Antimicrobial resistance (AMR) in Enterococcus poses an emerging One Health concern due to its capacity for environmental persistence and cross-sector transmission. This two-year longitudinal study investigated the spatial and seasonal distribution of Enterococcus across interconnected animal, environmental, and human wastewater compartments in a peri-urban region of western Canada. We aimed to i.) characterize Enterococcus species across varying stages of dairy cow maturity, farm environments and nearby municipal wastewater systems and ii.) determine the AMR profiles and genomic relationships of E. faecalis and E. faecium. Among the 1133 Enterococcus isolates collected, over 24 species were identified, with E. hirae (n = 319) being the most prevalent followed by E. faecium (n = 256) and E. faecalis (n = 223). Species distributions varied significantly by season across most sample types. Antimicrobial susceptibility testing of E. faecalis (n = 58) and E. faecium (n = 61) isolates revealed four, multi-drug resistance E. faecalis isolates, with resistance to tetracycline (18%) and quinupristin-dalfopristin (18%) most common. Genomic analysis of E. faecalis (n = 158) and E. faecium (n = 171) isolates revealed 36 resistance determinants, with aac(6')-Ii, lsa(A), msr(C), tet(M) and tet(L) being the predominant resistance genes. Phylogenomic analysis revealed high diversity within both species, comprising 88 sequence types (STs) for E. faecalis and 100 for E. faecium, including 46 novel STs. Most of the closely related isolates (≤10 SNPs) originated from the same sample type and site (>74%), reflecting localized transmission dynamics. Evidence of cross-domain exchange and seasonal persistence was confined to organic dairy farms, while no genomic relatedness was observed between livestock-associated isolates and those from wastewater. These findings show that dairy-associated Enterococcus populations are diverse yet spatially contained, with limited connectivity to the human wastewater domain. Targeted One Health surveillance can help identify localized points to guide AMR mitigation in regions of intensive dairy production.

The Joint Programming Initiative on Antimicrobial Resistance (JPIAMR) networks ‘Seq4AMR’ and ‘B2B2B AMR Dx’ were established to promote collaboration between microbial whole genome sequencing (WGS) and antimicrobial resistance (AMR) stakeholders. A key topic discussed was the frequent variability in results obtained between different microbial WGS-related AMR gene prediction workflows. Further, comparative benchmarking studies are difficult to perform due to differences in AMR gene prediction accuracy and a lack of agreement in the naming of AMR genes (semantic conformity) for the results obtained. To illustrate this problem, and as a capacity-building exercise to encourage stakeholder involvement, a comparative Galaxy-based BenchAMRking platform was developed and validated using datasets from bacterial species with PCR-verified AMR gene presence or absence information from abritAMR. The Galaxy-based BenchAMRking platform ( https://erasmusmc-bioinformatics.github.io/benchAMRking/ ) specifically focusses on the steps involved in identifying AMR genes from raw reads and sequence assemblies. The platform currently comprises four well-characterised and published workflows that have previously been used to identify AMR genes using WGS data from several different bacterial species. These four workflows, which include the ISO certified abritAMR workflow, make use of different computational tools (or tool versions), and interrogate different AMR gene sequence databases. By utilising their own data, users can investigate potential AMR gene-calling problems associated with their own in silico workflows/protocols, with a potential use case outlined in this publication. BenchAMRking is a Galaxy-based comparison platform where users can access, visualise, and explore some of the major discrepancies associated with AMR gene prediction from microbial WGS data.

