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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.

Shiga toxin-producing Escherichia coli (STEC) O157:H7/nonmotile and some non-O157 STEC strains are foodborne pathogens. In response to pork-associated O157 STEC outbreaks in Canada, we investigated the occurrence of STEC in Canadian retail raw ground pork during the period of 1 November 2014 to 31 March 2016. Isolated STEC strains were characterized to determine the Shiga toxin gene (stx) subtype and the presence of virulence genes encoding intimin (eae) and enterohemorrhagic E. coli hemolysin (hlyA). O157 STEC and non-O157 STEC strains were isolated from 1 (0.11%) of 879 and 13 (2.24%) of 580 pork samples, respectively. STEC virulence gene profiles containing both eae and hlyA were found only in the O157 STEC (stx2a, eae, hlyA) isolate. The eae gene was absent from all non-O157 STEC isolates. Of the 13 non-O157 STEC isolates, two virulence genes of stx1a and hlyA were found in four (30.8%) O91:H14 STEC isolates, whereas one virulence gene of stx2e, stx1a, and stx2a was identified in five (38.5%), two (15.4%), and one (7.7%) STEC isolates, respectively, of various serotypes. The remaining non-O157 STEC isolate carried stx2, but the subtype is unknown because this isolate could not be recovered for sequencing. O91:H14 STEC (stx1a, hlyA) was previously reported in association with diarrheal illnesses, whereas the other non-O157 STEC isolates identified in this study are not known to be associated with severe human illnesses. Virulence gene profiles identified in this study indicate that the occurrence of non-O157 STEC capable of causing severe human illness is rare in Canadian retail pork. However, O157 STEC in ground pork can occasionally occur; therefore, education regarding the potential risks associated with STEC contamination of pork would be beneficial for the public and those in the food industry to help reduce foodborne illnesses. HIGHLIGHTS

Next Generation Sequencing technologies significantly impact the field of Antimicrobial Resistance (AMR) detection and monitoring, with immediate uses in diagnosis and risk assessment. For this application and in general, considerable challenges remain in demonstrating sufficient trust to act upon the meaningful information produced from raw data, partly because of the reliance on bioinformatics pipelines, which can produce different results and therefore lead to different interpretations. With the constant evolution of the field, it is difficult to identify, harmonise and recommend specific methods for large-scale implementations over time. In this article, we propose to address this challenge through establishing a transparent, performance-based, evaluation approach to provide flexibility in the bioinformatics tools of choice, while demonstrating proficiency in meeting common performance standards. The approach is two-fold: first, a community-driven effort to establish and maintain “live” (dynamic) benchmarking platforms to provide relevant performance metrics, based on different use-cases, that would evolve together with the AMR field; second, agreed and defined datasets to allow the pipelines’ implementation, validation, and quality-control over time. Following previous discussions on the main challenges linked to this approach, we provide concrete recommendations and future steps, related to different aspects of the design of benchmarks, such as the selection and the characteristics of the datasets (quality, choice of pathogens and resistances, etc.), the evaluation criteria of the pipelines, and the way these resources should be deployed in the community.

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 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.

Heidelberg is among the top three Salmonella enterica serovars associated with human foodborne illness in Canada. Traditional culture and antimicrobial susceptibility testing techniques can be time-consuming to identify Salmonella Heidelberg resistant to cephalosporins and fosfomycin. Rapid and accurate detection of such antibiotic-resistant Salmonella Heidelberg isolates is essential to adopt appropriate control measures. In this study, 15 Salmonella Heidelberg strains isolated from feces of Canadian broiler chickens were characterized by whole genome sequencing. Salmonella Heidelberg genomes had an average coverage of greater than 80-fold, an average of 4,761 protein-coding genes, and all belonged to multilocus sequence type ST15. Genome sequences were compared with genomes in the National Center for Biotechnology Information Pathogen Detection database ( www.ncbi.nlm.nih.gov/pathogens/ ), including human outbreak isolates. The Canadian broiler isolates clustered with chicken isolates from the United States and an equine clinical isolate from Ontario, Canada. In agreement with their antimicrobial resistance phenotypes, several chromosomally encoded specific antimicrobial resistance genes including fosA7 and multidrug resistance efflux pump determinants were detected. An AmpC-like β-lactamase gene, blaCMY-2, linked with a quaternary ammonium compound resistance gene, sugE, on a replicon type IncI1 plasmid was detected in all 15 broiler Salmonella Heidelberg isolates. Of the 205,031 published Salmonella genomes screened in silico, 4,954 (2.4%) contained blaCMY-2, 8,143 (4.0%) contained fosA7, and 919 (0.4%) contained both resistance genes. The combination of both resistance genes (fosA7 and blaCMY-2) was detected in 64% of the Heidelberg genomes and in a small proportion of various other serovars. A PCR method was developed to detect Salmonella Heidelberg in pure culture and chicken feces based on specific primers targeting genes conferring fosfomycin (fosA7) and third-generation cephalosporin (blaCMY-2) resistance as well as the Salmonella-specific invA gene and the universal 16S rRNA genes. The PCR assay was specific and sensitive for blaCMY-2 and fosA7 harboring Salmonella Heidelberg. However, some other Salmonella serovars containing these two resistance genes could also be detected by the developed PCR method.

