes, AMR gene families, and plasmid genes across all sample types. For the detection of critically important extended spectrum beta-lactamase (ESBL) genes the bait capture method had a higher overall positivity rate (44%) compared to shotgun metagenomics (26%), and a culture-based method (29%). Overall, the results support the use of bait-capture for the identification of low abundance genes such as AMR genes from food samples.

Salmonella enterica Outbreaks Linked to the Consumption of Tahini and Tahini-Based Products

Salmonella is a leading cause of bacterial foodborne illness in the world. Although typically associated with foods of animal origin, low-moisture foods, such as tahini, are quickly gaining recognition as an important vehicle of Salmonella exposure. This review offers the Canadian perspective on the issue of Salmonella in tahini and tahini-based products. A summary of several recent food product recalls and foodborne outbreaks related to the presence of Salmonella in tahini and tahini-based products such as halva are presented. The properties of the food vehicles, their production practices, and potential routes of contamination are discussed. Particular focus is placed on the ecology of Salmonella in the tahini production continuum, including its survival characteristics and response to intervention technologies.

Choice of DNA extraction method affects detection of bacterial taxa from retail chicken breast.

BACKGROUND: Sequence-based methods for the detection of bacteria such as 16S rRNA amplicon sequencing and metagenomics can provide a comprehensive view of the bacterial microbiome of food. These methods rely on the detection of gene sequences to indicate the presence of viable bacteria. This indirect form of detection can be prone to experimental artefacts. Sample handling and processing are key sources of variation that require standard approaches. Extracting sufficient quantities of high quality DNA from food matrices is challenging because target bacterial species are usually minor components of the microbiota and foods contain an array of compounds that are inhibitory to downstream DNA applications. Here, three DNA extraction methods are compared for their ability to extract high quality bacterial DNA from retail chicken breast rinses, with or without enrichment. Method performance was assessed by comparing ease of use, DNA yield, DNA quality, PCR amplicon yield, and the detection of bacterial taxa by 16S rRNA amplicon sequencing. RESULTS: All three DNA extraction methods yielded DNA of sufficient quantity and quality to perform quantitative PCR and 16S rRNA amplicon sequencing. The extraction methods differed in ease of use, with the two commercial kits (PowerFood, PowerSoil) offering considerable time and cost savings over a hybrid method that used laboratory reagents for lysis and commercial column based kits for further purification. Bacterial richness as determined by 16S rRNA amplicon sequencing was similar across the three DNA extraction methods. However, differences were noted in the relative abundance of bacterial taxa, with significantly higher abundance of Gram-positive genera detected in the DNA samples prepared using the PowerFood DNA extraction kit. CONCLUSION: The choice of DNA extraction method can affect the detection of bacterial taxa by 16S rRNA amplicon sequencing in chicken meat rinses. Investigators should be aware of this procedural bias and select methods that are fit for the purposes of their investigation.

Phylogenomic Analysis of Salmonella enterica subsp. enterica Serovar Bovismorbificans from Clinical and Food Samples Using Whole Genome Wide Core Genes and kmer Binning Methods to Identify Two Distinct Polyphyletic Genome Pathotypes

Salmonella enterica subsp. enterica serovar Bovismorbificans has caused multiple outbreaks involving the consumption of produce, hummus, and processed meat products worldwide. To elucidate the intra-serovar genomic structure of S. Bovismorbificans, a core-genome analysis with 2690 loci (based on 150 complete genomes representing Salmonella enterica serovars developed as part of this study) and a k-mer-binning based strategy were carried out on 95 whole genome sequencing (WGS) assemblies from Swiss, Canadian, and USA collections of S. Bovismorbificans strains from foodborne infections. Data mining of a digital DNA tiling array of legacy SARA and SARB strains was conducted to identify near-neighbors of S. Bovismorbificans. The core genome analysis and the k-mer-binning methods identified two polyphyletic clusters, each with emerging evolutionary properties. Four STs (2640, 142, 1499, and 377), which constituted the majority of the publicly available WGS datasets from >260 strains analyzed by k-mer-binning based strategy, contained a conserved core genome backbone with a different evolutionary lineage as compared to strains comprising the other cluster (ST150). In addition, the assortment of genotypic features contributing to pathogenesis and persistence, such as antimicrobial resistance, prophage, plasmid, and virulence factor genes, were assessed to understand the emerging characteristics of this serovar that are relevant clinically and for food safety concerns. The phylogenomic profiling of polyphyletic S. Bovismorbificans in this study corresponds to intra-serovar variations observed in S. Napoli and S. Newport serovars using similar high-resolution genomic profiling approaches and contributes to the understanding of the evolution and sequence divergence of foodborne Salmonellae. These intra-serovar differences may have to be thoroughly understood for the accurate classification of foodborne Salmonella strains needed for the uniform development of future food safety mitigation strategies.

