Phylogenomic and phenotypic profiling of carbapenem-resistant Pseudomonas aeruginosa clinical isolates reveals lineage-specific resistance mechanisms and adaptive responses

Carbapenem resistance in Pseudomonas aeruginosa is a growing public health concern. Multiple mechanisms of antimicrobial resistance have been described. While surveillance often focuses on carbapenemase detection, non-carbapenemase mechanisms and their interplay with the genomic background remain underexplored. This study aimed to characterize how genomic background influences carbapenem resistance mechanisms and adaptive responses in carbapenem-resistant P. aeruginosa (CRPA). A total of 136 CRPA clinical isolates collected from 28 healthcare centres across Mexico were analysed through core genome phylogeny, sequence type (ST) assignment, resistome profiling, oprD variant analysis, bacterial growth kinetics under imipenem and meropenem exposure and qRT-PCR-based expression of oprD , mexA , mexC , mexE and mexY genes. Isolates clustered into PAO1 and PA14 phylogroups. ST309 was the most prevalent ST (29/136). Pseudomonas paraeruginosa lineage was also identified within these isolates. Phylogenetic clustering of antibiotic resistance genes was observed across phylogroups. In 36% (49/146) of the isolates, β -lactamases ( bla VIM 16%, bla GES 11% and bla IMP 11%) were identified with carbapenemase activity previously reported. Potentially inactivating oprD variants were observed in 75% (102/136) of isolates, with nonsense and frameshift variants associated with resistance phenotypes. Isolates harbouring carbapenemase-encoding genes (CEGs) exhibited stable lag phases regardless of antibiotic exposure, whereas isolates lacking CEGs showed significantly prolonged lag phases. Overexpression of mexA, mexC and mexY genes was observed in 39% (7/18), 17% (3/18) and 39% (7/18) of isolates, respectively, under antibiotic-free condition, and increased under carbapenem exposure. mexA expression was significantly higher in PAO1 isolates (6/18) under antibiotic-free condition and in PA14 isolates under imipenem exposure (5/18). Carbapenem resistance in P. aeruginosa is shaped by both phylogenetic background and antibiotic-driven stress responses. This study provides an integrated analysis of resistance mechanisms – including gene expression and physiological adaptation – across major phylogenetic lineages in clinical isolates recovered in Mexico, underscoring the importance of considering non-carbapenemase resistance pathways in surveillance and treatment strategies.