Evaluation of methods for identifying poultry wing rinses with Salmonella concentrations greater than or equal to 10 CFU/mL.

In the United States, the Proposed Regulatory Framework to Reduce Salmonella Illnesses Attributable to Poultry published by the Food Safety and Inspection Service (FSIS) has highlighted the need for simple, rapid methods that identify poultry wing rinse samples harboring Salmonella concentrations ≥ 10 CFU/mL. One of eight cold-stressed and nutrient starved Salmonella strains were inoculated into post-chill two-joint poultry wing rinses (48 turkey and 72 chicken) at levels from 0.22 to 3.79 log CFU/mL; and then measured by 3-tube Most Probable Number (MPN), BioMerieux GENE-UP QUANT, Hygiena BAX SalQuant, and novel threshold methods. The MPN lower limit of quantification (LLQ) for Salmonella was -0.96 log CFU/mL. MPN overestimated the inoculated Salmonella level by 0.05 ± 0.35 log CFU/mL. The GENE-UP QUANT Salmonella method (LLQ = 1.00 log CFU/mL) underestimated the inoculated Salmonella level by 0.05 ± 0.51 log CFU/mL. The BAX SalQuant method (LLQ = 0.00 log CFU/mL) underestimated the inoculated Salmonella level by 1.21 ± 0.78 log CFU/mL. Threshold test methods with Poisson probabilities of 0.95 (PiLOT-95), 0.86 (PiLOT-86), 0.63 (PiLOT-63), and 0.50 (PiLOT-50) were developed to identify poultry wing rinses harboring Salmonella levels ≥ 10 CFU. MPN was 93.1%, accurate for determining if Salmonella levels in poultry wing rinses were ≥ 10 CFU/mL, but MPN costs and time requirements can be prohibitive for most laboratories. GENE-UP quantification was 86.1% accurate, but the GENE-UP method requires equipment and technical expertise that some food safety laboratories may not possess. BAX quantification had the lowest accuracy, 58.4%. PiLOT threshold test accuracies ranged from 83.2% for PiLOT-50 to 93.1% for PiLOT-86. The PiLOT threshold tests are simple and can be adapted to identify many environmental or food samples containing Salmonella exceeding any user defined concentration threshold.

Digital PCR assay for the specific detection and estimation of Salmonella contamination levels in poultry rinse.

Strains of Salmonella are a frequent cause of foodborne illness and are known to contaminate poultry products. Most Salmonella testing methods can qualitatively detect Salmonella and cannot quantify or estimate the Salmonella load in samples. Therefore, the aim of this study was to standardize and validate a partitioned-based digital PCR (dPCR) assay for the detection and estimation of Salmonella contamination levels in poultry rinses. Pure culture Salmonella strains were cultured, enumerated, cold-stressed for 48 h, and used to inoculate whole carcass chicken rinse (WCCR) at 1-4 log CFU/30 mL and enriched at 37 °C for 5 h. Undiluted DNA samples with primer and probes targeting the Salmonella-specific invA gene were used for the dPCR assay. The dPCR assay was highly specific, with a limit of detection of 0.001 ng/µL and a limit of quantification of 0.01 ng/µL. The dPCR assay further showed no PCR reaction inhibition up to 5 µg of crude DNA extract. The assays accurately detected all cold-stressed Salmonella in inoculated WCCR samples following a 5-h enrichment. Most importantly, when converted to log, the dPCR copies/µL values accurately estimated the inoculated Salmonella levels. The dPCR assay standardized in this study is a robust method for the detection and estimation of Salmonella concentration in contaminated food samples. This approach can allow same-day decision-making for poultry processors attempting to maintain limits and controls on Salmonella contamination.

Impact of intense sanitization procedures on bacterial communities recovered from floor drains in pork processing plants.

