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Pseudomonas aeruginosa is an opportunistic pathogen displaying high intrinsic antimicrobial resistance and the ability to thrive in different ecological environments. In this study, the ability of P. aeruginosa to develop simultaneous resistance to multiple antibiotics and disinfectants in different natural niches were investigated using strains collected from clinical samples, veterinary samples, and wastewater. The correlation between biocide and antimicrobial resistance was determined by employing principal component analysis. Molecular mechanisms linking biocide and antimicrobial resistance were interrogated by determining gene expression using RT-qPCR and identifying a potential genetic determinant for co- and cross-resistance using whole-genome sequencing. A subpopulation of P. aeruginosa isolates belonging to three sequence types was resistant against the common preservative benzalkonium chloride and showed cross-resistance to fluoroquinolones, cephalosporins, aminoglycosides, and multidrug resistance. Of these, the epidemiological high-risk ST235 clone was the most abundant. The overexpression of the MexAB-OprM drug efflux pump resulting from amino acid mutations in regulators MexR, NalC, or NalD was the major contributing factor for cross-resistance that could be reversed by an efflux pump inhibitor. This is the first comparison of antibiotic-biocide cross-resistance in samples isolated from different ecological niches and serves as a confirmation of laboratory-based studies on biocide adapted isolates. The isolates from wastewater had a higher incidence of multidrug resistance and biocide-antibiotic cross …