Characterization of MLST-99 Salmonella Typhimurium and the monophasic variant I:4,[5],12:i:- isolated from Canadian Atlantic coast shellfish.

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.

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

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

Resistance determinants and their genetic context in enterobacteria from a longitudinal study of pigs reared under various husbandry conditions.

Pigs are major reservoirs of resistant Enterobacteriaceae that can reach humans through consumption of contaminated meat or vegetables grown in manure-fertilized soil. Samples were collected from sows during lactation and their piglets at five time points spanning the production cycle. Cefotaxime-resistant bacteria were quantified and isolated from feed, feces, manures and carcasses of pigs reared with penicillin-using or antibiotic-free husbandries. The isolates were characterized by antibiotic susceptibility testing, whole genome sequencing and conjugation assays. The extended spectrum ß-lactamase (ESBL) phenotype was more frequent in isolates originating from antibiotic-free animals, while the bacteria isolated from penicillin-using animals were on average resistant to a greater number of antibiotics. The ESBL-encoding genes identified were bla CTX-M-1, bla CTX-M-15 and bla CMY-2 and they co-localised on plasmids with various genes encoding resistance to ß-lactams, co-trimoxazole, phenicols and tetracycline, all antibiotics used in pig production. Groups of genes conferring the observed resistance and the mobile elements disseminating multidrug resistance were determined. The observed resistance to ß-lactams was mainly due to the complementary actions of penicillin-binding proteins, an efflux pump and ß-lactamases. Most resistance determinants were shared by animals raised with or without antimicrobials. This suggests a key contribution of indigenous enterobacteria maternally transmitted along the sow lineage, regardless of antimicrobial use. It is unclear if the antimicrobial resistance observed in the enterobacteria populations of the commercial pig herds studied were present before the use of antibiotics, or the extent to which historical antimicrobial use exerted a selective pressure defining the resistant bacterial populations in farms using penicillin prophylaxis.Importance: Antimicrobial resistance is a global threat that needs to be fought on numerous fronts along the One Health continuum. Vast quantities of antimicrobials are used in agriculture to ensure animal welfare and productivity, and are arguably a driving force for the persistence of environmental and food-borne resistant bacteria. This study evaluated the impact of conventional, organic and other antibiotic-free husbandry practices on the frequency and nature of antimicrobial resistance genes and multidrug resistant enterobacteria. It provides knowledge about the relative contribution of specific resistance determinants to observed antibiotic resistance. It also showed the clear co-selection of genes coding for extended-spectrum beta-lactamases and genes coding for the resistance to antibiotics commonly used for prophylaxis or in curative treatments in pig operations.

Local genes for local bacteria: Evidence of allopatry in the genomes of transatlantic Campylobacter populations.

The genetic structure of bacterial populations can be related to geographical locations of isolation. In some species, there is a strong correlation between geographical distance and genetic distance, which can be caused by different evolutionary mechanisms. Patterns of ancient admixture in Helicobacter pylori can be reconstructed in concordance with past human migration, whereas in Mycobacterium tuberculosis it is the lack of recombination that causes allopatric clusters. In Campylobacter, analyses of genomic data and molecular typing have been successful in determining the reservoir host species, but not geographical origin. We investigated biogeographical variation in highly recombining genes to determine the extent of clustering between genomes from geographically distinct Campylobacter populations. Whole-genome sequences from 294 Campylobacter isolates from North America and the UK were analysed. Isolates from within the same country shared more recently recombined DNA than isolates from different countries. Using 15 UK/American closely matched pairs of isolates that shared ancestors, we identify regions that have frequently and recently recombined to test their correlation with geographical origin. The seven genes that demonstrated the greatest clustering by geography were used in an attribution model to infer geographical origin which was tested using a further 383 UK clinical isolates to detect signatures of recent foreign travel. Patient records indicated that in 46 cases, travel abroad had occurred <2 weeks prior to sampling, and genomic analysis identified that 34 (74%) of these isolates were of a non-UK origin. Identification of biogeographical markers in Campylobacter genomes will contribute to improved source attribution of clinical Campylobacter infection and inform intervention strategies to reduce campylobacteriosis.