Background: Pork processing plants in the United States (US) cease operations for 24-48 h every six or twelve months to perform intense sanitization (IS) using fogging, foaming, and further antimicrobial treatments to disrupt natural biofilms that may harbor pathogens and spoilage organisms. The impact such treatments have on short-term changes in environmental microorganisms is not well understood, nor is the rate at which bacterial communities return. Methods: Swab samples were collected from floor drains to provide representative environmental microorganisms at two US pork processing plants before, during, and after an IS procedure. Samples were collected from four coolers where finished carcasses were chilled and from four locations near cutting tables. Each sample was characterized by total mesophile count (TMC), total psychrophile count (TPC), and other indicator bacteria; their biofilm-forming ability, tolerance of the formed biofilm to a quaternary ammonium compound (300 ppm, QAC), and ability to protect co-inoculated Salmonella enterica. In addition, bacterial community composition was determined using shotgun metagenomic sequencing. Results: IS procedures disrupted bacteria present but to different extents depending on the plant and the area of the plant. IS reduced TPC and TMC, by up to 1.5 Log10 CFU only to return to pre-IS levels within 2-3 days. The impact of IS on microorganisms in coolers was varied, with reductions of 2-4 Log10, and required 2 to 4 weeks to return to pre-IS levels. The results near fabrication lines were mixed, with little to no significant changes at one plant, while at the other, two processing lines showed 4 to 6 Log10 reductions. Resistance to QAC and the protection of Salmonella by the biofilms varied between plants and between areas of the plants as well. Community profiling of bacteria at the genus level showed that IS reduced species diversity and the disruption led to new community compositions that in some cases did not return to the pre-IS state even after 15 to 16 weeks. Discussion: The results found here reveal the impact of using IS to disrupt the presence of pathogen or spoilage microorganisms in US pork processing facilities may not have the intended effect.

Antibiotic Resistance and Disinfectant Resistance Among Escherichia coli Isolated During Red Meat Production.

Escherichia coli commonly found in the gastrointestinal tracts of food animals include Shiga toxin-producing E. coli (STEC, stx+, eae-), Enterohemorrhagic E. coli (EHEC, stx+, eae+), Enteropathogenic E. coli (EPEC, stx-, eae+), and "nondiarrheagenic" E. coli (NDEC, stx-, eae-). EHEC, EPEC, and STEC are associated with foodborne disease outbreaks. During meat processing, disinfectants are employed to control various bacteria, including human pathogens. Concerns exist that E. coli resistant to antibiotics are less susceptible to disinfectants used during meat processing. Since EHEC, EPEC, and STEC with reduced susceptibility to disinfectants are potential public health risks, the goal of this study was to evaluate the association of antibiotic resistant (ABR) E. coli with increased tolerance to 4% lactic acid (LA) and 150 ppm quaternary ammonium compounds (QACs). A pool of 3,367 E. coli isolated from beef cattle, veal calves, swine, and sheep at various processing stages was screened to identify ABR E. coli. Resistance to ≥1 of the six antibiotics examined was identified in 27.9%, 36.1%, 54.5%, and 28.7% among the NDEC (n = 579), EHEC (n = 693), EPEC (n = 787), and STEC (n = 1308) isolates evaluated, respectively. Disinfectant tolerance did not differ (P > 0.05) between ABR and antibiotic susceptible EHEC isolates. Comparable frequencies (P > 0.05) of biofilm formation or congo red binding were observed between ABR and antibiotic susceptible strains of E. coli. Understanding the frequencies of ABR and disinfectant tolerance among E. coli present in food-animal is a critically important component of meat safety.

Pathogenomes and virulence profiles of representative big six non-O157 serogroup Shiga toxin-producing Escherichia coli.

Shiga toxin (Stx)-producing Escherichia coli (STEC) of non-O157:H7 serotypes are responsible for global and widespread human food-borne disease. Among these serogroups, O26, O45, O103, O111, O121, and O145 account for the majority of clinical infections and are colloquially referred to as the "Big Six." The "Big Six" strain panel we sequenced and analyzed in this study are reference type cultures comprised of six strains representing each of the non-O157 STEC serogroups curated and distributed by the American Type Culture Collection (ATCC) as a resource to the research community under panel number ATCC MP-9. The application of long- and short-read hybrid sequencing yielded closed chromosomes and a total of 14 plasmids of diverse functions. Through high-resolution comparative phylogenomics, we cataloged the shared and strain-specific virulence and resistance gene content and established the close relationship of serogroup O26 and O103 strains featuring flagellar H-type 11. Virulence phenotyping revealed statistically significant differences in the Stx-production capabilities that we found to be correlated to the strain's individual stx-status. Among the carried Stx1a, Stx2a, and Stx2d phages, the Stx2a phage is by far the most responsive upon RecA-mediated phage mobilization, and in consequence, stx2a + isolates produced the highest-level of toxin in this panel. The availability of high-quality closed genomes for this "Big Six" reference set, including carried plasmids, along with the recorded genomic virulence profiles and Stx-production phenotypes will provide a valuable foundation to further explore the plasticity in evolutionary trajectories in these emerging non-O157 STEC lineages, which are major culprits of human food-borne disease.