Genomic and phenotypic analysis of SspH1 identifies a new Salmonella effector, SspH3.

Salmonella is a major foodborne pathogen and is responsible for a range of diseases. Not all Salmonella contributes to severe health outcomes as there is a large degree of genetic heterogeneity among the 2,600 serovars within the genus. This variability across Salmonella serovars is linked to numerous genetic elements that dictate virulence. While several genetic elements encode virulence factors with well-documented contributions to pathogenesis, many genetic elements implicated in Salmonella virulence remain uncharacterized. Many pathogens encode a family of E3 ubiquitin ligases that are delivered into the cells that they infect using a Type 3 Secretion System (T3SS). These effectors, known as NEL-domain E3s, were first characterized in Salmonella. Most Salmonella encodes the NEL-effectors sspH2 and slrP, whereas only a subset of Salmonella encodes sspH1. SspH1 has been shown to ubiquitinate the mammalian protein kinase PKN1, which has been reported to negatively regulate the pro-survival program Akt. We discovered that SspH1 mediates the degradation of PKN1 during infection of a macrophage cell line but that this degradation does not impact Akt signaling. Genomic analysis of a large collection of Salmonella genomes identified a putative new gene, sspH3, with homology to sspH1. SspH3 is a novel NEL-domain effector.

Isolation of third generation cephalosporin resistant Enterobacteriaceae from retail meats and detection of extended spectrum beta-lactamase activity.

Various methods have been described to isolate third generation cephalosporin (3GC) resistant Enterobacteriaceae from foods, but it is not known how comparable they are between studies. Here, the performance of five enrichment broths and two selective agars are compared for their ability to isolate 3GC resistant Enterobacteriaceae from retail chicken, beef, pork, and veal samples. The results showed equivalence between Enterobacteriaceae enrichment broth (EE), lauryl sulfate broth (LST), and modified typtone soy broth (mTSB). Lower isolation rates were observed when LST and mTSB were supplemented with the 3GC antibiotic cefotaxime. The overall performance of MacConkey agar supplemented with cefotaxime and a proprietary selective agar (ESBL CHROMagar) was equivalent, although differences linked to the microbiota of specific meat commodities were noted. Regardless of the isolation method, further screening was required to confirm the taxonomy and resistance of the presumptive positive strains. Approximately 40% of confirmed 3GC resistant foodborne Enterobacteriaceae strains tested positive for extended spectrum beta-lactamase (ESBL) activity. Strains that were resistant to ceftriaxone and susceptible to cefoxitin were more likely to test positive for ESBL activity, as were strains that possessed either of two ESBL genes (blaSHV or blaTEM). Based on our results, we recommend using an antibiotic-free enrichment broth, two selective agars, and an isolate screening strategy to isolate 3GC resistant Enterobacteriaceae from retail meats. Antibiotic susceptibility testing and/or PCR screening for blaSHV or blaTEM can then be used to identify ESBL producing strains among the 3GC resistant meat isolates. The adoption of this approach by the research community will enable more effective monitoring of antibiotic resistance rates and trends among foodborne Enterobacteriaceae over time and across jurisdictions.

Microbiological analysis of frozen profiteroles and mini chocolate eclairs implicated in a national salmonellosis outbreak.