Escherichia coli is a Gram-negative bacterium that is ubiquitous in animals and humans, with some strains capable of causing disease. The aim of this study was to perform a comparative genomic analysis of 2732 generic E. coli isolates that were recovered from poultry samples collected from six regions in Canada as part of the National Microbiological Baseline study in Broiler Chicken. Isolates were subjected to whole genome sequencing and a subset (1122/2732) were tested for phenotypic resistance to 15 antimicrobials. These E. coli isolates were highly diverse, representing 376 serotypes, 236 sequence types and 21 pathotypes, of which 19 were hybrid pathotypes. A high concordance (>85%) between resistance phenotype and the presence of antimicrobial resistance genes and point mutations (resistance determinants) was observed for 13/15 antimicrobials. Over 95% of the β-lactam, fluoroquinolone, and phenicol resistance genes were predicted to be plasmid-borne. The number of resistance determinants per genome was highest in Quebec, while resistance genes associated with β-lactam resistance were more frequently detected in isolates from British Columbia. Generic E. coli in Canadian poultry are highly diverse, can carry pathotype-associated virulence factors and resistance determinants of clinical significance with a risk of emerging into pathogenic strains.

Towards fostering a more sustainable food production system in face of the climate change challenge, alternative protein meat-substitute products that are plant-based and free of animal by-products have been gaining attractions from both food manufacturers and consumers. With these so-called plant-based meat analogues (PBMAs) becoming increasingly available at supermarkets, there is very little known about their microbial properties. In this short report, we characterized the bacterial composition of raw plant-based ground meat imitation retail products using 16S rRNA gene amplicon sequencing. Despite the observed bacterial community dissimilarity between sample brands, a total of 18 shared genera (dominated by Bacilli and Gammaproteobacteria classes) were identified as the core constituents of the bacterial microbiota of these PBMA products. Within the scope of food safety testing, to gain insights on the dynamics of the enrichment process for E. coli O157:H7 in accordance with the Health Canada reference method MFHPB-10, bacterial taxonomic analyses were conducted at different stages of the prescribed cultural procedures. Using both control and E. coli O157:H7-inoculated PBMA samples it was revealed that, independent of the presence of E. coli O157:H7, off-target bacteria of the Clostridium sensu stricto 1 genus were significantly enriched from the uncultured samples. Additionally, the abundance of Hafnia-Obesumbacterium bacteria in the PBMA samples was also increased in the enrichment products, but only when E. coli O157:H7 was absent. Consistent with the spread-plating results indicating that the inoculated E. coli O157:H7 cells were capable of reaching a high density (> 108 CFU/ml) in the resultant enrichment cultures, the significant enrichment of bacterial 16S rRNA gene sequences belonging to the targeted genus of Escherichia, but not Hafnia-Obesumbacterium. This further highlights the competitive nature of the selective enrichment for E. coli O157:H7 against specific background bacteria associated with the PBMA products.

Antimicrobial resistance (AMR) is a global public health threat, but the role of foods in its dissemination is poorly understood. We examined the incidence of foodborne bacteria carrying AMR genes considered high-priority research targets by the World Health Organization. Frozen, ready-to-eat, avocado, coconut, mango, and peach (n = 161) were tested for bacteria encoding extended-spectrum β-lactamases (ESBLs) and carbapenemases. Over 600 presumptive-positive isolates were recovered and analyzed with a pooled sequencing (Pool-seq) strategy. Coconut samples exhibited the highest bacterial loads and prevalence/diversity of AMR genes. Isolates harbouring the β-lactamase genes blactx-m, blatem, and blashv, identified in 14 coconut and 2 mango samples, were further characterized by whole-genome sequencing and antimicrobial susceptibility testing. The most common gene was blactx-m-15, detected in 20 unique strains. Two carbapenemase-producing strains were isolated from coconut: Enterobacter roggenkampii encoding blandm-1 and Escherichia coli encoding blandm-5. Subsequent quantitative PCR (qPCR) analysis of enrichments for blactx-m/blandm indicated a potentially higher prevalence of these genes than observed by colony screening. This study presents a practical method for recovering ESBL- and carbapenemase-producing bacteria from foods. Mapping their distribution in food products is crucial to assessing the role of foods in the global spread of AMR and developing effective public health interventions.

