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 gentamicinand 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.

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 sequence (WGS) analyses of 1279 well-characterized L. monocytogenes isolates from a variety of foods and food manufacturing environments and identified the bcrABC gene cassette associated with BC resistance in 41.5% of isolates. Of particular interest was the finding that all but one of 177 clonal complex (CC) 321 isolates, representing one of the most commonly occurring CCs found in foods and food-production environments, harbored the intact bcrABC cassette. Thirty-nine (38.6) percent of isolates recovered from foods representing 67 different CCs, and 59.2% of strains 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 were resistant to BC. Determination of bcrABC in colony isolates could be achieved using both polymerase chain reaction and whole genome sequencing techniques, providing food testing laboratories with options for the characterization of isolates. The ability to detect bcrABC provides risk managers with a valuable tool to assess the potential for persistent contamination of the food manufacturing environment, which in turn may indicate the need for more targeted surveillance to ensure the efficacy of mitigation actions.

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 (MDR) bacteria harboring resistance to medically important antibiotics. Using a MDR 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 37°C 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.

Bacterial carbapenem resistance is a major public health concern since these antimicrobials are often the last resort to treat serious human infections. To evaluate methodologies for detection of carbapenem resistance, carbapenem-tolerant bacteria were isolated from wastewater treatment plants in Toronto, Ottawa, and Arnprior, Ontario. A total of 135 carbapenem-tolerant bacteria were recovered. Polymerase chain reaction (PCR) indicated the presence of carbapenem hydrolysing enzymes KPC ( n = 10), GES ( n = 5), VIM ( n = 7), and IMP ( n = 1), and β-lactamases TEM ( n = 7), PER ( n = 1), and OXA-variants ( n = 16). A subset of 46 isolates were sequenced and analysed using ResFinder and CARD-RGI. Both programs detected carbapenem resistance genes in 35 sequenced isolates and antimicrobial resistance genes (ARGs) conferring resistance to multiple class of other antibiotics. Where β-lactamase resistance genes were not initially identified, lowering the thresholds for ARG detection enabled identification of closely related β-lactamases. However, no known carbapenem resistance genes were found in seven sequenced Pseudomonas spp. isolates. Also of note was a multi-drug-resistant Klebsiella pneumoniae isolate from Ottawa, which harboured resistance to seven antimicrobial classes including β-lactams. These results highlight the diversity of genes encoding carbapenem resistance in Ontario and the utility of whole genome sequencing over PCR for ARG detection where resistance may result from an assortment of genes.