Proportions and Serogroups of Enterohemorrhagic Shiga Toxin-producing Escherichia coli in Feces of Fed and Cull Beef and Cull Dairy Cattle at Harvest.

Cattle are considered a primary reservoir of Shiga toxin (stx)-producing Escherichia coli that cause enterohemorrhagic disease (EHEC), and contaminated beef products are one vehicle of transmission to humans. However, animals entering the beef harvest process originate from differing production systems: feedlots, dairies, and beef breeding herds. The objective of this study was to determine if fed cattle, cull dairy, and or cull beef cattle carry differing proportions and serogroups of EHEC at harvest. Feces were collected via rectoanal mucosal swabs (RAMSs) from 1,039 fed cattle, 1,058 cull dairy cattle, and 1,018 cull beef cattle at harvest plants in seven U.S. states (CA, GA, NE, PA, TX, WA, and WI). The proportion of the stx gene in feces of fed cattle (99.04%) was not significantly different (P > 0.05) than in the feces of cull dairy (92.06%) and cull beef (91.85%) cattle. When two additional factors predictive of EHEC (intimin and ecf1 genes) were considered, EHEC was significantly greater (P < 0.05) in fed cattle (77.29%) than in cull dairy (47.54%) and cull beef (38.51%) cattle. The presence of E. coli O157:H7 and five common non-O157 EHEC of serogroups O26, O103, O111, O121, and O145 was determined using molecular analysis for single nucleotide polymorphisms (SNPs) followed by culture isolation. SNP analysis identified 23.48%, 17.67%, and 10.81% and culture isolation confirmed 2.98%, 3.31%, and 3.00% of fed, cull dairy, and cull beef cattle feces to contain one of these EHEC, respectively. The most common serogroups confirmed by culture isolation were O157, O103, and O26. Potential EHEC of fourteen other serogroups were isolated as well, from 4.86%, 2.46%, and 2.01% of fed, cull dairy, and cull beef cattle feces, respectively; with the most common being serogroups O177, O74, O98, and O84. The identification of particular EHEC serogroups in different types of cattle at harvest may offer opportunities to improve food safety risk management.

Pathogenomes of Shiga Toxin Positive and Negative Escherichia coli O157:H7 Strains TT12A and TT12B: Comprehensive Phylogenomic Analysis Using Closed Genomes.

Shiga toxin-producing Escherichia coli are zoonotic pathogens that cause food-borne human disease. Among these, the O157:H7 serotype has evolved from an enteropathogenic O55:H7 ancestor through the displacement of the somatic gene cluster and recurrent toxigenic conversion by Shiga toxin-converting bacteriophages. However, atypical strains that lack the Shiga toxin, the characteristic virulence hallmark, are circulating in this lineage. For this study, we analyzed the pathogenome and virulence inventories of the stx+ strain, TT12A, isolated from a patient with hemorrhagic colitis, and its respective co-isolated stx- strain, TT12B. Sequencing the genomes to closure proved critical to the cataloguing of subtle strain differentiating sequence and structural polymorphisms at a high-level of phylogenetic accuracy and resolution. Phylogenomic profiling revealed SNP and MLST profiles similar to the near clonal outbreak isolates. Their prophage inventories, however, were notably different. The attenuated atypical non-shigatoxigenic status of TT12B is explained by the absence of both the ΦStx1a- and ΦStx2a-prophages carried by TT12A, and we also recorded further alterations in the non-Stx prophage complement. Phenotypic characterization indicated that culture growth was directly impacted by the strains' distinct lytic phage complement. Altogether, our phylogenomic and phenotypic analyses show that these intimately related isogenic strains are on divergent Stx(+/stx-) evolutionary paths.