The One Health (OH) approach recognizes the interconnectedness of the health of people, animals, plants/crops and ecosystems, and is central to addressing antimicrobial resistance (AMR). The 7th Environmental Dimension of Antimicrobial Resistance Conference (EDAR7), held in Montreal in May 2024, exemplified this approach by convening international experts and stakeholders to discuss AMR research and policy progress. EDAR7 workshop #8 focused on (1) barriers to establishing effective OH AMR research programs, (2) gaps in OH AMR research priorities, and (3) potential solutions/approaches or "tools" to ensure programs develop in accordance with OH principles and generate insightful data that maximizes limited resources. Key workshop outcomes included identifying critical principles for OH AMR research programs and highlighting the pivotal role of sustainable data management strategies. Additionally, the importance of considering AMR policy and risk assessment needs when planning and designing research was emphasized. Discussions explored specific tools and approaches that support the standardized and harmonized collection and analysis of data, and associated challenges of integrating genomics data into current risk assessments and models. Synthesis of the workshop's discussions outlined critical considerations that interdisciplinary OH AMR research programs and networks should prioritize to enhance the impact of their outputs.

Linking outbreaks of Shigella spp. to specific foods is challenging due to poor selectivity of current enrichment media. We have previously shown that enrichment media, tailored to the genomically-predicted antimicrobial resistance (AMR) of Shiga toxigenic E. coli strains, enhances their isolation from foods. This study investigates the application of this approach for Shigella isolation. The AMR gene profiles of 21,908 published S. sonnei genomes indicated a high prevalence of genes conferring resistance to streptomycin (aadA, aph(3”)-Ib, aph(6)-Id , 92.8%), sulfonamides (sul1, sul2, 74.8%) and/or trimethoprim (dfrA, 96.2%). Genomic analysis and antibiotic susceptibility testing conducted with a panel of 17 outbreak-associated S. sonnei strains confirmed the correlation of AMR gene detection with resistance phenotypes. Supplementation of Shigella Broth (SB) with up to 400 µg/mL of trimethoprim or sulfadiazine did not supress growth of sensitive strains, whereas 100 µg/mL of streptomycin increased the selectivity of this broth. All three antibiotics increased the selectivity of modified Tryptone Soya Broth (mTSB). Based on these results, supplemented media formulations were developed and assessed by measuring the relative growth of S. sonnei in cultures co-inoculated with a strain of bacteriocin-producing E. coli that is inhibitory to Shigella growth. S. sonnei was not recovered from co-cultures grown in SB or mTSB without antibiotics. In contrast, media supplemented with streptomycin at 50 and 100 µg/mL, trimethoprim at 25 and 50 µg/mL, and sulfadiazine at 100 µg/mL increased the relative proportion of S. sonnei in post-enrichment cultures. The enhanced recovery of resistant S. sonnei strains achieved in this study indicates that, in cases where genomic data is available for clinical S. sonnei isolates, customization of selective enrichment media based on AMR gene detection could be a valuable tool for supporting the investigation of foodborne shigellosis outbreaks.

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.

Salmonella enterica subsp. enterica Typhimurium and its monophasic variant I 1;4,[5],12:i:- (MVST) are responsible for thousands of reported cases of salmonellosis each year in Canada, and countries worldwide. We investigated S. Typhimurium and MVST isolates recovered from raw shellfish harvested in Atlantic Canada by the Canadian Food Inspection Agency (CFIA) over the past decade, to assess the potential impact of these isolates on human illness and to explore possible routes of shellfish contamination. Whole-genome sequence analysis was performed on 210 isolates of S. Typhimurium and MVST recovered from various food sources, including shellfish. The objective was to identify genetic markers linked to ST-99, a sequence type specifically associated with shellfish, which could explain their high prevalence in shellfish. We also investigated the genetic similarity amongst CFIA ST-99 isolates recovered in different years and geographical locations. Finally, the study aimed to enhance the molecular serotyping of ST-99 isolates, as they are serologically classified as MVST but are frequently misidentified as S. Typhimurium through sequence analysis. To ensure recovery of ST-99 from shellfish was not due to favourable growth kinetics, we measured the growth rates of these isolates relative to other Salmonella and determined that ST-99 did not have a faster growth rate and/or shorter lag phase than other Salmonella evaluated. The CFIA ST-99 isolates from shellfish were highly clonal, with up to 81 high-quality single nucleotide variants amongst isolates. ST-99 isolates both within the CFIA collection and those isolated globally carried numerous unique deletions, insertions and mutations in genes, including some considered important for virulence, such as gene deletions in the type VI secretion system. Interestingly, several of these genetic characteristics appear to be unique to North America. Most notably was a large genomic region showing a high prevalence in genomes from Canadian isolates compared to those from the USA. Although the functions of the majority of the proteins encoded within this region remain unknown, the genes umuC and umuD, known to be protective against UV light damage, were present. While this study did not specifically examine the effects of mutations and insertions, results indicate that these isolates may be adapted to survive in specific environments, such as ocean water, where wild birds and/or animals serve as the natural hosts. Our hypothesis is reinforced by a global phylogenetic analysis, which indicates that isolates obtained from North American shellfish and wild birds are infrequently connected to isolates from human sources. These findings suggest a distinct ecological niche for ST-99, potentially indicating their specialization and adaptation to non-human hosts and environments, such as oceanic habitats.