Enteritidis and Typhimurium are among the top Salmonella enterica serovars implicated in human salmonellosis worldwide. This study examined the individual and combined roles of catecholate-iron and hydroxamate-iron transporters in the survival in meat of Salmonella Enteritidis and Typhimurium. Catecholate–iron-III (Fe3+) and hydroxamate-Fe3+ transporter genes fepA, iroN, and fhuACDB were deleted in isolates of these serovars to generate single, double, and triple mutants. Growth rate in high- and low-iron media was compared among mutants, complements, and their wild-type parents. Susceptibility to 14 antibiotics, the ability to produce and utilize siderophores, and survival on cooked chicken breast were evaluated. In iron-poor liquid media, differences were observed between the growth characteristics of mutant Salmonella Enteritidis and Typhimurium. The double ΔiroNΔfepA and the triple ΔfhuΔiroNΔfepA mutants of Salmonella Enteritidis exhibited prolonged lag phases (λ = 9.72 and 9.53 h) and a slow...

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 β-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. HIGHLIGHTS

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. HIGHLIGHTS

Following two O121 STEC outbreaks linked to wheat flour, this study was conducted to gain baseline information on the occurrence of bacterial pathogens and levels of indicator organisms in wheat flour in Canada. A total of 347 pre-packaged wheat flour samples were analyzed for Salmonella spp., Shiga Toxin-Producing Escherichia coli (STEC), Listeria monocytogenes ( L. monocytogenes ) , aerobic colony count (ACC), total coliforms, and generic Escherichia coli ( E. coli ) . Salmonella spp. and O157 STEC were not detected in any of the samples. L. monocytogenes was identified in two samples (0.6%) at levels below the limit of detection (

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.

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.

This study investigated virulence potential of poultry antimicrobial resistant extraintestinal pathogenic E. coli (ExPEC). A total of 46 E. coli isolates from poultry meat, feces, or humans were sequenced and identified as ExPEC. Based on their characteristics, eight of these ExPEC isolates were evaluated for their potentials using a Caenorhabditis elegans infection model. The life-span of C. elegans in response to these eight isolates was examined in three life-span experiments: 1) E. coli OP 50 (negative control), K88+ enterotoxigenic E. coli strain JG280 (positive control), and an ExPEC isolate from human urinal tract infection; 2) three ExPEC isolates from chicken and turkey retail meats; 3) four ExPEC isolates from chicken feces with different antimicrobial resistance (AMR) profiles or a various number of virulence genes (VGs). All 46 isolates belonged to 24 serotypes among which 6 were of serotype O25:H4 Sequence Type 131 (ST131). Interestingly, all ST 131 isolates from chicken or turkey retail meats clustered with a human UTI isolate belonging to the similar serotype and ST type. The types and numbers of AGRs and VGs varied among the eight selected isolates for C. elegans model. The human ExPEC induced a similar effect as the JG280 on reducing (P < 0.05) survival of C. elegans. Interestingly, chicken and turkey meat ExPEC isolates, caused similar negative impacts on the survival of worms as the human ExPEC. Additionally, fecal ExPEC isolates reduced (P < 0.05) the survival of C. elegans compared to OP50. However, the survival of C. elegans was not reduced with an increasing number of VGs and did not seem to be affected by AMR profiles. This study indicated the virulence potential of ExPEC isolates from retail poultry meat or feces. The relationship between specific AMR profiles and/or numbers of VGs with pathogenicity in these E. coli isolates deserves further investigations.

