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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.

Whole genome sequencing (WGS) is rapidly replacing other molecular techniques for identifying and subtyping bacterial isolates. The resolution or discrimination offered by WGS is significantly higher than that offered by other molecular techniques, and WGS readily allows infrequent differences that occur between 2 closely related strains to be found. In this investigation, WGS was used to identify the changes that occurred in the genomes of 13 strains of bacterial foodborne pathogens after 100 serial subcultures. Pure cultures of Shiga-toxin-producing Escherichia coli, Salmonella enterica, Listeria monocytogenes, and Vibrio parahaemolyticus were subcultured daily for 100 successive days. The 1st and 100th subcultures were whole-genome sequenced using short-read sequencing. Single nucleotide polymorphisms (SNPs) were identified between the 1st and final culture using 2 different approaches, and multilocus sequence typing of the whole genome was also performed to detect any changes at the allelic level. The number of observed genomic changes varied by strain, species, and the SNP caller used. This study provides insight into the genomic variation that can be detected using next-generation sequencing and analysis methods after repeated subculturing of 4 important bacterial pathogens.