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 labor-intensive, leading to increasing interest in exploring culture-independent diagnostic tests (CIDTs) for pathogen detection. However, few studies quantify the relative sensitivity and reliability of these CIDTs compared to current approaches. To address this issue, we conducted a comparison of the limit of detection (LOD50) for Salmonella between a culture-based method and three CIDTs: qPCR (targeting invA and stn), metabarcode (16S) sequencing, and shotgun metagenomic sequencing. Samples of chicken feed and chicken caecal contents were spiked with S. 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 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. However, Salmonella reads were detected in some unspiked feed samples, suggesting false-positive detection of Salmonella. In addition, the LOD50 in feeds was three logs lower than in caecal contents, underscoring the impact of background microbiota on Salmonella detection using all methods. IMPORTANCE:The appeal of culture-independent diagnostic tests (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. This study provides quantitative data on the sensitivity of CIDTs relative to current approaches, which is essential for determining the feasibility of implementing these methods in pathogen surveillance programs.

Shigella spp. are Gram-negative gastrointestinal bacterial pathogens that cause bacillary dysentery or shigellosis in humans. Isolation of Shigella from outbreak -associated foods is often problematic due to the lack of selectivity of cultural enrichment broths. To facilitate Shigella recovery from foods, we have developed strain -speci fic enrichment media based on the genomically-predicted antimicrobial resistance (AMR) features of an outbreak -associated Shigella sonnei strain harboring resistance genes for streptomycin (STR) and trimethoprim (TMP). To assess performance of the method, baby carrots were arti ficially contaminated with the S. sonnei strain at low (2.4 CFU), medium (23.5 CFU), and high levels (235 CFU) along with 10 -fold higher levels of a Shigella -inhibiting Escherichia coli strain. The target S. sonnei strain was successfully recovered from arti ficially -contaminated baby carrots when enriched in modi fied Tryptone Soya Broth (mTSB) supplemented with TMP, whereas Shigella was not recovered from Shigella broth (SB) or SB supplemented with STR. Quantitative PCR analysis indicated that supplementation of the enrichment broths with TMP or STR increased the relative proportion of S. sonnei in enrichment cultures, except at the lowest inoculation level for STR. Microbiome pro filing of the baby carrot enrichment cultures conducted by 16S rRNA gene sequencing indicated that both SB-STR and mTSB-TMP repressed the growth of competing Enterobacteriaceae in the enrichment cultures, relative to SB without supplementation. Overall, improved Shigella recovery was achieved with the addition of the appropriate custom selective agent during cultural enrichments demonstrating that genomically informed custom selective enrichment of Shigella could be a valuable tool for supporting future foodborne shigellosis outbreak investigations.

