International Journal of Food Microbiology

Listeria monocytogenes is known to colonize food production environments and cross-contaminate finished foods. We investigated 30 L. monocytogenes collected from artisan cheese production facilities in Vermont from 2006-2008 for sanitizer tolerance, biofilm formation capacity, biofilm architecture, and tolerance to sanitizers of mature biofilms. Sixteen of these isolates represented a putatively persistent ribotype (DUP-1042B) found in one facility over two years. Isolates of the putatively persistent ribotype all aligned into ST191 and were 0-6 SNPs different, confirming they represented a persistent strain. We found no significant differences in sanitizer tolerance or crystal assay-based attachment capacity between persistent and non-persistent strains. However, using scanning electron microscopy, we found that isolates FML-10 and FML-19 formed substantially denser biofilms after 10 days on stainless steel. Ten-day old biofilms were highly resistant to sanitizers; neither quaternary ammonium nor sodium hypochlorite-based sanitizers achieved an EPA-recommended 6-log reduction. More EPS was found in low-nutrient biofilm conditions; thus, non-food contact surfaces in cheese environments may induce formation of biofilms with high sanitizer tolerance. Our results highlight the importance of regular environmental testing and strain typing for rapid detection of L. monocytogenes colonization attempts while they can still be removed without major renovations or equipment replacement. HighlightsO_LIIsolates from persistent ribotype DUP-1042B/ST191 were within 6 SNPs of each other C_LIO_LITwo isolates from ST191 made dense biofilms in nutrient rich conditions C_LIO_LIMore EPS was produced in nutrient-poor conditions C_LIO_LIMature biofilms of all isolates were highly resistant to QAC and SH sanitizers C_LI ImportanceThis study identifies strategies used by a set of persistently colonizing L. monocytogenes isolated from an artisanal cheese producer in Vermont, finding that some persistently colonizing isolates had high biofilm forming capacity, which may have contributed to their persistence.

Non-Typhoidal Salmonella (NTS) continues to be a leading cause of foodborne illness worldwide. Food manufacturers implement hurdle technology by combining more than one approach to control food safety and quality, including preservatives such as organic acids, refrigeration, and heating. We assessed the variation in survival in stresses of genotypically diverse isolates of Salmonella enterica to identify genotypes with potential elevated risk to sub-optimal processing or cooking. Sub-lethal heat treatment, survival in desiccated conditions and growth in the presence of NaCl or organic acids were investigated. S. Gallinarum strain 287/91 was most sensitive to all stress conditions. While none of the strains replicated in a food matrix at 4{degrees}C, S. Infantis strain S1326/28 retained the greatest viability, and six strains exhibited a significantly reduced viability. A S. Kedougou strain exhibited the greatest resistance to incubation at 60{degrees}C in a food matrix that was significantly greater than S. Typhimurium U288, S Heidelberg, S. Kentucky, S. Schwarzengrund and S. Gallinarum strains. Two isolates of monophasic S. Typhimurium, S04698-09 and B54 Col9 exhibited the greatest tolerance to desiccation that was significantly more than for the S. Kentucky and S. Typhimurium U288 strains. In general, the presence of 12mM acetic acid or 14mM citric acid resulted in a similar pattern of decreased growth in broth, but this was not observed for S. Enteritidis, and S. Typhimurium strains ST4/74 and U288 S01960-05. Acetic acid had a moderately greater effect on growth despite the lower concentration tested. A similar pattern of decreased growth was observed in the presence of 6% NaCl, with the notable exception that S. Typhimurium strain U288 S01960-05 exhibited enhanced growth in elevated NaCl concentrations. An understanding of the molecular basis of phenotypic variation in response to stress has the potential to improve process validation during food challenge tests, improve processing, and result in more reliable risk assessments in the food industry.