Campylobacter species in animal, food, and environmental sources, and relevant testing programs in Canada.

Campylobacter species, particularly thermophilic campylobacters, have emerged as a leading cause of human foodborne gastroenteritis worldwide, with Campylobacter jejuni, Campylobacter coli, and Campylobacter lari responsible for the majority of human infections. Although most cases of campylobacteriosis are self-limiting, campylobacteriosis represents a significant public health burden. Human illness caused by infection with campylobacters has been reported across Canada since the early 1970s. Many studies have shown that dietary sources, including food, particularly raw poultry and other meat products, raw milk, and contaminated water, have contributed to outbreaks of campylobacteriosis in Canada. Campylobacter spp. have also been detected in a wide range of animal and environmental sources, including water, in Canada. The purpose of this article is to review (i) the prevalence of Campylobacter spp. in animals, food, and the environment, and (ii) the relevant testing programs in Canada with a focus on the potential links between campylobacters and human health in Canada.

Strain-specific Recovery of S. sonnei from Artificially Contaminated Baby Carrots: Enhancing Food-safety Investigations with a Customized Shigella Detection Method Based on Genomically predicted Antibiotic Resistance Traits.

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-specific 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 artificially 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 artificially-contaminated baby carrots when enriched in modified 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 profiling 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.

Overcoming Microbial Inhibition of S. Sonnei Through the Exploitation of Genomically Predicted Antibiotic Resistance Profiles for the Development of Food Enrichment Media.

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 suppress the 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 coinoculated with a strain of bacteriocin-producing E. coli that is inhibitory to Shigella growth. S. sonnei was not recovered from cocultures 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 postenrichment cultures. The enhanced recovery of resistant S. sonnei strains achieved in this study indicates that, in cases where genomic data are 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.

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

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

Exploiting a targeted resistome sequencing approach in assessing antimicrobial resistance in retail foods.

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.

Virulence potential of antimicrobial-resistant extraintestinal pathogenic Escherichia coli from retail poultry meat in a Caenorhabditis elegans model.

Healthy poultry can be a reservoir for extraintestinal pathogenic Escherichia coli (ExPEC), some of which could be multidrug resistant to antimicrobials. These ExPEC strains could contaminate the environment and/or food chain representing thus, food safety and human health risk. However, few studies have shown the virulence of poultry-source antimicrobial-resistant (AMR) ExPEC in humans. This study characterized AMR ExPEC and investigated the virulence potential of some of their isolates in a Caenorhabditis elegans infection model. A total of 46 E. coli isolates from poultry (chicken, n = 29; turkey, n = 12) retail meats and chicken feces (n = 4), or humans (n = 1) were sequenced and identified as ExPEC. Except eight, all remaining 38 ExPEC isolates were resistant to at least one antibiotic and carried corresponding antimicrobial resistance genes (ARGs). About 27 of the 46 ExPEC isolates were multidrug-resistant (≥3 antibiotic classes). Seven ExPEC isolates from chicken or turkey meats were of serotype O25:H4 and sequence type (ST) 131 which clustered with an isolate from a human urinary tract infection (UTI) case having the same serotype and ST. The C. elegans challenge model using eight of studied ExPEC isolates harboring various ARGs and virulence genes (VGs) showed that regardless of their ARG or VG numbers in tested poultry meat and feces, ExPEC significantly reduced the life span of the nematode (P < 0.05) similarly to a human UTI isolate. This study indicated the pathogenic potential of AMR ExPEC from retail poultry meat or feces, but more studies are warranted to establish their virulence in poultry and human. Furthermore, relationships between specific resistance profiles and/or VGs in these E. coli isolates for their pathogenicity deserve investigations.

Modulation of toxin production by the flagellar regulon in Clostridium difficile.

