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

Carbapenem resistance in <i>Pseudomonas aeruginosa</i> 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 <i>P. aeruginosa</i> (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, <i>oprD</i> variant analysis, bacterial growth kinetics under imipenem and meropenem exposure and qRT-PCR-based expression of <i>oprD</i>, <i>mexA</i>, <i>mexC</i>, <i>mexE</i> and <i>mexY</i> genes. Isolates clustered into PAO1 and PA14 phylogroups. ST309 was the most prevalent ST (29/136). <i>Pseudomonas paraeruginosa</i> lineage was also identified within these isolates. Phylogenetic clustering of antibiotic resistance genes was observed across phylogroups. In 36% (49/146) of the isolates, <i>β</i>-lactamases (<i>bla</i> _VIM 16%, <i>bla</i> _GES11% and <i>bla</i> _IMP 11%) were identified with carbapenemase activity previously reported. Potentially inactivating <i>oprD</i> 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 <i>mexA, mexC</i> and <i>mexY</i> 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. <i>mexA</i> 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 <i>P. aeruginosa</i> 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.