Listeria monocytogenes is a significant concern for the food industry due to its ability to persist in the food processing environment. Decreased susceptibility to disinfectants is one of the factors that contribute to the persistence of L. monocytogenes. The objective of this study was to explore the diversity of L. monocytogenes susceptibility to quaternary ammonium compounds (QACs) using 1,671 L. monocytogenes isolates. This was used to determine the phenotype-genotype concordance and characterize genomes of the QAC sensitive and tolerant isolates for stress resistance, virulence and plasmid replicon genes. Distribution of QAC tolerance genes among 37,897 publicly available L. monocytogenes genomes were also examined. The minimum inhibitory concentration to QACs was determined by the broth microdilution method and non-sequenced isolates (n=1,244) were whole genome sequenced. Genotype-phenotype concordance was 99% for benzalkonium chloride, DDAC and a commercial QAC based sanitizer. Prevalence of QAC tolerance genes was 23% and 28% in our L. monocytogenes collection and in the global dataset, respectively. qacH was the most prevalent gene in our collection (61%), with 19% prevalence in the global dataset. Notably, bcrABC was most common (72%) globally, while 25% in our collection. Prevalence of emrC and emrE was comparable in both datasets, 7% and 2%, respectively. Replicon genes, indicative of plasmid harborage, were detected in 44% of the isolates and associated with the QAC tolerant phenotype. The presented analysis is based on the biggest L. monocytogenes collection in diversity and quantity for characterization of the L. monocytogenes QAC tolerance at both phenotypic and genomic levels. IMPORTANCEContamination of Listeria monocytogenes within the food processing environment is of concern to the food industry due to challenges in eradicating the pathogen once it becomes persistent in the environment. Genetic markers associated with increased tolerance to disinfectants have been identified, which alongside factors favor the persistence of L. monocytogenes in the production environment. By employing a comprehensive large-scale phenotypic testing and genomic analysis our study significantly enhances the understanding of the prevalence of quaternary ammonium compound (QAC) tolerant L. monocytogenes and the genetic determinants associated with the increased tolerance. Furthermore, we report on the prevalence of QAC tolerance genes among 37,897 publicly available L. monocytogenes sequences and their distribution within clonal complexes, isolation sources and geographical locations. As the propagation of QAC tolerance showed not be evenly distributed globally this highlights that understanding the development of L. monocytogenes disinfectant tolerance can be monitored using publicly available WGS data.

Presence of and selection for Listeria spp. and Listeria monocytogenes that are less effectively inactivated by quaternary ammonium compounds (such as benzalkonium chloride [BC]) is a food safety concern, including in fresh produce environments. An initial MIC assay on 67 produce-associated Listeria strains showed that strains carrying BC resistance genes bcrABC (n=10) and qacH (n=1) showed higher MIC (4-6 mg/L BC) compared to strains lacking these genes (MIC of 1-2 mg/L BC). Serial passaging experiments that exposed the 67 strains to increasing BC concentrations revealed that 62/67 strains showed growth in BC concentrations above their parent MIC (range of 4-20 mg/L). Two serially passaged isolates were obtained for each parent strain and substreaked onto BHI agar in the absence of BC for seven rounds; 105/134 substreaked isolates showed higher substreaked MIC (range of 4 - 6 mg/L) compared to parent MIC. These results suggested isolates acquired genetic adaptations that confer BC resistance. Substreaked isolates were characterized by a combination of whole genome sequencing and Sanger sequencing of fepR, a local repressor of the MATE family efflux pump FepA. These data identified nonsynonymous fepR mutations in 48/67 isolates including 24 missense, 16 nonsense, and 8 frameshift mutations. The mean inactivation of substreaked isolates after exposure to use level concentrations of BC (300 mg/L) was 4.48 log, which was not significantly different from inactivation observed in parent strains. Serial passage experiments performed on cocultures of Listeria strains containing bcrABC or qacH did not yield growth at higher BC concentrations than monoculture experiments. IMPORTANCEListeria resistance to quaternary ammonium compounds has been raised as a concern with regards to Listeria persistence in food environments, which can increase risk of product contamination, food recalls and foodborne illness outbreaks. Findings from our study show that individual Listeria strains can acquire genetic adaptations that confer resistance to low concentrations of benzalkonium chloride, but these genetic adaptations dont increase Listeria survival when exposed to concentrations of benzalkonium chloride used for food contact surface sanitation (300 mg/L). Our study also suggests that there is limited risk of benzalkonium chloride resistance genes bcrABC and qacH spreading through horizontal gene transfer and conferring enhanced resistance of L. monocytogenes to benzalkonium chloride. Overall, our study suggests that emergence of benzalkonium chloride resistant Listeria strains in food processing environments is of limited concern, as even strains adapted to gain higher MIC in vitro maintain full sensitivity to use level concentrations of benzalkonium chloride.

