A global medical crisis is unfolding as antibiotics lose effectiveness against a growing number of bacterial pathogens. Horizontal gene transfer (HGT) contributes significantly to the rapid spread of resistance, yet the transmission dynamics of genes that confer antibiotic resistance are poorly understood. Multiple mechanisms of HGT liberate genes from normal vertical inheritance. Conjugation by plasmids, transduction by bacteriophages, and natural transformation by extracellular DNA each allow genetic material to jump between strains and species. Thus, HGT adds an important dimension to infectious disease whereby an antibiotic resistance gene (ARG) can be the agent of an outbreak by transferring resistance to multiple unrelated pathogens. Here, we review the small number of cases where HGT has been detected in clinical environments. We discuss differences and synergies between the spread of plasmid-borne and chromosomal ARGs, with a special consideration of the difficulties of detecting transduction and transformation by routine genetic diagnostics. We highlight how 11 of the top 12 priority antibiotic-resistant pathogens are known or predicted to be naturally transformable, raising the possibility that this mechanism of HGT makes significant contributions to the spread of ARGs. HGT drives the evolution of untreatable “superbugs” by concentrating ARGs together in the same cell, thus HGT must be included in strategies to prevent the emergence of resistant organisms in hospitals and other clinical settings.

The best whole genome assemblies are currently built from a combination of highly accurate short-read sequencing data and long-read sequencing data that can bridge repetitive and problematic regions. Oxford Nanopore Technologies (ONT) produce long-read sequencing platforms and they are continually improving their technology to obtain higher quality read data that is approaching the quality obtained from short-read platforms such as Illumina. As these innovations continue, we evaluated how much ONT read coverage produced by the Rapid Barcoding Kit v14 (SQK-RBK114) is necessary to generate high-quality hybrid and long-read-only genome assemblies for a panel of carbapenemase-producing Enterobacterales bacterial isolates. We found that 30× long-read coverage is sufficient if Illumina data are available, and that more (at least 100× long-read coverage is recommended for long-read-only assemblies. Illumina polishing is still improving single nucleotide variants (SNVs) and INDELs in long-read-only assemblies. We also examined if antimicrobial resistance genes could be accurately identified in long-read-only data, and found that Flye assemblies regardless of ONT coverage detected >96% of resistance genes at 100% identity and length. Overall, the Rapid Barcoding Kit v14 and long-read-only assemblies can be an optimal sequencing strategy (i.e., plasmid characterization and AMR detection) but finer-scale analyses (i.e., SNV) still benefit from short-read data.

Salmonella Pathogenicity Island 1 (SPI-1) encodes a type three secretion system (T3SS), effector proteins, and associated transcription factors that together enable invasion of epithelial cells in animal intestines. The horizontal acquisition of SPI-1 by the common ancestor of all Salmonella is considered a prime example of how gene islands potentiate the emergence of new pathogens with expanded niche ranges. However, the evolutionary history of SPI-1 has attracted little attention. Here, we apply phylogenetic comparisons across the family Enterobacteriaceae to examine the history of SPI-1, improving the resolution of its boundaries and unique architecture by identifying its composite gene modules. SPI-1 is located between the core genes fhlA and mutS, a hotspot for the gain and loss of horizontally acquired genes. Despite the plasticity of this locus, SPI-1 demonstrates stable residency of many tens of millions of years in a host genome, unlike short-lived homologous T3SS and effector islands including Escherichia ETT2, Yersinia YSA, Pantoea PSI-2, Sodalis SSR2, and Chromobacterium CPI-1. SPI-1 employs a unique series of regulatory switches, starting with the dedicated transcription factors HilC and HilD, and flowing through the central SPI-1 regulator HilA. HilA is shared with other T3SS, but HilC and HilD may have their evolutionary origins in Salmonella. The hilA, hilC, and hilD gene promoters are the most AT-rich DNA in SPI-1, placing them under tight control by the transcriptional repressor H-NS. In all Salmonella lineages, these three promoters resist amelioration towards the genomic average, ensuring strong repression by H-NS. Hence, early development of a robust and well-integrated regulatory network may explain the evolutionary stability of SPI-1 compared to T3SS gene islands in other species.