We show in this study that toxin production in Clostridium difficile is altered in cells which can no longer form flagellar filaments. The impact of inactivation of fliC, CD0240, fliF, fliG, fliM, and flhB-fliR flagellar genes upon toxin levels in culture supernatants was assessed using cell-based cytotoxicity assay, proteomics, immunoassay, and immunoblotting approaches. Each of these showed that toxin levels in supernatants were significantly increased in a fliC mutant compared to that in the C. difficile 630 parent strain. In contrast, the toxin levels in supernatants secreted from other flagellar mutants were significantly reduced compared with that in the parental C. difficile 630 strain. Transcriptional analysis of the pathogenicity locus genes (tcdR, tcdB, tcdE, and tcdA) revealed a significant increase of all four genes in the fliC mutant strain, while transcription of all four genes was significantly reduced in fliM, fliF, fliG, and flhB-fliR mutants. These results demonstrate that toxin transcription in C. difficile is modulated by the flagellar regulon. More significantly, mutant strains showed a corresponding change in virulence compared to the 630 parent strain when tested in a hamster model of C. difficile infection. This is the first demonstration of differential flagellum-related transcriptional regulation of toxin production in C. difficile and provides evidence for elaborate regulatory networks for virulence genes in C. difficile.

Microbial Antagonism in Food-Enrichment Culture: Inhibition of Shiga Toxin-Producing Escherichia coli and Shigella Species.

Bacterial pathogens, such as Shiga toxin-producing Escherichia coli (STEC) and Shigella spp., are important causes of foodborne illness internationally. Recovery of these organisms from foods is critical for food safety investigations to support attribution of illnesses to specific food commodities; however, isolation of bacterial cultures can be challenging. Methods for the isolation of STEC and Shigella spp. from foods typically require enrichment to amplify target organisms to detectable levels. Yet, during enrichment, target organisms can be outcompeted by other bacteria in food matrices due to faster growth rates, or through production of antimicrobial agents such as bacteriocins or bacteriophages. The purpose of this study was to evaluate the occurrence of Shigella and STEC inhibitors produced by food microbiota. The production of antimicrobial compounds in cell-free extracts from 200 bacterial strains and 332 food-enrichment broths was assessed. Cell-free extracts produced by 23 (11.5%) of the strains tested inhibited growth of at least one of the five Shigella and seven STEC indicator strains used in this study. Of the 332 enrichment broths tested, cell-free extracts from 25 (7.5%) samples inhibited growth of at least one of the indicator strains tested. Inhibition was most commonly associated with E. coli recovered from meat products. Most of the inhibiting compounds were determined to be proteinaceous (34 of the 48 positive samples, 71%; including 17 strains, 17 foods) based on inactivation by proteolytic enzymes, indicating presence of bacteriocins. The cell-free extracts from 13 samples (27%, eight strains, five foods) were determined to contain bacteriophages based on the observation of plaques in diluted extracts and/or resistance to proteolytic enzymes. These results indicate that the production of inhibitors by food microbiota may be an important challenge for the recovery of foodborne pathogens, particularly for Shigella sonnei. The performance of enrichment media for recovery of Shigella and STEC could be improved by mitigating the impact of inhibitors produced by food microbiota during the enrichment process.

Virulence Genotype and Phenotype of Multiple Antimicrobial-Resistant Escherichia coli Isolates from Broilers Assessed from a "One-Health" Perspective.

ABSTRACT: Extraintestinal pathogenic Escherichia coli (ExPEC) include several serotypes that have been associated with colibacillosis in poultry and with urinary tract infections (UTIs) and newborn meningitis in humans. In this study, 57 antimicrobial-resistant E. coli from apparently healthy broiler chickens were characterized for their health and safety risks. These isolates belonged to 12 serotypes, and isolates of the same serotype were clonal based on single nucleotide variant analysis. Most of the isolates harbored plasmids; IncC and IncFIA were frequently detected. The majority of the resistant isolates harbored plasmid-mediated resistance genes, including aph(3″)-Ib, aph(6)-Id, blaCMY-2, floR, sul1, sul2, tet(A), and tet(B), in agreement with their resistant phenotypes. The class 1 integron was detected in all E. coli serotypes except O124:H25 and O7:H6. Of the 57 broiler E. coli isolates, 27 were avian pathogenic, among which 18 were also uropathogenic E. coli and the remainder were other ExPEC. The two isolates of serotype O161:H4 (ST117) were genetically related to the control avian pathogenic strains and a clinical isolate associated with UTIs. A strain of serotype O159:H45 (ST101) also was closely related to a UTI isolate. The detected virulence factors included adhesins, invasins, siderophores, type III secretion systems, and toxins in combination with other virulence determinants. A broiler isolate of serotype O7:H18 (ST38) carried the ibeA gene encoding a protein involved in invasion of brain endothelium on a 102-kbp genetic island. This isolate moderately adhered and invaded Caco-2 cells and induced mortality (42.5%) in a day-old-chick infection model. The results of this study suggest that multiple antimicrobial-resistant E. coli isolates recovered from apparent healthy broilers can be pathogenic and act as reservoirs for antimicrobial resistance genes, highlighting the necessity of their assessment in a "One-Heath" context.