Between November 2018 and May 2019, Canada experienced a nationwide salmonellosis outbreak linked to the presence of Salmonella enterica ser. Enteritidis in frozen profiteroles. Analysis of the implicated food products revealed low levels of Salmonella ranging from 0.2 to 0.7 MPN/100g. Water activity and pH of the food samples ranged from 0.9479 to 0.9867 and 4.6-6.8 respectively indicating conditions conducive to bacterial growth. Higher levels of the hygiene indicators Enterobacteriaceae and coliforms were associated with Salmonella positive samples compared to Salmonella negative samples. Investigation of the relationship between storage conditions, temperature, and pathogen levels during thawing revealed that the profiteroles reached temperatures permissive to pathogen growth (≥5 °C) much sooner than pathogen growth was observed and that the composition of the food matrix can influence bacterial levels upon thawing. Collectively these data can be used to inform guidance to minimize the risk of infection from the consumption of contaminated cream-filled frozen desserts.

Salmonella enterica serovars associated with bacteremia in Canada, 2006-2019.

BACKGROUND: Members of the bacterial genus Salmonella cause salmonellosis, a disease with a spectrum of clinical presentations from a self-limiting gastroenteritis to more severe bacteremia, organ failure and sepsis. The genus consists of over 2,600 serological variants (serovars). Important differences in the pathogenesis of Salmonella serovars have been noted. OBJECTIVE: The purpose of this study was to determine which Salmonella serovars were more likely to be associated with bacteremia in Canada. METHODS: Information on the total number of Salmonella infections and blood isolations reported to the National Enteric Surveillance Program (NESP) from 2006 to 2019 was extracted for each serovar. The risk (proportion) and likelihood (odds) of bacteremia were calculated for all serovars. RESULTS: Of the 96,082 Salmonella cases reported to the NESP during the 14-year study period, 4.4% (95% CI: 4.3%-4.6%) were bacteremic. Twenty nontyphoidal Salmonella (NTS) serovars were associated with lower rates of bacteremia compared to all NTS serovars, and 19 NTS serovars were identified as having higher rates. Heidelberg, Oranienburg, Schwarzengrund, Virchow, Panama and Poona among the top 25 most commonly reported serovars in Canada during the study period. CONCLUSION: The identification of serovars associated with Salmonella bacteremia in Canada is a first step towards understanding differences in pathogenesis and disease presentation.

The Salmonella enterica Plasmidome as a Reservoir of Antibiotic Resistance.

The emergence of multidrug-resistant bacterial strains worldwide has become a serious problem for public health over recent decades. The increase in antimicrobial resistance has been expanding via plasmids as mobile genetic elements encoding antimicrobial resistance (AMR) genes that are transferred vertically and horizontally. This study focuses on Salmonella enterica, one of the leading foodborne pathogens in industrialized countries. S. enterica is known to carry several plasmids involved not only in virulence but also in AMR. In the current paper, we present an integrated strategy to detect plasmid scaffolds in whole genome sequencing (WGS) assemblies. We developed a two-step procedure to predict plasmids based on i) the presence of essential elements for plasmid replication and mobility, as well as ii) sequence similarity to a reference plasmid. Next, to confirm the accuracy of the prediction in 1750 S. enterica short-read sequencing data, we combined Oxford Nanopore MinION long-read sequencing with Illumina MiSeq short-read sequencing in hybrid assemblies for 84 isolates to evaluate the proportion of plasmid that has been detected. At least one scaffold with an origin of replication (ORI) was predicted in 61.3% of the Salmonella isolates tested. The results indicated that IncFII and IncI1 ORIs were distributed in many S. enterica serotypes and were the most prevalent AMR genes carrier, whereas IncHI2A/IncHI2 and IncA/C2 were more serotype restricted but bore several AMR genes. Comparison between hybrid and short-read assemblies revealed that 81.1% of plasmids were found in the short-read sequencing using our pipeline. Through this process, we established that plasmids are prevalent in S. enterica and we also substantially expand the AMR genes in the resistome of this species.

Similar yet different: phylogenomic analysis to delineate Salmonella and Citrobacter species boundaries.