Salmonella enterica subsp. enterica Typhimurium and its monophasic variant I 1;4,[5],12:i:- (MVST) are responsible for thousands of reported cases of salmonellosis each year in Canada, and countries worldwide. We investigated S. Typhimurium and MVST isolates recovered from raw shellfish harvested in Atlantic Canada by the Canadian Food Inspection Agency (CFIA) over the past decade, to assess the potential impact of these isolates on human illness and to explore possible routes of shellfish contamination. Whole- genome sequence analysis was performed on 210 isolates of S. Typhimurium and MVST recovered from various food sources, including shellfish. The objective was to identify genetic markers linked to ST- 99, a sequence type specifically associated with shellfish, which could explain their high prevalence in shellfish. We also investigated the genetic similarity amongst CFIA ST- 99 isolates recovered in different years and geographical locations. Finally, the study aimed to enhance the molecular serotyping of ST- 99 isolates, as they are serologically classified as MVST but are frequently misidentified as S. Typhimurium through sequence analysis. To ensure recovery of ST- 99 from shellfish was not due to favourable growth kinetics, we measured the growth rates of these isolates relative to other Salmonella and determined that ST- 99 did not have a faster growth rate and/or shorter lag phase than other Salmonella evaluated. The CFIA ST- 99 isolates from shellfish were highly clonal, with up to 81 high- quality single nucleotide variants amongst isolates. ST- 99 isolates both within the CFIA collection and those isolated globally carried numerous unique deletions, insertions and mutations in genes, including some considered important for virulence, such as gene deletions in the type VI secretion system. Interestingly, several of these genetic characteristics appear to be unique to North America. Most notably was a large genomic region showing a high prevalence in genomes from Canadian isolates compared to those from the USA. Although the functions of the majority of the proteins encoded within this region remain unknown, the genes umuC and umuD, known to be protective against UV light damage, were present. While this study did not specifically examine the effects of mutations and insertions, results indicate that these isolates may be adapted to survive in specific environments, such as ocean water, where wild birds and/or animals serve as the natural hosts. Our hypothesis is reinforced by a global phylogenetic analysis, which indicates that isolates obtained from North American shellfish and wild birds are infrequently connected to isolates from human sources. These findings suggest a distinct ecological niche for ST- 99, potentially indicating their specialization and adaptation to non- human hosts and environments, such as oceanic habitats.

BACKGROUND:Culture-independent diagnostic tests are gaining popularity as tools for detecting pathogens in food. Shotgun sequencing holds substantial promise for food testing as it provides abundant information on microbial communities, but the challenge is in analyzing large and complex sequencing datasets with a high degree of both sensitivity and specificity. Falsely classifying sequencing reads as originating from pathogens can lead to unnecessary food recalls or production shutdowns, while low sensitivity resulting in false negatives could lead to preventable illness. RESULTS:We used simulated and published shotgun sequencing datasets containing Salmonella-derived reads to explore the appearance and mitigation of false positive results using the popular taxonomic annotation softwares Kraken2 and Metaphlan4. Using default parameters, Kraken2 is sensitive but prone to false positives, while Metaphlan4 is more specific but unable to detect Salmonella at low abundance. We then developed a bioinformatic pipeline for identifying and removing reads falsely identified as Salmonella by Kraken2 while retaining high sensitivity. Carefully considering software parameters and database choices is essential to avoiding false positive sample calls. With well-chosen parameters plus additional steps to confirm the taxonomic origin of reads, it is possible to detect pathogens with very high specificity and sensitivity.