Genomic Markers for Quaternary Ammonium Compound Resistance as a Persistence Indicator for Listeria monocytogenes Contamination in Food Manufacturing Environments.

ABSTRACT: Persistent contamination of food manufacturing environments by Listeria monocytogenes is an important public health risk, because such contamination events defy standard sanitization protocols, for example, the application of quaternary ammonium compounds such as benzalkonium chloride (BC), providing a source for prolonged dissemination of the bacteria in food products. We performed whole genome sequencing analyses of 1,279 well-characterized L. monocytogenes isolates from various foods and food manufacturing environments and identified the bcrABC gene cassette associated with BC resistance in 531 (41.5%) isolates. The bcrABC cassette was significantly associated with L. monocytogenes isolates belonging to clonal complex (CC) 321, CC155, CC204, and CC199, which are among the 10 most prevalent genotypes recovered from foods and food production environments. All but 1 of the 177 CC321 isolates harbored the bcrABC cassette. In addition, 384 (38.6%) of the 994 isolates recovered from foods representing 67 different CCs and 119 (59.2%) of isolates from food manufacturing environmental samples representing 26 different CCs were found to harbor the intact bcrABC cassette. A representative set of 69 isolates with and without bcrABC was assayed for the ability to grow in the presence of BC, and 34 of 35 isolates harboring the bcrABC cassette exhibited MICs of ≥10 µg/mL BC. Determination of bcrABC in isolates could be achieved using both PCR and whole genome sequencing techniques, providing food testing laboratories with options for the characterization of isolates. The ability to determine markers of quaternary ammonium compound resistance such as bcrABC and epidemiologic lineage may provide risk managers with a tool to assess the potential for persistent contamination of the food manufacturing environment and the need for more targeted surveillance to ensure the efficacy of mitigation actions.

Systematic Evaluation of Whole-Genome Sequencing Based Prediction of Antimicrobial Resistance in Campylobacter jejuni and C. coli.

The increasing prevalence of antimicrobial resistance (AMR) in Campylobacter spp. is a global concern. This study evaluated the use of whole-genome sequencing (WGS) to predict AMR in Campylobacter jejuni and C. coli. A panel of 271 isolates recovered from Canadian poultry was used to compare AMR genotype to antimicrobial susceptibility testing (AST) results (azithromycin, ciprofloxacin, erythromycin, gentamicin, tetracycline, florfenicol, nalidixic acid, telithromycin, and clindamycin). The presence of antibiotic resistance genes (ARGs) was determined for each isolate using five computational approaches to evaluate the effect of: ARG screening software, input data (i.e., raw reads, draft genome assemblies), genome coverage and genome assembly software. Overall, concordance between the genotype and phenotype was influenced by the computational pipelines, level of genome coverage and the type of ARG but not by input data. For example, three of the pipelines showed a 99% agreement between detection of a tet(O) gene and tetracycline resistance, whereas agreement between the detection of tet(O) and TET resistance was 98 and 93% for two pipelines. Overall, higher levels of genome coverage were needed to reliably detect some ARGs; for example, at 15X coverage a tet(O) gene was detected in >70% of the genomes, compared to <60% of the genomes for bla(OXA). No genes associated with florfenicol or gentamicin resistance were found in the set of strains included in this study, consistent with AST results. Macrolide and fluoroquinolone resistance was associated 100% with mutations in the 23S rRNA (A2075G) and gyrA (T86I) genes, respectively. A lower association between a A2075G 23S rRNA gene mutation and resistance to clindamycin and telithromycin (92.8 and 78.6%, respectively) was found. While WGS is an effective approach to predicting AMR in Campylobacter, this study demonstrated the impact that computational pipelines, genome coverage and the genes can have on the reliable identification of an AMR genotype.