Cytotoxic Bacillus cereus group strains are common causes of foodborne illness including diarrhea. However, our ability to assess food safety risks associated with the exposure to cytotoxic B. cereus group strains via contaminated food is limited due to the lack of predictive tools. In this study, we experimentally quantified the growth of 17 cytotoxic B. cereus group strains, representing six phylogenetic groups, in skim milk broth and used the growth data to develop an exposure assessment model. While none of the tested strains showed detectable growth in HTST milk at 4 or 6{degrees}C, 15 of the 17 strains showed growth at 10{degrees}C, 1 of the 17 strains showed growth at 8{degrees}C, and all strains grew at [≥]14{degrees}C. Growth data for 16 strains allowed us to generate linear secondary growth models, which were then used to develop the exposure assessment model. We simulated a five-stage supply chain with up to 35 consumer storage days, as that was the timing when distinguishable variations in percent milk containers over 105 CFU/m with different B. cereus genotypes were observed. When the initial contamination level of the HTST milk is set at an average of 100 CFU/mL, the model predicts that, on consumer home storage day 21 and 35, 2.81{+/-}0.66 and 4.13{+/-}2.53 % (mean {+/-} standard deviation) of the milk containers would exceed B. cereus group concentrations of 105 CFU/mL; these data represent the average across all strains. Sensitivity analysis showed that variation in the input parameter Q0, the initial physiological state of cells, has the largest effect on models prediction for 1 of 4 group II isolates, 1 of 6 group IV isolates and both group V isolates, suggesting the need to better characterize the growth parameters of these isolates. What-if scenario analysis showed that increased mean and variability in storage temperature at the consumers home both have substantial influence on final predicted B. cereus group concentration in milk containers. This model introduces an initial tool designed to facilitate risk-based food safety decision making for products that are contaminated with low B. cereus group levels.

Molecular-based microbiological approaches have become valuable tools for the food industry. However, even the most advanced molecular techniques are limited in their ability to differentiate based on viability creating the potential for biased results when applied to the food industry. The objective of this study was to generate a viable microbial bio-map of a commercial ready-to-eat (RTE) meat manufacturing process and assess its utility as a diagnostic tool. Product samples were collected from a commercial RTE meat manufacturing facility at various locations throughout processing. Samples were homogenized and aliquoted for culture-based microbial isolation and 16S rRNA gene sequencing. Homogenates were split into pairs and subject to either no treatment (Control) or treated with 25 M PMAxx (PMA) to remove free and non-viable cellular DNA. Overall, PMA treatment resulted in a less rich microbial community compared to Control samples. Paired analysis revealed that the impact of PMA varied by location with the greatest effects being observed at the beginning and end of manufacturing. Both Control and PMA treated samples identified a shift in the microbial population after thermal processing; however, only PMA treated samples identified a secondary shift in the microbial population occurring after slicing. Taxonomic analysis identified Lactobacillus as a predominant genera in sliced and packaged products. These results were further confirmed by the identification of Lactobacillus sakei on packaged product using a culture-based approach. These results suggest PMA treatment provides a higher level of sequencing resolution by removing background DNA. ImportanceMicrobial bio-mapping is a valuable tool for the meat and poultry industry to assess process control and evaluate the efficacy of intervention systems. In recent years it has become more common to incorporate the use of molecular techniques, such as qPCR and 16S rRNA, to quantitatively track target pathogens and gain a more holistic understanding of the microbial community throughout processing. One major limitation we face when applying these DNA-based techniques to the food industry is their inability to differentiate between DNA from viable versus non-viable cells, which may result in the false identification of pathogenic or spoilage microorganisms and bias microbiota results. To practically apply this technology in a ready-to-eat meat manufacturing setting, it is crucial to develop and validate strategies that are capable of differentiating between viable and non-viable cellular DNA.

Pathogen cross-contamination during food production is controlled through sanitation. However, sanitizer efficacy is often studied in bench-scale experiments (e.g., submerged coupons in static or stirred sanitizer) which poorly approximate fluid dynamics. This limits our understanding of how effective sanitization is in commercial application. This study paired computational fluid dynamic (CFD) estimates of shear stress during spray application of sanitizer with measurements of Listeria innocua reduction on stainless steel by 100 ppm hypochlorite sanitizer under various application methods. Static submersion of inoculated coupons for 3 s resulted in a log reduction of 2.3 {+/-} 0.1 log CFU. Bench-scale spray application for 3 s had the largest microbial reduction at the point of sanitizer spray impingement (7.5 {+/-} 0.5 log CFU) and directly adjacent to the impingement point (6.4 {+/-} 0.7 log CFU) where shear stress was the highest. Surface locations below the impingement point that only received fluid film sanitizer run-off had a significantly lower microbial reduction of 0.4 {+/-} 0.1 log CFU (p < 0.05). At the pilot scale, sanitizer spray manually applied by operators achieved a 2.5 {+/-} 0.4 log CFU reduction, which was significantly lower than what was achieved during bench-scale spray application (p < 0.05). Microbial reduction from manual operation of spray equipment was also significantly different among operators (p < 0.05). Discrepancies between bench-scale spraying, pilot-scale spraying, and submerged coupons underscores the need for sanitizer validation under realistic conditions to better understand the risk reduction achieved through sanitation programs during food processing.