Salmonella enterica subsp. enterica Typhimurium and its monophasic variant I 1;4,[5],12:i:- (MVST) are responsible for thousands of reported cases of salmonellosis each year in Canada, and countries worldwide. We investigated S. Typhimurium and MVST isolates recovered from raw shellfish harvested in Atlantic Canada by the Canadian Food Inspection Agency (CFIA) over the past decade, to assess the potential impact of these isolates on human illness and to explore possible routes of shellfish contamination. Whole-genome sequence analysis was performed on 210 isolates of S. Typhimurium and MVST recovered from various food sources, including shellfish. The objective was to identify genetic markers linked to ST-99, a sequence type specifically associated with shellfish, which could explain their high prevalence in shellfish. We also investigated the genetic similarity amongst CFIA ST-99 isolates recovered in different years and geographical locations. Finally, the study aimed to enhance the molecular serotyping of ST-99 isolates, as they are serologically classified as MVST but are frequently misidentified as S. Typhimurium through sequence analysis. To ensure recovery of ST-99 from shellfish was not due to favourable growth kinetics, we measured the growth rates of these isolates relative to other Salmonella and determined that ST-99 did not have a faster growth rate and/or shorter lag phase than other Salmonella evaluated. The CFIA ST-99 isolates from shellfish were highly clonal, with up to 81 high-quality single nucleotide variants amongst isolates. ST-99 isolates both within the CFIA collection and those isolated globally carried numerous unique deletions, insertions and mutations in genes, including some considered important for virulence, such as gene deletions in the type VI secretion system. Interestingly, several of these genetic characteristics appear to be unique to North America. Most notably was a large genomic region showing a high prevalence in genomes from Canadian isolates compared to those from the USA. Although the functions of the majority of the proteins encoded within this region remain unknown, the genes umuC and umuD, known to be protective against UV light damage, were present. While this study did not specifically examine the effects of mutations and insertions, results indicate that these isolates may be adapted to survive in specific environments, such as ocean water, where wild birds and/or animals serve as the natural hosts. Our hypothesis is reinforced by a global phylogenetic analysis, which indicates that isolates obtained from North American shellfish and wild birds are infrequently connected to isolates from human sources. These findings suggest a distinct ecological niche for ST-99, potentially indicating their specialization and adaptation to non-human hosts and environments, such as oceanic habitats.

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.

Shiga toxigenic Escherichia coli (STEC) have been implicated in major foodborne outbreaks worldwide. The STEC family of pathogens is biochemically diverse, and current microbiological methods for detecting STEC are limited by the lack of a universal selective enrichment approach and prone to interference by high levels of background microbiota associated with certain types of foods. A novel approach has been developed for the recovery of foodborne illness outbreak strains during outbreak investigations based on the analysis of whole genome sequence data of implicated clinical isolates to determine antimicrobial resistance (AMR) genes. The presence of certain AMR genes in STEC has been correlated with the ability to grow in the presence of a specific antibiotic, which can be used to supplement enrichment broths to improve the recovery of a target strain. The enhanced recovery of STEC strains with different AMR profiles from various food types (beef, sprouts, leafy greens, and raw milk cheese) containing high levels of background microbiota was demonstrated using AMR predictions for nine different antibiotics. This genomically informed custom selective enrichment approach increases the availability of analytical options and improves the reliability of food microbiological analyses in confirming food vehicles implicated in outbreak events and defining the scope of product contamination to support risk assessment and risk management actions.

Background With the escalating risk of antimicrobial resistance (AMR), there are limited analytical options available that can comprehensively assess the burden of AMR carried by clinical/environmental samples. Food can be a potential source of AMR bacteria for humans, but its significance in driving the clinical spread of AMR remains unclear, largely due to the lack of holistic-yet-sensitive tools for surveillance and evaluation. Metagenomics is a culture-independent approach well suited for uncovering genetic determinants of defined microbial traits, such as AMR, present within unknown bacterial communities. Despite its popularity, the conventional approach of non-selectively sequencing a sample’s metagenome (namely, shotgun-metagenomics) has several technical drawbacks that lead to uncertainty about its effectiveness for AMR assessment; for instance, the low discovery rate of resistance-associated genes due to their naturally small genomic footprint within the vast metagenome. Here, we describe the development of a targeted resistome sequencing method and demonstrate its application in the characterization of the AMR gene profile of bacteria associated with several retail foods. Result A targeted-metagenomic sequencing workflow using a customized bait-capture system targeting over 4,000 referenced AMR genes and 263 plasmid replicon sequences was validated against both mock and sample-derived bacterial community preparations. Compared to shotgun-metagenomics, the targeted method consistently provided for improved recovery of resistance gene targets with a much-improved target detection efficiency (> 300-fold). Targeted resistome analyses conducted on 36 retail-acquired food samples (fresh sprouts, n = 10; ground meat, n = 26) and their corresponding bacterial enrichment cultures (n = 36) reveals in-depth features regarding the identity and diversity of AMR genes, most of which were otherwise undetected by the whole-metagenome shotgun sequencing method. Furthermore, our findings suggest that foodborne Gammaproteobacteria could be the major reservoir of food-associated AMR genetic determinants, and that the resistome structure of the selected high-risk food commodities are, to a large extent, dictated by microbiome composition. Conclusions For metagenomic sequencing-based surveillance of AMR, the target-capture method presented herein represents a more sensitive and efficient approach to evaluate the resistome profile of complex food or environmental samples. This study also further implicates retail foods as carriers of diverse resistance-conferring genes indicating a potential impact on the dissemination of AMR.