BACKGROUND: Salmonella enterica is a leading cause of foodborne illness worldwide resulting in considerable public health and economic costs. Testing for the presence of this pathogen in food is often hampered by the presence of background microflora that may present as Salmonella (false positives). False positive isolates belonging to the genus Citrobacter can be difficult to distinguish from Salmonella due to similarities in their genetics, cell surface antigens, and other phenotypes. In order to understand the genetic basis of these similarities, a comparative genomic approach was used to define the pan-, core, accessory, and unique coding sequences of a representative population of Salmonella and Citrobacter strains. RESULTS: Analysis of the genomic content of 58 S. enterica strains and 37 Citrobacter strains revealed the presence of 31,130 and 1540 coding sequences within the pan- and core genome of this population. Amino acid sequences unique to either Salmonella (n = 1112) or Citrobacter (n = 195) were identified and revealed potential niche-specific adaptations. Phylogenetic network analysis of the protein families encoded by the pan-genome indicated that genetic exchange between Salmonella and Citrobacter may have led to the acquisition of similar traits and also diversification within the genera. CONCLUSIONS: Core genome analysis suggests that the Salmonella enterica and Citrobacter populations investigated here share a common evolutionary history. Comparative analysis of the core and pan-genomes was able to define the genetic features that distinguish Salmonella from Citrobacter and highlight niche specific adaptations.

Systematic Evaluation of Whole Genome Sequence-Based Predictions of Salmonella Serotype and Antimicrobial Resistance.

Whole-genome sequencing (WGS) is used increasingly in public-health laboratories for typing and characterizing foodborne pathogens. To evaluate the performance of existing bioinformatic tools for in silico prediction of antimicrobial resistance (AMR) and serotypes of Salmonella enterica, WGS-based genotype predictions were compared with the results of traditional phenotyping assays. A total of 111 S. enterica isolates recovered from a Canadian baseline study on broiler chicken conducted in 2012-2013 were selected based on phenotypic resistance to 15 different antibiotics and isolates were subjected to WGS. Both SeqSero2 and SISTR accurately determined S. enterica serotypes, with full matches to laboratory results for 87.4 and 89.2% of isolates, respectively, and partial matches for the remaining isolates. Antimicrobial resistance genes (ARGs) were identified using several bioinformatics tools including the Comprehensive Antibiotic Resistance Database - Resistance Gene Identifier (CARD-RGI), Center for Genomic Epidemiology (CGE) ResFinder web tool, Short Read Sequence Typing for Bacterial Pathogens (SRST2 v 0.2.0), and k-mer alignment method (KMA v 1.17). All ARG identification tools had ≥ 99% accuracy for predicting resistance to all antibiotics tested except streptomycin (accuracy 94.6%). Evaluation of ARG detection in assembled versus raw-read WGS data found minimal observable differences that were gene- and coverage- dependent. Where initial phenotypic results indicated isolates were sensitive, yet ARGs were detected, repeat AMR testing corrected discrepancies. All tools failed to find resistance-determining genes for one gentamicin- and two streptomycin-resistant isolates. Further investigation found a single nucleotide polymorphism (SNP) in the nuoF coding region of one of the isolates which may be responsible for the observed streptomycin-resistant phenotype. Overall, WGS-based predictions of AMR and serotype were highly concordant with phenotype determination regardless of computational approach used.

Effectiveness of Preparation Practices on the Inactivation of Salmonella enterica Serovar Enteritidis in Frozen Breaded Chicken Strips.

ABSTRACT: Over the past 15 years, multiple foodborne outbreaks have occurred in Canada due to the presence of Salmonella enterica in frozen breaded chicken products. These chicken products were raw and required cooking in conventional household ovens to inactivate any pathogens that they may have been harboring. During the course of food safety investigations associated with these outbreaks, many consumers reported using alternative household appliances such as air fryers to cook these products. The effectiveness of these appliances for the inactivation of pathogens in food is not known. Here, we compare the ability of a toaster oven, air fryer, deep fryer, and conventional oven to inactivate a cocktail of Salmonella Enteritidis in frozen breaded chicken strips. Deep frying was the most effective cooking method, demonstrating a median 7-log reduction; the conventional oven was next with a median 6-log reduction. Both the air fryer and toaster oven performed poorly, with respective median 4- and 3-log reductions. Overall, the results of this study suggest the revision of cooking instructions is required for the safe household use of toaster ovens and air fryers.