Pathogenic L. monocytogenes may inhabit dairy processing environments, increasing the risk for cross-contamination of foods. Using biocontrol microorganisms that inhibit or outcompete L. monocytogenes to complement sanitation of dairy processing facilities may enhance the control of L. monocytogenes. However, it remains unknown whether the resident microbiota of dairy processing facilities affects the antilisterial activity of biocontrol strains. Here, two lactic acid bacteria (LAB) strains (Enterococcus PS01155 and PS01156) were tested for their biocontrol potential in the context of microbiomes collected from three ice cream processing facilities (A, B, and C). Antilisterial ability was assessed by co-culturing LABs with 8-L. monocytogenes strains in the presence of microbiota for 3 days at 15{degrees}C, followed by quantification of the most probable number of attached L. monocytogenes. L. monocytogenes concentration increased by 0.38{+/-}0.77 log10 MPN/sample in treatments containing microbiota from facility A, while it decreased by 0.99{+/-}1.13 and 2.54{+/-}0.84 log10 MPN/sample in treatments with microbiota from facilities B and C, respectively. The attachment of LAB to an abiotic surface was assessed by co-culturing LABs in with the microbiomes at 15{degrees}C for 3 days, followed by characterization of attached microbiota composition using amplicon sequencing. All samples containing microbiomes from facilities A and B had high relative abundance of Pseudomonas, while samples with facility C microbiome had high relative abundance of Enterococcus. Overall, we show that microbiota composition of ice cream processing facilities affected the antilisterial ability of LABs. IMPORTANCEAntilisterial lactic acid bacteria strains had been proposed as biological pathogen control agents for application in food processing environments. However, the effect of resident food processing environment microbiota on the performance on antilisterial lactic acid bacteria strains is poorly understood. Our study shows that the composition of the microbiota collected from ice cream processing facilities environmental surfaces can affect the attachment and inhibitory effect of lactic acid bacteria strains against L. monocytogenes. Further studies are therefore needed to evaluate whether individual microbial taxa affect antilisterial properties of lactic acid bacteria strains and to characterize the underlying mechanisms.

Bacillus cereus sensu lato (s.l.) encompasses strains with diverse impacts, ranging from foodborne illness and anthrax to beneficial applications in agriculture and industry. While the risk of anthrax and emetic intoxication can be reliably predicted by the presence of specific virulence genes, predicting diarrheal foodborne illness risk based solely on enterotoxin gene presence has proven unreliable. In this study, we evaluated cytotoxicity against Caco-2 human gut cells using a diverse collection of B. cereus s.l. isolates representing all eight panC phylogenetic groups and conducted genomic analyses to identify predictive markers of cytotoxicity. Isolates from panC groups I, IV, and V exhibited significantly higher cytotoxicity compared to other groups, although individual isolates from other panC groups have also been linked to illness. Logistic and random forest regression models revealed that while the presence of enterotoxin genes was a sensitive indicator of cytotoxicity, it lacked specificity. Logistic regression analysis identified 21 nonsynonymous single nucleotide polymorphisms (SNPs) within enterotoxin (Nhe and Hbl) gene sequences that were more effective predictors of cytotoxicity, providing higher specificity with comparable sensitivity. These SNPs achieved accuracy and precision values exceeding 0.7. Random forest models highlighted the importance of panC group, enterotoxin gene SNPs, and the presence of the full hbl operon as key predictors of cytotoxicity. The strong sensitivity, specificity, and biological relevance of these SNPs position them as promising markers for improving strain-based risk assessment of B. cereus s.l. IMPORTANCEEnterotoxin genes have been associated with B. cereus sensu lato (s.l.) diarrheal foodborne illness; however, their mere presence in a genome is an unreliable predictor of an isolates cytotoxicity towards human gut epithelial cells. To improve food safety risk assessment, more specific markers of cytotoxicity are required. In this study, we identified nonsynonymous SNPs within the coding sequences of the enterotoxins Nhe and Hbl. These SNPs offer potential targets for rapid molecular tests to identify B. cereus s.l. isolates with an elevated food safety risk due to their capacity to inflict cytotoxic damage on human gut epithelial cells. Implementation of such markers upon validation could improve consumer safety while reducing food waste.

Salmonella is a frequent zoonotic foodborne pathogen, with swine and pork meats the most common source of human infection. In Chiang Mai and Lamphun Province in northern Thailand, there has been a high prevalence of salmonellosis for over a decade. Infection is usually with several dominant S. enterica serotypes, including serotypes Rissen and Monophasic Typhimurium. However, several less common serotypes also contribute to disease. Whole genome sequencing of 43 of these less common S. enterica serotypes isolated from the pork production chain through 2011-2014 were used to evaluate their genetic diversity and virulence potential. Salmonella contamination at local retail markets represented cross-contamination from multiple sources, including decontaminated foodstuff. Previous studies have highlighted the importance of host cell adhesion, invasion and intracellular survival for the development of clinical salmonellosis. We screened our dataset for known virulence genes and antimicrobial resistance genes, identifying at least 10 antimicrobial resistance genes in all isolates. These results indicate that these less common S. enterica serotypes also pose a significant public health risk. Our findings support the need for appropriate surveillance of food products going to market to reduce public exposure to highly pathogenic, multi-drug resistant Salmonella. Surveillance throughout the pork production chain would motivate stakeholders to reinforce sanitation standards and help reduce the risk of salmonellosis in humans.