Impact of intense sanitization on environmental biofilm communities and the survival of Salmonella enterica at a beef processing plant.

Salmonella enterica is a leading cause of foodborne illness in the U.S. In the meat industry, one action taken to address pathogen contamination incidence is an intense sanitization (IS) of the entire processing plant that many large processors perform annually or semiannually. However, this procedure's immediate and long-term impact on environment microbial community and pathogen colonization are unknown. Here we investigated the impact of IS procedure on environmental biofilms and the subsequent S. enterica colonization and stress tolerance. Environmental samples were collected from floor drains at various areas 1 week before, 1 week, and 4 weeks after the IS procedure at a beef plant with sporadic S. enterica prevalence. Biofilm formation by microorganisms in the drain samples without S. enterica presence was tested under processing temperature. The ability of the biofilms to recruit and/or protect a co-inoculated S. enterica strain from quaternary ammonium compound (QAC) treatment was determined. The community structure of each drain sample was elucidated through 16S rRNA amplicon community sequencing. Post-IS samples collected from 8 drains formed significantly stronger biofilms than the respective pre-IS samples. S. enterica colonization was not different between the pre- and post-IS biofilms at all drain locations. S. enterica survival in QAC-treated pre- and post-IS mixed biofilms varied depending upon the drain location but a higher survival was associated with a stronger biofilm matrix. The 16S rRNA amplicon gene community sequencing results exhibited a decrease in community diversity 1 week after IS treatment but followed by a significant increase 4 weeks after the treatment. The IS procedure also significantly altered the community composition and the higher presence of certain species in the post-IS community may be associated with the stronger mixed biofilm formation and Salmonella tolerance. Our study suggested that the IS procedure might disrupt the existing environmental microbial community and alter the natural population composition, which might lead to unintended consequences as a result of a lack of competition within the multispecies mixture. The survival and recruitment of species with high colonizing capability to the post-IS community may play crucial roles in shaping the ensuing ecological dynamics.

Salmonella-induced microbiome profile in response to sanitation by quaternary ammonium chloride.

Salmonella enterica is a prominent cause of foodborne disease in the United States. However, the mechanism and route of pathogen transmission that leads to Salmonella infection in commercial processing plants are poorly understood. This study aimed to investigate the effect of mixed-species biofilms on S. enterica survival and persistence under sanitizer stress [Quaternary ammonium compounds (QACs)] by analyzing 78 floor drain samples from a meat processing facility and three S. enterica strains (serovars Cerro, Montevideo, and Typhimurium) isolated from that facility and an unrelated source. The four test groups were as follows: control, QAC treatment, Salmonella addition, and QAC treatment with Salmonella addition. DNAs were extracted, and 16S rRNA gene based on the variable region V4 amplicon sequencing was performed to analyze the relative abundance, core microbiome, and Alpha and Beta diversity using the qiime2 pipeline. At the genus level, the Brochothrix (45.56%), Pseudomonas (38.94%), Carnobacterium (6.18%), Lactococcus (4.68%), Serratia (3.14%), and Staphylococcus (0.82%) were shown to be the most prevalent in all drain samples. The results demonstrate that the relative abundance of different bacterial genera was affected by both QAC treatment and Salmonella addition, with some genera showing increases or decreases in abundance. Notably, the correlation network was constructed to understand the relationships between the different bacteria. Nitrospira had the greatest number of connections in the floor drain environment network, with two negative and eight positive correlations. The results suggest that Nitrospira in the mixed-species biofilm community may play a role in converting ammonium in the QAC sanitizer into nitrites. Thus, Nitrospira could be a potentially important genus in providing sanitizer resistance to pathogen-encompassed mixed-species biofilms.IMPORTANCESalmonella contamination in meat processing facilities can lead to foodborne illness outbreaks. Our study characterized the microbiome dynamics in beef facility drains and their response to Salmonella addition and common sanitizer (QAC). Nitrospira could be an important genus in providing sanitizer resistance to pathogen-encompassed mixed-species biofilms. The results provide insight into the impact of mixed-species biofilms on Salmonella survival and persistence under sanitizer stress in meat processing facilities. The results highlight the need to consider mixed-species biofilm effects when developing targeted interventions to enhance food safety.