This study investigated effects of in-feed encapsulated cinnamaldehyde (CIN) and citral (CIT) alone or in combination (CIN+CIT) on antimicrobial resistance (AMR) phenotypes and genotypes of E. coli isolated from feces of 6-, 16-, 23- and 27-days old broiler chickens. Five dietary treatments included the basal diet (NC), the basal diet supplemented with 55 ppm bacitracin (BAC), 100 ppm encapsulated CIN, CIT, or CIN+CIT. Antimicrobial susceptibility using a Sensititre method of 240 E. coli isolates showed that the most frequent resistances were against b-lactam, aminoglycoside, sulfonamide and tetracycline, however, the prevalence of AMR decreased (P < 0.05) when birds aged. The prevalence of resistance to amoxicillin-clavulanic acid, ceftiofur, ceftriaxone, cefoxitin, gentamicin, and sulfonamide was lower (P < 0.05) in isolates from CIN or CIN+CIT compared to those from NC or BAC. The whole-genome sequencing analysis of 227 of the 240 isolates detected 26 AMR genes (ARGs) and 19 plasmids but the prevalence of some ARGs and plasmid numbers were lower (P < 0.05) in E. coli isolated from CIN or CIN+CIT than NC or BAC. The most prevalent resistance genes included tet(A) (n = 108), aac3_Vla (n = 91), aadA1 (n = 86), blaCMY-2 (n = 78), sul1 (n = 77), aph3_lb (n = 58), aph6_ld (n = 58), and sul2 (n = 24). Interestingly, the number of most virulence genes (VGs) increased (P < 0.05) over time from 6 to 27 days of age. The prevalence of isolates of serotype O21:H16 was lower (P < 0.05) in CIN and CIN+CIT while colibacillosis-associated multi-locus sequence typing (ST117) was the most prevalent in isolates from day 23. A whole genome-based phylogenetic tree revealed a close relationship of 25 of 227 isolates to human or broiler extraintestinal pathogenic E. coli. This study indicates that AMR and virulence genotypes of E. coli could be modulated by encapsulated CIN or CIN+CIT feed supplementations and prompt further investigations on the involved mechanisms.

A previous soil metagenomics study recovered a novel cephalosporin resistance determinant, pbpTET A6, for which the exact resistance mechanism was unclear. This study used a three-dimensional structure-guided mutagenesis approach to demonstrate that PBPTET A6 is likely to be a class A penicillin-binding protein (PBP), and that its ability to confer cephalosporin resistance is directly linked to the functional integrity of its transpeptidase (TP) catalytic core. Screening of a library of PBPTET A6 variants carrying randomly introduced point mutations revealed additional residue modifications that compromised resistance, all of which were proximal to the TP active site except one which was found in a 29-amino-acid-long superstructure (α6-α7 loop) absent in other class A PBP homologues. Based on the site-specific mutagenesis results, it is hypothesized that residue arginine-400 plays an important role in limiting the access of certain cephalosporin compounds to the enzymatic core of the TP domain of PBPTET A6. Using a combination of adaptive evolution assays and whole-genome sequencing, the potential impact of PBPTET A6 on promoting the development of resistance in the clinically significant opportunistic pathogen Pseudomonas aeruginosa was investigated. Under the selective pressure of serial ceftazidime exposures, the pbpTET A6-expressing P. aeruginosa population readily evolved by excluding a ~400-kbp chromosomal element to acquire additional resistance against cephalosporins, suggesting that PBPTET A6 has a catalytic effect on facilitating antibiotic-resistance-associated genome adaptation. Overall, the soil environment contains genes conferring resistance to critically important antibiotics by cryptic mechanisms. Understanding what impact anthropogenic activities might have on the abundance and evolution of these genes should be a priority.