Antimicrobial Resistance Phenotypes and Genotypes of Escherichia coli Isolates from Broiler Chickens Fed Encapsulated Cinnamaldehyde and Citral.

ABSTRACT: This study was conducted to investigate the effects of in-feed encapsulated cinnamaldehyde (CIN) and citral (CIT) alone or in combination on antimicrobial resistance (AMR) phenotypes and genotypes of Escherichia coli isolates recovered from feces of 6-, 16-, 23-, and 27-day-old broiler chickens. The five dietary treatments including the basal diet (negative control [NC]) and the basal diet supplemented with 55 ppm of bacitracin (BAC), 100 ppm of encapsulated CIN, 100 ppm of encapsulated CIT, or 100 ppm each of encapsulated CIN and encapsulated CIT (CIN+CIT). Antimicrobial susceptibility testing of 240 E. coli isolates revealed that the most common resistance was to ß-lactams, aminoglycosides, sulfonamides, and tetracycline; however, the prevalence of AMR decreased (P < 0.05) as birds aged. The prevalence of resistance to amoxicillin-clavulanic acid, ceftiofur, ceftriaxone, cefoxitin, gentamicin, and sulfonamide was lower (P < 0.05) in isolates from the CIN or CIN+CIT groups than in isolates from the NC or BAC groups. Whole genome sequencing of 227 of the 240 isolates revealed 26 AMR genes and 19 plasmids, but the prevalence of some AMR genes and the number of plasmids were lower (P < 0.05) in E. coli isolated from CIN or CIN+CIT birds than in isolates from NC or BAC birds. The most prevalent resistance genes were tet(A) (108 isolates), aac(3)-VIa (91 isolates), aadA1 (86 isolates), blaCMY-2 (78 isolates), sul1 (77 isolates), aph(3)-Ib (58 isolates), aph(6)-Id (58 isolates), and sul2 (24 isolates). The numbers of most virulence genes carried by isolates increased (P < 0.05) in chickens from 6 to 27 days of age. The prevalence of E. coli O21:H16 isolates was lower (P < 0.05) in CIN and CIN+CIT, and the colibacillosis-associated multilocus sequence type (ST117) was most prevalent in isolates from 23-day-old chickens. A phylogenetic tree of whole genome sequences revealed a close relationship between 25 of the 227 isolates and human or broiler extraintestinal pathogenic E. coli strains. These findings indicate that AMR and virulence genotypes of E. coli could be modulated by providing encapsulated CIN or CIN+CIT feed supplements, but further investigation is needed to determine the mechanisms of the effects of these supplements.

A comparison of fourteen fully characterized mammalian-associated Campylobacter fetus isolates suggests that loss of defense mechanisms contribute to high genomic plasticity and subspecies evolution.