ABSTRACT This study was to assess the gene diversity and characterize a large set of plasmids harboring extended β-lactamase (ESBL) genes from raw and digested dairy manure. A total of eighty-four plasmids that were captured in this E. coli recipient were sequenced using Illumina MiSeq sequencing technology. Twenty-four plasmids of interest were subsequently sequenced using MinION technology in order that a hybrid assembly could be performed on short-and long-read sequences to circularize and complete these plasmids. The size of sequenced plasmids ranged between 40 and 260 kb with various incompatibility groups: IncC, IncI1, IncN, IncY, IncB/O/K/Z, IncX1, IncHI2, IncHI2A, IncFIB(K), IncFII. A variety of extended β-lactamase genes were identified: bla CTXM -1 , bla CTXM -14 , bla CTXM -15 , bla CTXM-27 , bla CTXM-55 , bla CTXM-61 , bla PER-1, bla IMP-27 . Interestingly, the bla IMP-27 gene, a novel metallo-β-lactamase discovered in the last decade, was found located on an integrated region in the host chromosome. And one plasmid carrying the bla CMY-2 gene, an AmpC gene, also expressed ESBL phenotype. Four virulence factors, including cia, cib, traT and terC, were detected on some of these plasmids. In addition, six type-2 toxin-antitoxin systems were detected: MazF/E, PemK/I, HipA/B, YdcE/D, RelB/E and HigB/A. Twenty-two out of twenty-four complete plasmids carried putative prophage regions; and most of prophage hits were marked as incomplete, except that the largest plasmid pT525A and the IncY plasmid pT415A had prophage hits with higher scores. IMPORTANCE The widespread of antibiotic resistant bacteria is largely due to the exchange of mobile genetic elements such as plasmids. Plasmids harboring extended β-lactamase (ESBL) genes originated from dairy manure potentially become entrained in manured soil, which subsequently enter the human food chain. Currently there is a lack of detailed information on these plasmids in the environment, specifically in dairy manure. This study unveils the abundance and diversity of ESBL-carrying plasmids from both raw and digested manures which were captured in gfp- labelled E. coli CV601. In addition, the study provides insightful information of plasmid characteristics including incompatibility groups, ESBL genes combined with other resistance genes, mobile genetic elements (transposons, insertion sequence), toxin-antitoxin systems, virulence factors and prophage sequences.

Background Culture-independent diagnostic tests (CIDTs) are gaining popularity as tools for detecting pathogens in food. Shotgun sequencing holds substantial promise for food testing as it provides abundant information on microbial communities, but the challenge is in analyzing large and complex sequencing datasets with a high degree of both sensitivity and specificity. Falsely classifying sequencing reads as originating from pathogens can lead to unnecessary food recalls or production shutdowns, while low sensitivity resulting in false negatives could lead to preventable illness. Results We have developed a bioinformatic pipeline for identifying Salmonella as a model pathogen in metagenomic datasets with very high sensitivity and specificity. We tested this pipeline on mock communities of closely related bacteria and with simulated Salmonella reads added to published metagenomic datasets. Salmonella -derived reads could be found at very low abundances (high sensitivity) without false positives (high specificity). Carefully considering software parameters and database choices is essential to avoiding false positive sample calls. With well-chosen parameters plus additional steps to confirm the taxonomic origin of reads, it is possible to detect pathogens with very high specificity and sensitivity.