Antimicrobial resistance (AMR) represents an existential threat to the function of modern medicine. Genomics and machine learning methods are being increasingly used to analyze and predict AMR. This type of surveillance is very important to try to reduce the impact of AMR. Machine learning models are typically trained using genomic data, but the aspects of the genomes that they use to make predictions are rarely analyzed. In this work, we showed how, by using different types of machine learning models and performing this analysis, it is possible to identify the key genes underlying AMR in nontyphoidal Salmonella (NTS). NTS is among the leading cause of foodborne illness globally; however, AMR in NTS has not been heavily studied within the food chain itself. Therefore, in this work we performed a broad-scale analysis of the AMR in NTS isolates from commercial chicken farms and identified some priority AMR genes for surveillance., Nontyphoidal Salmonella (NTS) is a leading global cause of bacterial foodborne morbidity and mortality. Our ability to treat severe NTS infections has been impaired by increasing antimicrobial resistance (AMR). To understand and mitigate the global health crisis AMR represents, we need to link the observed resistance phenotypes with their underlying genomic mechanisms. Broiler chickens represent a key reservoir and vector for NTS infections, but isolates from this setting have been characterized in only very low numbers relative to clinical isolates. In this study, we sequenced and assembled 97 genomes encompassing 7 serotypes isolated from broiler chicken in farms in British Columbia between 2005 and 2008. Through application of machine learning (ML) models to predict the observed AMR phenotype from this genomic data, we were able to generate highly (0.92 to 0.99) precise logistic regression models using known AMR gene annotations as features for 7 antibiotics (amoxicillin-clavulanic acid, ampicillin, cefoxitin, ceftiofur, ceftriaxone, streptomycin, and tetracycline). Similarly, we also trained “reference-free” k-mer-based set-covering machine phenotypic prediction models (0.91 to 1.0 precision) for these antibiotics. By combining the inferred k-mers and logistic regression weights, we identified the primary drivers of AMR for the 7 studied antibiotics in these isolates. With our research representing one of the largest studies of a diverse set of NTS isolates from broiler chicken, we can thus confirm that the AmpC-like CMY-2 β-lactamase is a primary driver of β-lactam resistance and that the phosphotransferases APH(6)-Id and APH(3″-Ib) are the principal drivers of streptomycin resistance in this important ecosystem. IMPORTANCE Antimicrobial resistance (AMR) represents an existential threat to the function of modern medicine. Genomics and machine learning methods are being increasingly used to analyze and predict AMR. This type of surveillance is very important to try to reduce the impact of AMR. Machine learning models are typically trained using genomic data, but the aspects of the genomes that they use to make predictions are rarely analyzed. In this work, we showed how, by using different types of machine learning models and performing this analysis, it is possible to identify the key genes underlying AMR in nontyphoidal Salmonella (NTS). NTS is among the leading cause of foodborne illness globally; however, AMR in NTS has not been heavily studied within the food chain itself. Therefore, in this work we performed a broad-scale analysis of the AMR in NTS isolates from commercial chicken farms and identified some priority AMR genes for surveillance.

Citrobacter braakii and Citrobacter freundii are Gram-negative opportunistic pathogens associated with many infectious diseases, including septicemia, in humans and animals. Here, we report the draft genome sequences of seven C. braakii strains and one C. freundii strain isolated from Canadian wastewater treatment facilities.

Exposure to antimicrobial resistant (AMR) bacteria is a major public health issue which may, in part, have roots in food production practices that are conducive to the selection of AMR bacteria ultimately impacting the human microbiome through food consumption. Of particular concern is the prophylactic use of antibiotics in animal husbandry, such as the medication of feeds with sulfonamides and other antibiotics not considered clinically relevant, but which may nonetheless co-select for multi-drug resistant bacteria harbouring resistance to medically-important antibiotics. Using a multi-drug resistant Klebsiella pneumoniae strain exhibiting resistance to sulfonamides and beta-lactams (including carbapenem) as a model, we examined the ability of non-medicated and commercially-medicated (sulfonamide) animal feeds to select for the model strain when inoculated at low levels by measuring its recovery along with key AMR markers, sul1(sulfonamide) and blaKPC-3 (meropenem), under different incubation conditions. When non-medicated feeds were supplemented with defined amounts of sulfadiazine the model strain was significantly enriched after incubation in Mueller Hinton Broth at 37oC overnight, or in same at room temperature for a week, with consistent detection of both the sul1 and blaKPC-3 markers as determined by polymerase chain reaction (PCR) techniques to screen colony isolates recovered on plating media. Significant recoveries of the inoculated strain and the sul1 and blaKPC-3 markers were observed with one of three commercially-medicated (sulfamethazine) feeds tested under various incubation conditions. These results demonstrate that under certain conditions the prophylactic use of so-called non-priority antibiotics in feeds can potentially lead to co-selection of environmental AMR bacteria with resistance to medically important antibiotics, which may have far-reaching implications for human health.