Plant-based cheese analogs have emerged as a novel global market trend driven by sustainability concerns for our planet. This study examines eleven soft ripened plant-based cheese analogs produced in Europe, primarily with bloomy rinds and cashew nuts as the main ingredient. First, we focused on exploring the macronutrients and salt content stated on the labels, as well a detailed fatty acid analysis of the samples. Compared to dairy cheeses, plant-based cheeses share similarities in lipid content, but their fatty acid profiles diverge significantly, with higher ratio of mono- and polyunsaturated fatty acids such as oleic and linoleic acids. We also investigated the microbiota of these analog products, employing a culture-dependent and -independent approaches. We identified a variety of microorganisms in the plant-based cheeses, with Lactococcus lactis and Leuconostoc mesenteroides being the dominant bacterial species, and Geotrichum candidum and Penicillium camemberti the dominant fungal species. Most of the species characterized are similar to those present in dairy cheeses, suggesting that they have been inoculated as culture starters to contribute to the sensorial acceptance of plant-based cheeses. However, we also identify several species that are possibly intrinsic to plant matrices or originate from the production environment, such as Pediococcus pentosaceus and Enterococcus spp. This coexistence of typical dairy-associated organisms with plant associated species highlights the potential microbial dynamics inherent in the production of plant-based cheese. These findings will contribute to a better understanding of plant-based cheese alternatives, enable the development of sustainable products, and pave the way for future research exploring the use of plant-based substrates in the production of cheese analogues.

In order to prevent the spread of foodborne illnesses, the presence of pathogens in the food chain is monitored by government agencies and food producers. The culture-based methods currently employed are sensitive but time-and labour-intensive, leading to increasing interest in exploring culture-independent diagnostic tests (CIDTs) for pathogen detection. However, sensitivity and reliability of these CIDTs relative to current approaches has not been well established. To address this issue, we conducted a comparison of the limit of detection (LOD50) for Salmonella between a culture-based method and three CIDT methods: qPCR (targeting invA and stn), metabarcode (16S) sequencing, and shotgun metagenomic sequencing. Samples of chicken feed and chicken caecal contents were spiked with Salmonella serovar Enteritidis and subjected to culture-and DNA-based detection methods. To explore the impact of non-selective enrichment on LOD50, all samples underwent both immediate DNA extraction and an overnight enrichment prior to gDNA extraction. In addition to this spike-in experiment, feed and caecal samples acquired from the field were tested with culturing, qPCR, and metabarcoding. In general, LOD50 was comparable between qPCR and shotgun sequencing methods. Overnight microbiological enrichment resulted in an improvement in LOD50 with up to a three log decrease, comparable to culture-based detection. However, Salmonella reads were detected in some unspiked feed samples, suggesting false-positive detection of Salmonella. Additionally, the LOD50 in feeds was three logs lower than in caecal contents, underscoring the impact of background microbiota on Salmonella detection using all methods. IMPORTANCEThe appeal of CIDTs is increased speed with lowered cost, as well as the potential to detect multiple pathogen species in a single analysis and to monitor other areas of concern such as antimicrobial resistance genes or virulence factors. Understanding the sensitivity of CIDTs relative to current approaches will help determine the feasibility of implementing these methods in pathogen surveillance programs.

Members of the family Lactobacillaceae, which now includes species formerly belonging to the genera Lactobacillus and Pediococcus, but also Leuconostocaceae, are of foremost importance in food fermentations and spoilage, but also as components of animal and human microbiota and as potentially pathogenic microorganisms. Knowledge of the ecological distribution of a given species and genus is important, among other things, for the inclusion in lists of microorganisms with a Qualified Presumption of Safety or with beneficial use. The objective of this work is to use the data in FoodMicrobionet database to obtain quantitative insights (in terms of both abundance and prevalence) on the distribution of these bacteria in foods and food environments. We first explored the reliability of taxonomic assignments using the SILVA v138.1 reference database with full length and partial sequences of the 16S rRNA gene for type strain sequences. Full length 16S rRNA gene sequences allow a reasonably good classification at the genus and species level in phylogenetic trees but shorter sequences (V1-V3, V3-V4, V4) perform much worse, with type strains of many species sharing identical V4 and V3-V4 sequences. Taxonomic assignment at the genus level of 16S rRNA genes sequences and the SILVA v138.1 reference database can be done for almost all genera of the family Lactobacillaceae with a high degree of confidence for full length sequences, and with a satisfactory level of accuracy for the V1-V3 regions. Results for the V3-V4 and V4 region are still acceptable but significantly worse. Taxonomic assignment at the species level for sequences for the V1-V3, V3-V4, V4 regions of the 16S rRNA gene of members of the family Lactobacillaceae is hardly possible and, even for full length sequences, and only 49.9% of the type strain sequences can be unambiguously assigned to species. We then used the FoodMicrobionet database to evaluate the prevalence and abundance of Lactobacillaceae in food samples and in food related environments. Generalist and specialist genera were clearly evident. The ecological distribution of several genera was confirmed and insights on the distribution and potential origin of rare genera (Dellaglioa, Holzapfelia, Schleiferilactobacillus) were obtained. We also found that combining Amplicon Sequence Variants from different studies is indeed possible, but provides little additional information, even when strict criteria are used for the filtering of sequences.