A novel method using a differential staining fluorescence microscopy (DSFM) to track the location of enteric pathogens within mixed-species biofilms.

This study developed a new tool, differential staining fluorescence microscopy (DSFM), to measure the biovolume and track the location of enteric pathogens in mixed-species biofilms which can pose a risk to food safety in beef processing facilities. DSFM was employed to examine the impact of pathogenic bacteria, Escherichia coli O157:H7 and three different Salmonella enterica strains on mixed-species biofilms of beef processing facilities. Fourteen floor drain biofilm samples from three beef processing plants were incubated with overnight BacLight stained enteric pathogens at 7 °C for 5 days on stainless steel surface then counter-stained with FM-1-43 biofilm stain and analyzed using fluorescence microscopy. Notable variations in biovolume of biofilms were observed across the fourteen samples. The introduction of E. coli O157:H7 and S. enterica strains resulted in diverse alterations of biofilm biovolume, suggesting distinct impacts on mixed-species biofilms by different enteric pathogens which were revealed to be located in the upper layer of the mixed-species biofilms. Pathogen strain growth curve comparisons and verification of BacLight Red Stain staining effectiveness were validated. The findings of this study show that the DSFM method is a promising approach to studying the location of enteric pathogens within mixed-species biofilms recovered from processing facilities. Understanding how foodborne pathogens interact with biofilms will allow for improved targeted antimicrobial interventions.

Shotgun-metagenomics reveals a highly diverse and communal microbial network present in the drains of three beef-processing plants.

Background: Multi-species biofilms pose a problem in various environments, especially food-processing environments. The diversity of microorganisms in these biofilms plays a critical role in their integrity and protection against external biotic and abiotic factors. Compared to single-species biofilms, mixed-species biofilms are more resistant to various stresses, including antimicrobials like sanitizers. Therefore, understanding the microbiome composition and diversity in biofilms and their metabolic potential is a priority when developing intervention techniques to combat foodborne pathogens in food processing environments. Methods: This study aimed to describe and compare the microbiome profile of 75 drain biofilm samples obtained from five different locations (Hotscale, Hotbox, Cooler, Processing, & Grind room) of three beef-processing plants (Plant A, B & C) taken over two timepoints 2017-18 (T1) and 2021 (T2) by shotgun sequencing. Results: Core microbiome analysis found Pseudomonas, Psychrobacter, and Acinetobacter to be the top three prevalent genera among the plants and locations. Alpha diversity analysis demonstrated a high diversity of microbiome present in all the plants and locations across the time points. Functional analysis showed the high metabolic potential of the microbial community with abundance of genes in metabolism, cell-adhesion, motility, and quorum sensing. Moreover, Quaternary Ammonium Compound (QAC) resistance genes were also observed, this is significant as QAC sanitizers are commonly used in many food processing facilities. Multi-functional genes such as transposases, polymerases, permeases, flagellar proteins, and Mobile Genetic Elements (MGEs) were found suggesting these are dynamic microbial communities that work together to protect themselves against environmental stresses through multiple defense mechanisms. Conclusion: This study provides a framework for understanding the collective microbial network spanning a beef processing system. The results can be used to develop intervention strategies to best control these highly communicative microbial networks.

Distribution of Extremely Heat-Resistant Escherichia coli in the Beef Production and Processing Continuum.