The determination of Shiga toxin (ST) subtypes can be an important element in the risk characterization of foodborne ST-producing Escherichia coli (STEC) isolates for making risk management decisions. ST subtyping methods include PCR techniques based on electrophoretic or pyrosequencing analysis of amplicons and in silico techniques based on whole genome sequence analysis using algorithms that can be readily incorporated into bioinformatics analysis pipelines for characterization of isolates by their genetic composition. The choice of technique will depend on the performance characteristics of the method and an individual laboratory's access to specialized equipment or personnel. We developed two whole genome sequence-based ST subtyping tools: (i) an in silico PCR algorithm requiring genome assembly to replicate a reference PCR-based method developed by the Statens Serum Institut (SSI) and (ii) an assembly-independent routine in which raw sequencing results are mapped to a database of known ST subtype sequence variants (V-Typer). These tools were evaluated alongside the SSI reference PCR method and a recently described PCR-based pyrosequencing technique. The V-Typer method results corresponded closely with the reference method in the analysis of 67 STEC cultures obtained from a World Health Organization National Reference Laboratory. In contrast, the in silico PCR method failed to detect ST subtypes in several cases, a result which we attribute to assembly-induced errors typically encountered with repetitive gene sequences. The V-Typer can be readily integrated into bioinformatics protocols used in the identification and characterization of foodborne STEC isolates.

Genomic antimicrobial resistance (AMR) prediction tools have the potential to support foodborne illness outbreak investigations through their application in the analysis of bacterial genomes from causative strains. The AMR marker profile of a strain of interest, initially identified in outbreak-associated clinical samples, may serve as the basis for customization of selective enrichment media, facilitating its recovery from samples in a food safety investigation. Different possibilities for AMR analyses include the use of comprehensive AMR gene databases such as the Comprehensive Antibiotic Resistance Database, which can be mined with in-house bioinformatics alignment tools (e.g., Antimicrobial Resistance Marker Identifier), or publicly available tools based on clinically relevant acquired AMR gene databases (e.g., ResFinder). In combination with a previously reported pipeline (SigSeekr) designed to identify specific DNA sequences associated with a particular strain for its rapid identification by PCR, it should be possible to deploy custom recovery and identification tools for the efficient detection of priority pathogens such as Shiga toxigenic Escherichia coli (STEC) outbreak strains within the time frame of an active investigation. Using a laboratory STEC strain as a model, trimethoprim resistance identified by both Antimicrobial Resistance Marker Identifier and ResFinder was used as the basis for its selective recovery against a background of commensal E. coli bacteria in ground beef samples. Enrichment in modified tryptic soy broth containing trimethoprim greatly enhanced the recovery of low numbers of model strain cells inoculated in ground beef samples, as verified by the enumeration of colonies on plating media using a strain-specific PCR method to determine the recovery efficiency for the target strain. We discuss the relative merits of different AMR marker prediction tools for this purpose and describe how such tools can be utilized to good effect in a typical outbreak investigation scenario.

Whole-genome sequencing (WGS) of bacterial pathogens is currently widely used to support public-health investigations. The ability to assess WGS data quality is critical to underpin the reliability of downstream analyses. Sequence contamination is a quality issue that could potentially impact WGS-based findings; however, existing tools do not readily identify contamination from closely-related organisms. To address this gap, we have developed a computational pipeline, ConFindr, for detection of intraspecies contamination. ConFindr determines the presence of contaminating sequences based on the identification of multiple alleles of core, single-copy, ribosomal-protein genes in raw sequencing reads. The performance of this tool was assessed using simulated and lab-generated Illumina short-read WGS data with varying levels of contamination (0-20% of reads) and varying genetic distance between the designated target and contaminant strains. Intraspecies and cross-species contamination was reliably detected in datasets containing 5% or more reads from a second, unrelated strain. ConFindr detected intraspecies contamination with higher sensitivity than existing tools, while also being able to automatically detect cross-species contamination with similar sensitivity. The implementation of ConFindr in quality-control pipelines will help to improve the reliability of WGS databases as well as the accuracy of downstream analyses. ConFindr is written in Python, and is freely available under the MIT License at github.com/OLC-Bioinformatics/ConFindr.