A Survey of Raw Frozen Breaded Chicken Products for Salmonella in British Columbia, Canada, and Phylogenetically Associated Illnesses.

ABSTRACT: The incidence of Salmonella enterica infection resulting from consumption of chicken products has historically been elevated in British Columbia compared with the rest of Canada. Raw frozen breaded chicken products are often implicated as the source of infection as there is a potential for consumers to not cook these products adequately. This occurs because the production process for these foods involves par-frying, a step which lends a cooked appearance to the product surface without reaching the internal temperatures required to fully inactivate potential pathogens. A survey of frozen chicken products from 10 retail stores of various sizes was conducted in order to determine the type and source of frozen chicken products that are available for purchase in British Columbia. Information on 391 individual products was collected and 50 were sampled for microbiological testing. Raw frozen breaded chicken products represented 59% of the frozen chicken products available to consumers at retail; 34% of these raw products were made by a single processor. The same processor was also found to have the highest proportion (33%) of samples testing positive for Salmonella. Whole genome sequencing of isolates obtained during this study revealed that majority of these isolates were phylogenetically related to clinical isolates of Salmonella. A substantial reduction of risk and increased consumer protection may be achieved by implementing a kill step (e.g., cook process that has been validated to achieve a 7-log reduction) during production of these products.

Enumeration and Survival of Salmonella enterica in Live Oyster Shellstock Harvested from Canadian Waters.

Since 2015, 11 recalls of live oyster shellstock have been issued in Canada due to the presence of Salmonella enterica. Six of those recalls took place in 2018. To understand this increase, fundamental information is needed on the relationship between S. enterica and oysters. The aims of this study were to address important data gaps concerning the levels of Salmonella in naturally contaminated oysters and the ability of this pathogen to survive in live oyster shellstock. Enumeration data were evaluated for five oyster and clam samples collected from the east coast of Canada from 2015 to 2018. The reported levels were <0.0015 to 0.064 most probable number per g of oyster tissue. The S. enterica isolates recovered from these animals belonged to serovars Typhimurium, Infantis, Enteritidis, and I 4,5:i:-. Filter feeding by the oysters was exploited to assess the Salmonella accumulation that would occur following a natural contamination event. Detectable levels of the pathogen were observed after 30 min of exposure and began to plateau at 60 min. A survival study in live oyster shellstock indicated that after 4 days of storage at ambient temperatures, the Salmonella level declined slightly from 4.3 to 3.7 log CFU/g. These data indicate that the levels of Salmonella found in naturally contaminated oysters are low and are not expected to increase between the point of harvest and the point of consumption. The changing ecology of shellfish environments requires continued monitoring and testing to safeguard public health. The data presented here will be useful for the evaluation and design of sampling plans and risk management approaches for the control of Salmonella in live oyster shellstock.

Changes detected in the genome sequences of Escherichia coli, Listeria monocytogenes, Vibrio parahaemolyticus, and Salmonella enterica after serial subculturing.

Whole genome sequencing (WGS) is rapidly replacing other molecular techniques for identifying and subtyping bacterial isolates. The resolution or discrimination offered by WGS is significantly higher than that offered by other molecular techniques, and WGS readily allows infrequent differences that occur between 2 closely related strains to be found. In this investigation, WGS was used to identify the changes that occurred in the genomes of 13 strains of bacterial foodborne pathogens after 100 serial subcultures. Pure cultures of Shiga-toxin-producing Escherichia coli, Salmonella enterica, Listeria monocytogenes, and Vibrio parahaemolyticus were subcultured daily for 100 successive days. The 1st and 100th subcultures were whole-genome sequenced using short-read sequencing. Single nucleotide polymorphisms (SNPs) were identified between the 1st and final culture using 2 different approaches, and multilocus sequence typing of the whole genome was also performed to detect any changes at the allelic level. The number of observed genomic changes varied by strain, species, and the SNP caller used. This study provides insight into the genomic variation that can be detected using next-generation sequencing and analysis methods after repeated subculturing of 4 important bacterial pathogens.

Relative response of populations of Escherichia coli and Salmonella enterica to exposure to thermal, alkaline and acidic treatments.