Understanding the dynamics of stress-resistant Escherichia coli (E. coli) across the meat production and processing continuum is important for tracking sources of such microbes and devising effective modes of control. The Locus of Heat Resistance (LHR) is a ∼14-19 Kb genetic element imparting extreme heat resistance (XHR) in Enterobacteriaceae. It has been hypothesized that thermal and antimicrobial interventions applied during meat processing may select for LHR+E. coli. Thus, our goal was to study the prevalence and molecular biology of LHR+E. coli among lots of beef cattle (n = 3) from production through processing. Two hundred thirty-two generic E. coli isolated from the same animals through seven stages of the beef processing continuum (cattle in feedyards to packaged strip loins) were examined. LHR+E. coli were rare (0.6%; 1 of 180) among the early stages of the beef continuum (feces and hides at feedlot, feces and hides at harvest, and preevisceration carcasses), whereas the prevalence of LHR+E. coli on final carcasses and strip loins was remarkably higher. Half (14 of 28) of the final carcass E. coli possessed the LHR, while 79.2% (19 of 24) of the strip loin E. coli did. Eighty-five percent (29 of 34) of the LHR+E. coli presented with the XHR phenotype. The selection or enrichment of LHR+E. coli from harvest steps to the final products appeared unlikely as the LHR+E. coli isolates were effectively controlled by antimicrobial interventions typically used during beef processing. Further, whole-genome sequencing of the isolates suggested LHR+E. coli are persisting in the chilled processing environment and that horizontal LHR transfer among E. coli isolates may take place.

Universal hydrolysis probe-based approach for specific detection and genotyping of foodborne pathogens.

Real-time PCR assays are the method of choice for the specific detection of DNA targets. Multiple real-time PCR chemistries are used for developing pathogen detection assays. Among them, a hydrolysis probe is a preferred choice for pathogen detection assays. Two known limitations of hydrolysis probes are high cost and limited storage life. Therefore, this study aimed to develop and validate a universal hydrolysis probe (UHP)-based approach with high-resolution melt (HRM) analysis capabilities. The approach can be used for the detection and genotyping of target DNA. The approach described in this study was validated by standardizing nine UHP assays for detecting seven Shiga toxin-producing Escherichia coli serogroups, Listeria monocytogenes, and Salmonella strains. These nine assays were validated with 141 pure culture bacterial strains. Additionally, the HRM capability of the developed approach was validated for three UHP assays targeting E. coli O26, O111, and O121 using 96 DNAs isolated from enriched food samples. The nine assays specifically detected the target bacterial strains, and the three assays showed single nucleotide polymorphism (SNP) identification capability and no cross-reactivity with non-target strains. The developed approach can be performed in singleplex or multiplex format and combined with HRM analysis. The data from this study demonstrate that the UHP real-time PCR approach is a robust method for detecting any deoxyribonucleic acid target.

A review of Shiga-toxin producing Escherichia coli (STEC) contamination in the raw pork production chain.

Epidemiological evidence of Shiga toxin-producing Escherichia coli (STEC) infections associated with the consumption of contaminated pork highlight the need for increased awareness of STEC as an emerging pathogen in the pork supply chain. The objective of this review is to contribute to our understanding of raw pork products as potential carriers of STEC into the food supply. We summarize and critically analyze primary literature reporting the prevalence of STEC in the raw pork production chain. The reported prevalence rate of stx-positive E. coli isolates in live swine, slaughtered swine, and retail pork samples around the world ranged from 4.4 % (22/500) to 68.3 % (82/120), 22 % (309/1395) to 86.3 % (69/80), and 0.10 % (1/1167) to 80 % (32/40), respectively, depending upon the sample categories, detection methods, and the hygiene condition of the slaughterhouses and retail markets. In retail pork, serogroup O26 was prevalent in the U.S., Europe, and Africa. Serogroup O121 was only reported in the U.S. Furthermore, serogroup O91 was reported in the U.S., Asia, and South American retail pork samples. The most common virulence gene combination in retail pork around the globe were as follows: the U.S.: serogroup O157 + stx, non-O157 + stx, unknown serogroups+stx + eae; Europe: unknown serogroups+(stx + eae, stx2 + eae, or stx1 + stx2 + eae); Asia: O157 + stx1 + stx2 + ehxA, Unknown+stx1 + eaeA + ehxA, or only eae; Africa: O157 + stx2 + eae + ehxA. STEC strains derived from retail pork in the U.S. fall under low to moderate risk categories capable of causing human disease, thus indicating the need for adequate cooking and prevention of cross contamination to minimize infection risk in humans.

Pathogenomes and variations in Shiga toxin production among geographically distinct clones of Escherichia coli O113:H21.