Studies investigating surface microbiota in food facilities often include estimates of relative abundance. However, obtaining accurate relative abundance values can be challenging. Differences among microbes can lead to different degrees of cell recovery from the surface and different DNA extraction yields that skew downstream relative abundance estimates. Here, we evaluated (1) the impact of cell recovery from surfaces using sponge swabs and (2) DNA extraction protocol on the relative abundance estimates of relative abundance on artificially inoculated surfaces. Our results showed that Escherichia coli (Gram-negative cell), Listeria monocytogenes (Gram-positive cell), Bacillus cereus (bacterial spore), Alicyclobacillus suci (bacterial spore), Exophiala phaeomuriformis (fungal cell), Aspergillus fischeri (fungal spore) differed significantly (p<0.05) in their recovery rates from stainless steel surfaces, ranging from 2.9%{+/-}3.0% recovery to 94.9%{+/-}3.0% recovery. Modification of the DNA extraction protocol by extending the bead-beating step by 10 min generally improved DNA yields, though the impact varied by organism. For example, DNA yields of E. coli increased from 70 to 84 ng/mL while that of L. monocytogenes increased only from 23.2 to 29.2 ng/mL. Amplicon sequencing results indicated that the differences in cell recovery and DNA extraction among microbial species skewed the relative abundance estimates from inoculated surfaces. For example, the estimated relative abundance of L. monocytogenes was 9-17%, which was lower than its actual relative abundance (25%). These results underscore the limitations of surface microbiota characterization in food facilities and highlighted the need to improve current recovery and DNA extraction methods. ImportanceAmplicon sequencing has been used to characterize microbial communities on facility surfaces. However, few studies have evaluated the accuracy of the amplicon sequencing workflow for quantifying spoilage and pathogenic organisms in these microbial communities. Here, we assessed the accuracy for amplicon sequencing to evaluate the relative abundance of spoilage and pathogenic organisms commonly found in food processing environments. The results revealed biases in relative abundances due to limitations in cell recover and DNA extraction methods. These findings revealed the potential biases in surface microbiota characterization in food facilities, and the need to refine current recovery and extraction methods to enhance the accuracy of microbiota characterization.

Salmonella infections are a leading cause of bacterial food-borne illness worldwide. Infections are highly associated with the consumption of contaminated food, and in particular, chicken meat. Understanding how management practices and environmental factors influence Salmonella populations in broiler chicken production may aid in reducing the risk of food-borne illness in humans. Utilizing whole genome sequencing with antimicrobial and heavy metal resistance, virulence factor and plasmid identification, we have characterized the genetic diversity of Salmonella enterica isolates (n = 55) obtained from broiler chicken litter. S. enterica isolates were recovered from the litter of broiler chickens over three consecutive flocks in four broiler houses on a single integrated farm in Georgia, USA. The chickens were raised under a newly adopted "No Antibiotics Ever" program and copper sulfate was administered via drinking water. In-silico serovar prediction identified three S. enterica serovars: Enteritidis (n = 12), Kentucky (n = 40) and Senftenberg (n = 3). Antimicrobial susceptibility testing revealed that only one S. Kentucky isolate was resistant to streptomycin, while the remaining isolates were susceptible to all antibiotics tested. Metal resistance operons, including copper and silver, were identified chromosomally and on plasmids in serovar Senftenberg and Kentucky isolates, respectively. Serovar Kentucky isolates harboring metal resistance operons were the only Salmonella isolates recovered from the litter of third flock cohort. These results suggest the addition of copper sulfate to drinking water may have selected for S. Kentucky isolates harboring plasmid-borne copper resistance genes and may explain their persistence in litter from flock to flock. ImportanceSalmonella foodborne illnesses are the leading cause of hospitalizations and deaths, resulting in a high economic burden on the healthcare system. Globally, chicken meat is one of the highest consumed meats and is a predominant source of foodborne illness. The severity of Salmonella infections depends on the presence of antimicrobial resistance genes and virulence factors. While there are many studies which have investigated Salmonella strains isolated from post-harvest chicken samples, there is a gap in our understanding of the prevalence and persistence of Salmonella in pre-harvest and in particular their makeup of antibiotic resistance genes, virulence factors and metal resistance genes. The objective of this study was to determine how on-farm management practices and environmental factors influence Salmonella persistence, as well as the antimicrobial resistance genes and virulence factors they harbor. In this study we demonstrate that broiler chickens raised without antibiotics are less likely to harbor antibiotic resistance, however the practice of adding acidified copper sulfate to drinking water may select for strains carrying metal resistant genes.