Campylobacter is the leading cause of food-borne bacterial disease in Canada and many developed countries. One of the most common sources of human campylobacteriosis is considered to be the consumption or handling of raw or undercooked poultry. To date, few Canadian studies have investigated both the prevalence of Campylobacter on retail poultry and its potential impact on human clinical cases. The objective of this study was to evaluate the prevalence of Campylobacter spp. at the retail level and the correlation between subtypes recovered from chicken and those recovered from human clinical cases within the province of Nova Scotia, Canada. From this study 354 human clinical isolates were obtained from provincial hospital laboratories and a total of 480 packages of raw poultry cuts were sampled from retail outlets, yielding 312 isolates (65%), of all which were subtyped using comparative genomic fingerprinting (CGF). Of the 312 chicken isolates, the majority of isolates were C. jejuni (91.7%), followed by C. coli (7.7%) and C. lari (0.6%). Using CGF to subtype C. jejuni and C. coli isolates, 99 and 152 subtypes were recovered from chicken and clinical cases, respectively. The most prevalent human and chicken subtypes found in NS are similar to those observed nationally; indicating that the Campylobacter from this study appear to reflect of the profile of Campylobacter subtypes circulating nationally. Of the subtypes observed, only 36 subtypes were common between the two groups, however, these subtypes represented 48.3% of the clinical isolates collected. The findings from this study provides evidence that in Nova Scotia, retail poultry can act as a reservoir for Campylobacter subtypes that have been implicated in human illness.

A 2016/2017 outbreak of Shiga toxin-producing Escherichia coli (STEC) O121 in Canada, was linked to wheat flour, milled at a single facility on three consecutive days in October 2016. Most Probable Number (MPN) estimates of the concentration of STEC O121 in the recalled flour were made using the results of qualitative testing conducted during the outbreak investigation and from analysis of 5 × 2.5 g, 5 × 25 g and 5 × 100 g analytical units of the recalled flour. The STEC O121 levels were estimated at 0.15 to 0.43 MPN/100 g, with no significant difference between production days and the two MPN estimates. The microbiota of the recalled flour, and eight retail flour samples, was enumerated by aerobic colony count, MacConkey agar and E. coli/Coliform petrifilm. The composition of the microbiota to a genus level was determined by identifying individual colonies with a Bruker Biotyper. All retail flour samples were negative for STEC in 5 × 100 g analytical units. There was no evidence of higher levels of organisms associated with fecal contamination in the recalled flour. The low levels of STEC O121 in the recalled flour indicate that a robust sampling plan, with multiple analytical units for a total of several hundred grams, may be required to reliably detect STEC in flour at levels observed in this outbreak.

Using a combination of Illumina paired-end sequencing, Pacific Biosciences RS II sequencing, and OpGen Argus whole-genome optical mapping, we report here the first complete genome sequence of Yersinia massiliensis . The completed genome consists of a 4.99-Mb chromosome, a 121-kb megaplasmid, and a 57-kb plasmid.

Listeria monocytogenes is a Gram-positive, rod-shaped, non-spore-forming bacterium which is an important foodborne bacterial pathogen for humans worldwide with high mortality rates. Here, we report a 2,964,284-bp draft genome sequence of Listeria monocytogenes strain ATCC 7644 (American Type Culture Collection).

The genetic structure of bacterial populations can be related to geographical locations of isolation. In some species, there is a strong correlation between geographical distances and genetic distances, 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 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 origins and 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 less than two weeks prior to sampling: genomic analysis indicated 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.

Enterohemorrhagic Escherichia coli serotype O157:H7 is a major cause of foodborne outbreaks and hemolytic-uremic syndrome. Here, we report the draft genome sequences of three antibiotic-resistant E. coli O157:H7 strains isolated from feedlot cattle. These draft genome sequences will aid in the development of sequence-based tools for the detection of virulence and antimicrobial resistance genotypes.