Infections with globally disseminated Shiga toxin-producing Escherichia coli (STEC) of the O113:H21 serotype can progress to severe clinical complications, such as hemolytic uremic syndrome (HUS). Two phylogeographically distinct clonal complexes have been established by multi locus sequence typing (MLST). Infections with ST-820 isolates circulating exclusively in Australia have caused severe human disease, such as HUS. Conversely, ST-223 isolates prevalent in the US and outside Australia seem to rarely cause severe human disease but are frequent contaminants. Following a genomic epidemiology approach, we wanted to gain insights into the underlying cause for this disparity. We examined the plasticity in the genome make-up and Shiga toxin production in a collection of 20 ST-820 and ST-223 strains isolated from produce, the bovine reservoir, and clinical cases. STEC are notorious for assembly into fragmented draft sequences when using short-read sequencing technologies due to the extensive and partly homologous phage complement. The application of long-read technology (LRT) sequencing yielded closed reference chromosomes and plasmids for two representative ST-820 and ST-223 strains. The established high-resolution framework, based on whole genome alignments, single nucleotide polymorphism (SNP)-typing and MLST, includes the chromosomes and plasmids of other publicly available O113:H21 sequences and allowed us to refine the phylogeographical boundaries of ST-820 and ST-223 complex isolates and to further identify a historic non-shigatoxigenic strain from Mexico as a quasi-intermediate. Plasmid comparison revealed strong correlations between the strains' featured pO113 plasmid genotypes and chromosomally inferred ST, which suggests coevolution of the chromosome and virulence plasmids. Our pathogenicity assessment revealed statistically significant differences in the Stx2a-production capabilities of ST-820 as compared to ST-223 strains under RecA-induced Stx phage mobilization, a condition that mimics Stx-phage induction. These observations suggest that ST-820 strains may confer an increased pathogenic potential in line with the strain-associated epidemiological metadata. Still, some of the tested ST-223 cultures sourced from contaminated produce or the bovine reservoir also produced Stx at levels comparable to those of ST-820 isolates, which calls for awareness and for continued surveillance of this lineage.

Characterization of Escherichia coli harboring colibactin genes (clb) isolated from beef production and processing systems.

Certain strains of Escherichia coli possess and express the toxin colibactin (Clb) which induces host mutations identical to the signature mutations of colorectal cancer (CRC) that lead to tumorigenic lesions. Since cattle are a known reservoir of several Enterobacteriaceae including E. coli, this study screened for clb amongst E. coli isolated from colons of cattle-at-harvest (entering beef processing facility; n = 1430), across the beef processing continuum (feedlot to finished subprimal beef; n = 232), and in ground beef (n = 1074). Results demonstrated that clb+ E. coli were present in cattle and beef. Prevalence of clb+ E. coli from colonic contents of cattle and ground beef was 18.3% and 5.5%, respectively. clb+ E. coli were found susceptible to commonly used meat processing interventions. Whole genome sequencing of 54 bovine and beef clb+ isolates showed clb occurred in diverse genetic backgrounds, most frequently in phylogroup B1 (70.4%), MLST 1079 (42.6%), and serogroup O49 (40.7%).

Complete Closed Genome Sequence of the Extremely Heat-Resistant Strain Escherichia coli AW1.7.

Escherichia coli isolate AW1.7 is an extremely heat-resistant bacterium and has been widely used as a reference strain in extreme heat resistance studies for almost a decade. Here, we report its complete closed genome sequence.

Locus of Heat Resistance (LHR) in Meat-Borne Escherichia coli: Screening and Genetic Characterization.