The food industry needs a straightforward, efficient, widely applicable and cost-efficient method to not only detect, but also determine serovar and potential sources of multiple strains in food and environment. While Whole Genome Sequencing (WGS) can generate complete genomic profile of food pathogens, it is a laborious, time-consuming, and expensive method that necessitates pure isolates. As a result, it is unsuitable for samples with complex background, limiting its widespread application by food industry. However, traditional multilocus sequence typing (MLST) approaches do not provide sufficient single nucleotide polymorphism (SNP) information to effectively track- and-trace sources of contamination. In contrast, ChapterDx MLSTnext-NGS (next-generation sequencing) Salmonella assay amplifies and sequences 47 polymorphic loci, evenly spanning the Salmonella enterica genome. As demonstrated in this study, the ChapterDx MLSTnext-NGS Salmonella can identify serovar with high resolution, distinguishing between various strains of the same serovar and resolve co-presence of different strains in a single sample. Additionally, this assay can analyze up to thousand samples in a single sequencing run within 22 hours, making it a highly efficient and scalable method for the food industry. Moreover, the cost per sample of the ChapterDx MLSTnext-NGS Salmonella assay is comparable to that of quantitative Polymerase Chain Reaction (qPCR), making it an affordable option. The assay uses ChapterDx amplification technology, allowing for the amplification of all 47 loci in a single-tube, single-step PCR reaction. This makes it one of the simplest NGS applications available. In summary, ChapterDx MLSTnext-NGS Salmonella assay can be implemented in many diagnostic laboratories to address increasingly complex food safety issues.

Nontyphoidal Salmonella (NTS) are major foodborne pathogens primarily transmitted to humans through contaminated poultry products. The increase in antibiotic-resistant NTS, including Salmonella Heidelberg, has recently become a public health issue. Current control measures are inadequate, emphasizing the need for novel approaches to mitigate NTS colonization in poultry and contamination of poultry products. We hypothesized that commensal Escherichia coli can reduce antibiotic-resistant NTS colonization in the chicken intestines by modulating the fitness, virulence, and antibiotic resistance (AMR) potential of Salmonella. To test this hypothesis, we co-cultured commensal E. coli 47-1826 and antibiotic-resistant S. Heidelberg 18-9079 strains isolated from poultry and analyzed their transcriptomes using RNA sequencing. Our analysis revealed 4,890 differentially expressed genes in S. Heidelberg 18-9079 when co-cultured with E. coli 47-1826. After filtering the expression data, we found 193 genes were significantly upregulated while 202 genes were downregulated. Notably, several genes involved in bacterial growth, pathogenicity and virulence, biofilm formation, metal-ion hemostasis, signal transduction and chemotaxis, stress response, transmembrane transport of xenobiotics, and cellular metabolism were down-regulated by as much as eighty-six folds in S. Heidelberg as compared to the control. Further, the study revealed the downregulation of genes associated with AMR and drug efflux in S. Heidelberg 18-9079 by up to twelvefold. These findings highlight that commensal E. coli 47-1826 may reduce the fitness, persistence, virulence, and antimicrobial resistance (AMR) dissemination of S. Heidelberg 18-9079, implying that E. coli strain 47-1826 could be utilized as a strategy to mitigate antibiotic-resistant S. Heidelberg in poultry, ultimately enhancing food safety. ImportanceNontyphoidal Salmonella, commonly transmitted to humans through contaminated poultry meat and eggs, is one of the most frequent causes of foodborne illness. Augmenting the situation, foodborne outbreaks of antibiotic-resistant NTS have become an additional food safety and public health concern. Evaluation of growth changes and transcriptomic profiling of antibiotic-resistant S. Heidelberg and commensal E. coli in a mixed culture of the two will provide insight into the ability of commensal E. coli to reduce S. Heidelberg colonization of chicken intestines and the genes involved in that change. Our study showed that commensal E. coli strain 47-1826 significantly reduced antibiotic-resistant S. Heidelberg 18-9079 counts and expression of Salmonella genes, which play a vital role in the growth and persistence of nontyphoidal Salmonella in poultry intestines. The study results suggest the potential use of commensal E. coli strain 47-1826 to control antibiotic-resistant S. Heidelberg colonization in poultry, leading to improved food safety through reduced NTS contamination of foods of poultry origin and reduced dissemination of antibiotic-resistant Salmonella and their resistant determinants to humans via the food chain.

