Bacterial infections pose a significant threat to global health, especially with the growing resistance to antibiotics. Among critical priority pathogens, Pseudomonas species are of particular concern due to their intrinsic resistance mechanisms and capacity to acquire novel resistance genes. Despite their clinical importance, relatively few studies have characterized antimicrobial resistance genes (ARGs) in environmental Pseudomonas strains, especially those encoding β-lactamases.

In this study, we analyzed a collection of 56 bacterial genomes isolated under carbapenem antibiotic selection from municipal and hospital wastewater systems across Ontario. Whole-genome sequencing revealed that 33 of these isolates belonged to 11 Pseudomonas species; further annotation in CARD and ResFinder databases identified the presence of known ARGs across all isolates. However, most of the carbapenem-resistant isolates did not show observable β-lactamases or carbapenemase gene determinants. To investigate the nature of the antibiotic resistance, we constructed cosmid-based genomic libraries using DNA extracted from highly resistant Pseudomonas isolates. After confirming carbapenem resistance, the positive clones were sequenced using Oxford Nanopore Technology. Further analysis showed a group of clones with overlapping regions when mapped to the reference genome, containing several uncharacterized coding sequences. Through functional screening of these clones, subcloning and Tn5 mutagenesis, we aim to identify and characterize novel resistance genes, with particular emphasis on uncharacterized β-lactamase and carbapenemase activities. This approach not only supports the detection of ARGs that may be overlooked by sequence-based methods but also enables the characterization of their phenotypic effects and potential clinical implications of ARGs found in non-clinical settings. Furthermore, it emphasizes the importance of monitoring wastewater as a reservoir system for emerging resistance threats.