Microbial resistance to processing treatments poses a food safety concern, as treatment tolerant pathogens can emerge. Occasional foodborne outbreaks caused by pathogenic Escherichia coli have led to human and economic losses. Therefore, this study screened for the extreme heat resistance (XHR) phenotype as well as one known genetic marker, the locus of heat resistance (LHR), in 4,123 E. coli isolates from diverse meat animals at different processing stages. The prevalences of XHR and LHR among the meat-borne E. coli were found to be 10.3% and 11.4%, respectively, with 19% agreement between the two. Finished meat products showed the highest LHR prevalence (24.3%) compared to other processing stages (0 to 0.6%). None of the LHR+E. coli in this study would be considered pathogens based on screening for virulence genes. Four high-quality genomes were generated by whole-genome sequencing of representative LHR+ isolates. Nine horizontally acquired LHRs were identified and characterized, four plasmid-borne and five chromosomal. Nine newly identified LHRs belong to ClpK1 LHR or ClpK2 LHR variants sharing 61 to 68% nucleotide sequence identity, while one LHR appears to be a hybrid. Our observations suggest positive correlation between the number of LHR regions present in isolates and the extent of heat resistance. The isolate exhibiting the highest degree of heat resistance possessed four LHRs belonging to three different variant groups. Maintenance of as many as four LHRs in a single genome emphasizes the benefits of the LHR in bacterial physiology and stress response.IMPORTANCE Currently, a "multiple-hurdle" approach based on a combination of different antimicrobial interventions, including heat, is being utilized during meat processing to control the burden of spoilage and pathogenic bacteria. Our recent study (M. Guragain, G. E. Smith, D. A. King, and J. M. Bosilevac, J Food Prot 83:1438-1443, 2020, https://doi.org/10.4315/JFP-20-103) suggests that U.S. beef cattle harbor Escherichia coli that possess the locus of heat resistance (LHR). LHR seemingly contributes to the global stress tolerance in bacteria and hence poses a food safety concern. Therefore, it is important to understand the distribution of the LHRs among meat-borne bacteria identified at different stages of different meat processing systems. Complete genome sequencing and comparative analysis of selected heat-resistant bacteria provide a clearer understanding of stress and heat resistance mechanisms. Further, sequencing data may offer a platform to gain further insights into the genetic background that provides optimal bacterial tolerance against heat and other processing treatments.

Rapid estimation of Salmonella enterica contamination level in ground beef - Application of the time-to-positivity method using a combination of molecular detection and direct plating.

Little progress has been made in decreasing the incidence rate of salmonellosis in the US over the past decade. Mitigating the contribution of contaminated raw meat to the salmonellosis incidence rate requires rapid methods for quantifying Salmonella, so that highly contaminated products can be removed before entering the food chain. Here we evaluated the use of Time-to-Positivity (TTP) as a rapid, semi-quantitative approach for estimating Salmonella contamination levels in ground beef. Growth rates of 14 Salmonella strains (inoculated at log 1 to -2 CFU/g) were characterized in lean ground beef mTSB enrichments and time-to-detection was determined using culture and molecular detection methods. Enrichments were sampled at five timepoints and results were used to construct a prediction model of estimated contamination level by TTP (superscript indicates time in hours) defined as TTP4: ≥5 CFU/g; TTP6: ≤5, ≥1 CFU/g; TTP8: ≤1, ≥0.01 CFU/g; with samples negative at 8 h estimated ≤0.01 CFU/g. Model performance measures showed high sensitivity (100%) and specificity (83% and 93% for two detection methods) for samples with a TTP4, with false negative rates of 0%.

Impact of mixed biofilm formation with environmental microorganisms on E. coli O157:H7 survival against sanitization.

Biofilm formation by foodborne pathogens is a serious threat to food safety and public health. Meat processing plants may harbor various microorganisms and occasional foodborne pathogens; thus, the environmental microbial community might impact pathogen survival via mixed biofilm formation. We collected floor drain samples from two beef plants with different E. coli O157:H7 prevalence history and investigated the effects of the environmental microorganisms on pathogen sanitizer tolerance. The results showed that biofilm forming ability and bacterial species composition varied considerably based on the plants and drain locations. E. coli O157:H7 cells obtained significantly higher sanitizer tolerance in mixed biofilms by samples from the plant with recurrent E. coli O157:H7 prevalence than those mixed with samples from the other plant. The mixed biofilm that best protected E. coli O157:H7 also had the highest species diversity. The percentages of the species were altered significantly after sanitization, suggesting that the community composition affects the role and tolerance level of each individual species. Therefore, the unique environmental microbial community, their ability to form biofilms on contact surfaces and the interspecies interactions all play roles in E. coli O157:H7 persistence by either enhancing or reducing pathogen survival within the biofilm community.