Ceviche is a traditional dish made from raw fish meat marinated in lime juice without any heat cooking step throughout its preparation process. Although the use of organic acids as antibacterial agents is well known; recent research indicates that lime juice actually can reduce the risk of V. parahemolyticus infections but it is ineffective against other potential pathogens. Despite the fact that fresh fish meat is safe; exposed organs including skin, gills and guts represent a potential source of bacterial contamination. In Mexico, diarrheal diseases are caused mainly by contaminated food; it is estimated that almost 67% of infections are due the presence of bacterial agents mainly in frozen and fresh fish. The main objective of this study was to estimate the taxonomic diversity of microbial species present in ready-to-eat ceviche using a metagenomic approach. Six samples of commercially available ceviche were subjected to DNA high throughput sequencing and bioinformatics analyses, we identified between 65,000 and 131,000 reads per sample. The predominant phyla identified through the samples were Proteobacteria, Bacteroidetes and Firmicutes. We discuss the factors involved in the microbiological quality of this kind of raw foods and how they influence the bacterial diversity within the analyzed samples.

The production of sheeps milk cheese has grown in recent years since it is a high value-added product with excellent properties. As such, it is necessary to provide data on the microbiota and organoleptic characteristics of this product, as well as the influence of these microorganisms on public health. Thus, the aim of the present study was to characterize the microbial community of different types of sheep cheeses using high-throughput sequencing of the 16S rRNA gene. The study was conducted with four groups of cheese: colonial, fresh, feta, and pecorino (n = 5 samples per group). The high-throughput 16S rRNA amplicon sequencing revealed 55 operational taxonomic units in the 20 samples, representing 9 genera of the two bacterial phyla Firmicutes and Proteobacteria. The predominant genera in the samples were Streptococcus and Lactobacillus. When evaluating alpha diversity by the indexes of Simpson, Chao1, Shannon, and Skew no significant differences were observed between the groups. Evaluating of the beta diversity using Bray-Curtis dissimilarity, the group of colonial cheeses presented a significant difference when compared to the feta (q = 0.030) and pecorino groups (q = 0.030). Additionally, the fresh group differed from the pecorino group (q = 0.030). The unweighted Unifrac distance suggests that the colonial cheese group differed from the others. Moreover, the feta cheese group differed from the fresh group. The distance-weighted Unifrac suggests that no significance exists between the groups. According to this information, the microbiota characterization of these cheese groups was useful in demonstrating the bacterial communities belonging to each group, its effects on processing, elaboration, maturation, and public health.

Listeria monocytogenes is ubiquitously found in nature and can easily enter food-processing facilities due to contaminations of raw materials. Several countermeasures are used to combat contamination of food products, for instance the use of disinfectants that contain quaternary ammonium compounds, such as benzalkonium chloride (BAC) and cetyltrimethylammonium bromide (CTAB). In this study, we assessed the potential of the commonly used wildtype strain EGD-e to adapt to BAC and CTAB under laboratory growth conditions. All BAC-tolerant suppressors exclusively carried mutations in fepR or its promoter region likely resulting in the overproduction of the efflux pump FepA. In contrast, CTAB- tolerance was associated with mutations in sugR, which regulates the expression of the efflux pumps SugE1 and SugE2. L. monocytogenes strains lacking either FepA or SugE1/2 could still acquire tolerance towards BAC and CTAB. Genomic analysis revealed that the overproduction of the remaining efflux system could compensate for the deleted one. Even in the absence of both efflux systems, tolerant strains could be isolated, which all carried mutations in the diacylglycerol kinase encoding gene lmo1753 (dgkB). DgkB converts diacylglycerol to phosphatidic acid, which is subsequently re-used for the synthesis of phospholipids suggesting that alterations in membrane composition could be the third adaptation mechanism. Originality-Significance StatementSurvival and proliferation of Listeria monocytogenes in the food industry is an ongoing concern, and while there are various countermeasures to combat contamination of food products, the pathogen still successfully manages to withstand the harsh conditions present in food-processing facilities, resulting in reoccurring outbreaks, subsequent infection, and disease. To counteract the spread of L. monocytogenes it is crucial to understand and elucidate the underlying mechanism that permit their successful evasion. We here present various adaptation mechanisms of L. monocytogenes to withstand two important quaternary ammonium compounds.