We evaluated the relative response of generic Escherichia coli (GEC), Shiga toxin-producing E. coli (STEC) and Salmonella enterica to heat, alkaline or acid treatments. GEC included strains from carcasses (n = 24) and trim (n = 25) at a small beef plant where no decontamination interventions are used and at a large plant where multiple decontamination interventions are used (carcass n = 25 and trim n = 25). STEC strains belonging to nine serogroups, included isolates from cattle (n = 53), beef (n = 16) and humans (n = 44). S. enterica strains belonging to 29 serotypes, included isolates from humans (n = 30), poultry (n = 26), pork (n = 10) and beef (n = 33). Strains were grown in Brain Heart Infusion (BHI) broth and subjected to the following treatments: 60 °C for 2 min, 5% lactic acid (pH 2.9) for 1 h at 4 °C, or NaOH (pH 11.0) for 2 h at 4 °C. Median log reductions of the GEC populations after heat, alkaline and acid treatment ranged from 2.3 to 3.8, 0.7 to 2.2 and 0.7 to 1.2 log CFU/mL, respectively. No statistically significant difference in reductions was observed between carcass GEC or trim GEC from the large or small plant, except for a greater reduction in trim GEC from the small plant. Median reductions of the STEC populations ranged from 3.3 to 3.5, 0.0 to 0.6, and 0.3 to 0.5 log CFU/mL after heat, alkaline and acid treatment, respectively. The median reductions were not dependent upon isolation source, except between STEC cattle and human isolates after alkaline treatment, where the reduction of the former was higher by 0.6 log unit. For the Salmonella populations, median log reductions ranged from 3.5 to 4.0, 1.7 to 2.4 and 3.7 to 4.1 log CFU/mL after heat, alkaline and acid treatment, respectively. The reductions were not isolation source related. The median log reductions were in the order GEC < STEC < Salmonella after heat treatment and STEC < GEC < Salmonella after alkaline or acid treatment. Overall, the relative response of GEC, STEC and Salmonella in the model system suggests that exposure to heat or pH-based decontamination interventions in meat plants is not associated with increased resistance among E. coli strains in these environments, and total E. coli counts on beef can be indicative of treatment efficacy for the control of Salmonella by heat, lactic acid and alkaline treatment and for the control of STEC subjected to heat.

Biofilm-Forming Capacity of Five Salmonella Strains and Their Fate on Postharvest Mini Cucumbers.

Salmonella enterica is one of the pathogens that is frequently identified as the cause of fresh produce-related outbreaks. Biofilm formation is a factor that can contribute to pathogen survival on produce surface. The goal of our current research was to investigate the survival of five S. enterica strains representing different serotypes (i.e., Typhimurium, Enteritidis, Daytona, Poona, and Newport) on whole mini cucumbers stored at refrigeration (4°C) and room temperature (22°C). We also determined the strains survival on glass slides and in phosphate-buffered saline at 4 and 22°C, as well as the ability to form biofilms on a solid-liquid interphase. A rapid decrease in cell density (>4-log reduction over 8 days) of all five tested strains was observed on glass slides, while a slower die-off (<1-log reduction in 8 days) was observed in PBS. No significant difference in the die-off rate was observed among the five strains at 4 or 22°C. The die-off rate on the surface of mini cucumbers at 4°C was significantly slower ( P < 0.02) for Salmonella Enteritidis LMFS-S-JF-005 compared with the remaining four strains. At 22°C, Salmonella Poona S306 was able to grow by more than 1.5 log units on whole mini cucumbers over a period of 8 days, while the cell density of the other four strains remained at the same level compared with day 0. At this temperature, Salmonella Poona S306 was also able to form significantly stronger biofilms on a solid-liquid interphase ( P < 0.01) and was the only strain that presented a red, dry, and rough morphotype on Congo red agar plates, indicating the formation of both curli fimbriae and cellulose. These results revealed that the fate of Salmonella on mini cucumbers is strain specific, which highlighted the need for tailored mitigation strategies, such as the effective control of temperature and moisture for limiting the survival or growth of high-risk Salmonella strains between harvest and consumption of fresh produce.