Foodborne illness attributed to enterohemorrhagic E. coli (EHEC), a highly pathogenic subset of Shiga toxin-producing E. coli (STEC), is increasingly recognized as a significant public health issue. Current microbiological methods for identification of EHEC in foods often use PCR-based approaches to screen enrichment broth cultures for characteristic gene markers [i.e., Shiga toxin (stx) and intimin (eae)]. However, false positives arise when complex food matrices, such as beef, contain mixtures of eae-negative STEC and eae-positive E. coli, but no EHEC with both markers in a single cell. To reduce false-positive detection of EHEC in food enrichment samples, a Multiplexed, Single Intact Cell droplet digital PCR (MuSIC ddPCR) assay capable of detecting the co-occurrence of the stx and eae genes in a single bacterial cell was developed. This method requires: (1) dispersal of intact bacteria into droplets; (2) release of genomic DNA (gDNA) by heat lysis; and (3) amplification and detection of genetic targets (stx and eae) using standard TaqMan chemistries with ddPCR. Performance of the method was tested with panels of EHEC and non-target E. coli. By determining the linkage (i.e., the proportion of droplets in which stx and eae targets were both amplified), samples containing EHEC (typically greater than 20% linkage) could be distinguished from samples containing mixtures of eae-negative STEC and eae-positive E. coli (0–2% linkage). The use of intact cells was necessary as this linkage was not observed with gDNA extracts. EHEC could be accurately identified in enrichment broth cultures containing excess amounts of background E. coli and in enrichment cultures derived from ground beef/pork and leafy-green produce samples. To our knowledge, this is the first report of dual-target detection in single bacterial cells using ddPCR. The application of MuSIC ddPCR to enrichment-culture screening would reduce false-positives, thereby improving the cost, speed, and accuracy of current methods for EHEC detection in foods.

Due to the public health importance of flagellar genes for typing, it is important to understand mechanisms that could alter their expression or presence. Phenotypic novelty in flagellar genes arise predominately through accumulation of mutations but horizontal transfer is known to occur. A linear plasmid termed pBSSB1 previously identified inSalmonellaTyphi, was found to encode a flagellar operon that can mediate phase variation, which results in the rare z66 flagella phenotype. The identification and tracking of homologs of pBSSB1 is limited because it falls outside the normal replicon typing schemes for plasmids. Here we report the generation of nine new pBSSB1-family sequences using Illumina and Nanopore sequence data. Homologs of pBSSB1 were identified in 154 genomes representing 25 distinct serotypes from 67,758Salmonellapublic genomes. Pangenome analysis of pBSSB1-family contigs was performed using Roary and we identified three core genes amenable to a minimal MLST scheme. Population structure analysis based on the newly developed MLST scheme identified three major lineages representing 35 sequence types, and the distribution of these sequence types was found to span multiple serovars across the globe. This MLST scheme has shown utility in tracking and subtyping pBSSB1-family plasmids and it has been incorporated into the plasmid MLST database under the name “pBSSB1-family”.

Campylobacter remains one of the most common bacterial causes of gastroenteritis worldwide. Tracking sources of this organism is challenging due to the large numbers of human cases, and the prevalence of this organism throughout the environment due to growth in a wide range of animal species. Many molecular subtyping methods have been developed to characterize Campylobacter species, but only a few are commonly used in molecular epidemiology studies. This review examines the applicability of these methods, as well as the role that emerging whole genome sequencing technologies will play in tracking sources of Campylobacter spp. infection.

Campylobacter jejuni and C. coli are the bacterial species most commonly associated with foodborne illness worldwide. Methods for isolation of these organisms from complex food matrices rely on conditions that allow for the growth of these organisms while inhibiting competitors. This chapter reviews the methodologies developed to isolate, identify, and characterize C. jejuni and C. coli. Several of the unique requirements for growth have been incorporated in the different methodologies available for isolation of Campylobacter spp. from foods. The incorporation of DNA-based identification methods has improved the specificity and the speed at which results are now available. These DNA-based methods are also responsible for the rapid development of typing protocols with high resolution for epidemiological studies. This Chapter emphasizes the most efficient methods for isolation of Campylobacter from foods, and for the typing of isolates for epidemiological studies.

The recovery of Campylobacter species from food and environmental sources is challenging due to the slow growth of these bacteria and the need to suppress competing organisms during the isolation procedures. The addition of multiple selective antimicrobials to growth media can negatively impact recovery of some Campylobacter spp. Here, we describe our current method for the isolation of thermotolerant Campylobacter species, mainly C. jejuni and C. coli, from food and environmental samples. We emphasize the use of membrane filtration during plating for the specific isolation of Campylobacter spp. and a reduced use of antimicrobial supplements throughout the whole isolation process.