Campylobacter fetus is currently classified into three main subspecies, but only two of these, C. fetus subspecies fetus and C. fetus subsp. venerealis originate principally from ruminants where they inhabit different niches and cause distinct pathogenicity. Their importance as pathogens in international trade and reporting is also different yet the criteria defining these properties have never been fully substantiated nor understood. The situation is further compromised because the ability to differentiate between these two closely related C. fetus subspecies has traditionally been performed by phenotypic characterisation of isolates, methods which are limited in scope, time-consuming, tedious, and often yield inconsistent results, thereby leading to isolate misidentification. The development of robust genetic markers that could enable rapid discrimination between C. fetus subsp. fetus and subsp. venerealis has also been challenging due to limited differences in the gene complement of their genomes, high levels of sequence repetition, the small number of closed genome sequences available and the lack of standardisation of the discriminatory biochemical tests employed for comparative purposes. To yield a better understanding of the genomic differences that define these C. fetus strains, seven isolates were exhaustively characterised phenotypically and genetically and compared with seven previously well characterised isolates. Analysis of these 14 C. fetus samples clearly illustrated that adaption by C. fetus subsp. venerealis to the bovine reproductive tract correlated with increasing genome length and plasticity due to the acquisition and propagation of several mobile elements including prophages, transposons and plasmids harbouring virulence factors. Significant differences in the repertoire of the CRISPR (clustered regularly interspersed short palindromic repeats)-cas system of all C. fetus strains was also found. We therefore suggest that a deficiency in this adaptive immune system may have permitted the emergence of extensive genome plasticity and led to changes in host tropism through gene disruption and/or changes in gene expression. Notable differences in the sub-species complement of DNA adenine methylase genes may also have an impact. These data will facilitate future studies to better understand the precise genetic differences that underlie the phenotypic and virulence differences between these animal pathogens and may identify additional markers useful for diagnosis and sub-typing.

"These Aren't the Strains You're Looking for": Recovery Bias of Common Campylobacter jejuni Subtypes in Mixed Cultures.

Microbiological surveillance of the food chain plays a critical role in improving our understanding of the distribution and circulation of food-borne pathogens along the farm to fork continuum toward the development of interventions to reduce the burden of illness. The application of molecular subtyping to bacterial isolates collected through surveillance has led to the identification of strains posing the greatest risk to public health. Past evidence suggests that enrichment methods for Campylobacter jejuni, a leading bacterial foodborne pathogen worldwide, may lead to the differential recovery of subtypes, obscuring our ability to infer the composition of a mixed-strain sample and potentially biasing prevalence estimates in surveillance data. To assess the extent of potential selection bias resulting from enrichment-based isolation methods, we compared enrichment and non-enrichment isolation of mixed subtype cultures of C. jejuni, followed by subtype-specific enumeration using both colony plate-counts and digital droplet PCR. Results differed from the null hypothesis that similar proportions of C. jejuni subtypes are recovered from both methods. Our results also indicated a significant effect of subtype prevalence on isolation frequency post-recovery, with the recovery of more common subtypes being consistently favored. This bias was exacerbated when an enrichment step was included in the isolation procedure. Taken together, our results emphasize the importance of selecting multiple colonies per sample, and where possible, the use of both enrichment and non-enrichment isolation procedures to maximize the likelihood of recovering multiple subtypes present in a sample. Moreover, the effects of subtype-specific recovery bias should be considered in the interpretation of strain prevalence data toward improved risk assessment from microbiological surveillance data.

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

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

Genomically Informed Strain-Specific Recovery of Shiga Toxin-Producing Escherichia coli during Foodborne Illness Outbreak Investigations.

Next-generation sequencing plays an important role in the characterization of clinical bacterial isolates for source attribution purposes during investigations of foodborne illness outbreaks. Once an illness cluster and a suspect food vehicle have been identified, food testing is initiated for confirmation and to determine the scope of a contamination event so that the implicated lots may be removed from the marketplace. For biochemically diverse families of pathogens such as Shiga toxin-producing Escherichia coli (STEC), the ability to detect specific strains may be hampered by the lack of a universal selective enrichment approach for their recovery against high levels of background microbiota. The availability of whole genome sequence data for a given outbreak STEC strain prior to commencement of food testing may provide food microbiologists an opportunity to customize selective enrichment techniques favoring the recovery of the outbreak strain. Here we demonstrate the advantages of using the publicly available ResFinder tool in the analysis of STEC model strains belonging to serotypes O111 and O157 to determine antimicrobial resistance traits that can be used in formulating strain-specific enrichment media to enhance recovery of these strains from microbiologically complex food samples. The improved recovery from ground beef of model STEC strains with various antimicrobial resistance profiles was demonstrated using three classes of antibiotics as selective agents, suggesting the universal applicability of this new approach in supporting foodborne illness investigations.