Carbapenems are considered last-resort antibiotics for the treatment of infections caused by multidrug-resistant Enterobacterales , but carbapenem resistance due to acquisition of carbapenemase genes is a growing threat that has been reported worldwide. Klebsiella pneumoniae carbapenemase ( bla _KPC ) is the most common type of carbapenemase in Canada and elsewhere; it can hydrolyze penicillins, cephalosporins, aztreonam, and carbapenems and is frequently found on mobile plasmids in the Tn 4401 transposon. This means that alongside clonal expansion, bla _KPC can disseminate through plasmid- and transposon-mediated horizontal gene transfer. We applied whole genome sequencing to characterize the molecular epidemiology of 829 bla _KPC carbapenemase-producing isolates collected by the Canadian Nosocomial Infection Surveillance Program from 2010 to 2021. Using a combination of short-read and long-read sequencing, we obtained 202 complete and circular bla _KPC -encoding plasmids. Using MOB-suite, 10 major plasmid clusters were identified from this data set which represented 87% (175/202) of the Canadian bla _KPC -encoding plasmids. We further estimated the genomic location of incomplete bla _KPC -encoding contigs and predicted a plasmid cluster for 95% (603/635) of these. We identified different patterns of carbapenemase mobilization across Canada related to different plasmid clusters, including clonal transmission of IncF-type plasmids (108/829, 13%) in K. pneumoniae clonal complex 258 and novel repE(pEh60-7) plasmids (44/829, 5%) in Enterobacter hormaechei ST316, and horizontal transmission of IncL/M (142/829, 17%) and IncN-type plasmids (149/829, 18%) across multiple genera. Our findings highlight the diversity of bla _KPC genomic loci and indicate that multiple, distinct plasmid clusters have contributed to bla _KPC spread and persistence in Canada.

Carbapenems are broad-spectrum antibiotics that are losing effectiveness against infections caused by multidrug-resistant Enterobacterales that have acquired carbapenemase genes. The New Delhi metallo-β-lactamase (bla _NDM) is one of the most common carbapenemases in Canada and around the globe. These genes are frequently found on conjugative plasmids, which can disseminate through horizontal gene transfer. We applied whole-genome sequencing to characterize 1,032 bla _NDM carbapenemase-producing Enterobacterales isolates collected by the Canadian Nosocomial Infection Surveillance Program from 2010 to 2023. Using a combination of short-read and long-read sequencing, we obtained 226 complete and circular bla _NDM-encoding plasmids. Unlike other carbapenemases in Canada, we found that bla _NDM plasmids were very diverse; there was a lack of dominant clusters identified using MOB-suite, and clustering methods were not able to accurately predict plasmid clusters for short-read-only data. The majority of bla _NDM plasmids were IncF-type (69.0%, 156/226). Both bla _NDM and bla _OXA-48-type carbapenemase genes were found in 11.4% (118/1,032) of isolates, and we identified several instances of both carbapenemase genes co-harboured on the same plasmid replicon (n=9). Our findings highlight that plasmid transfer has not played a major role in bla _NDM transmission across Canada and that long-read sequencing is essential for resolving bla _NDM plasmid structure and cluster membership.

Objectives: VRE resistant to potential therapeutic agents such as linezolid are an increasing concern in Canada and worldwide. Infections produced by these isolates have limited treatment options and are associated with poor clinical outcomes. We report an isolate of vancomycin-resistant Enterococcus faecium (VREfm); identified through the Canadian surveillance program, CANWARD, which exhibited linezolid resistance (LVREfm); and carried the optrA and cfr(D) genes on a linear plasmid. Methods: Antimicrobial susceptibility testing was performed by broth microdilution and MICs were interpreted by 2025 CLSI breakpoints. WGS was performed to characterize resistance determinants and the associated plasmid. Results: In 2021, a single LVREfm isolate was recovered from a blood culture of a 67-year-old male patient admitted to an ICU. The isolate was XDR but remained susceptible dose dependent to daptomycin (MIC = 2 mg/L). MLST identified the isolate as ST817, a sequence type not commonly found in our national collection. Vancomycin resistance was mediated by the vanA gene cluster, while linezolid resistance determinants included optrA, cfr(D) and the 23S rRNA gene mutation, G2576T. Vancomycin and linezolid resistance genes were co-located on a linear plasmid that exhibited over 94% sequence identity to previously described linear plasmids from India and the USA. Notably, the plasmid also encoded four toxin–antitoxin systems, potentially contributing to its stability and persistence. Conclusions: This case highlights the appearance of linezolid resistance in VRE mediated by linear plasmids in Canada and underscores the importance of ongoing genomic surveillance to track the dissemination of MDR determinants in clinical settings.