Noriega Medrano, Carla Daniela2026-07-032026-07-032026-06-092026-06-092026-07-02http://hdl.handle.net/1993/39835In Canada alone, 38% of patients with cystic fibrosis (CF) have experienced P. aeruginosa infections, highlighting this organism as a high-priority pathogen for the development of novel antibiotics. A major challenge in treating these infections is the intrinsic resistance to multiple antibiotics of P. aeruginosa, where resistance-nodulation-division efflux pumps, such as MexXY, play a central role. MexXY is an efflux pump that, usually, form a functional complex with the outer membrane protein OprM encoded in the mexAB-oprM operon; however, in certain strains, it associates instead with OprA, an alternative outer membrane protein encoded within the mexXY-oprA operon. While MexXY paired with OprM primarily confers resistance to aminoglycosides, when MexXY forms the complex with OprA exhibits an expanded substate profile that includes β-lactam antibiotics like sulbenicillin and carbenicillin. Despite structural similarities, OprM and OprA share only 46% amino acid identity, with significant differences located in their α-helical domains. In this work, determinants of substrate selection were studied using chimeric MexXY-OprM/OprA systems, where OprM α-helixes domains were substituted with the corresponding from OprA. Seven chimeric constructs were generated and integrated them into the chromosome of an efflux pump-deficient P. aeruginosa strain. Efflux activity was assessed using ethidium bromide susceptibility assays and broth microdilution MIC assays with various antibiotics. Among the seven strains with the chimera efflux pump complex, substitution of the α4 helix exhibited gain of function, restoring sulbenicillin efflux to levels comparable to the MexXY-OprA reference complex. Given the limited characterization of mexXY-oprA operon, its prevalence across Pseudomonas species remains unclear. This study screened whole-genome sequences using BLASTn against the RefSeq database to assess the presence of the operon within Pseudomonas spp. Phylogenetic relationships were validated through a rpoB-based analysis, and the genomic context of the operon was examined through a comparative gene cluster analysis within Pseudomonas spp.engEfflux pumpsAntibiotic multidrug resistancePseudomonas aeruginosaPseudomonas paraeruginosaMexXY-OprMMexXY-OprAPrevalence