Molecular and genomic characterization of biofilm associated antimicrobial resistance in clinical and resistant isolates of Pseudomonas aeruginosa in Iraq

Pseudomonas aeruginosa is a key opportunistic pathogen in healthcare-associated infections, driven by multidrug resistance and quorum-sensing-mediated biofilm formation, which promote persistence and spread of high-risk clones. This study characterizes clinical isolates phenotypically and molecularly, including biofilm formation and quorum-sensing gene prevalence ( lasI , lasR , rhlI , rhlR ), and performs whole-genome sequencing (WGS) to identify antimicrobial resistance and virulence determinants. A total of 630 clinical specimens were collected from tertiary and secondary hospitals in Iraq. P. aeruginosa was identified via conventional methods, as well as the VITEK 2 Compact system. Antimicrobial susceptibility, ESBL, and carbapenemase production were determined per CLSI guidelines. Biofilm formation was assessed using microtiter plate and sodium alginate bead assays. QS genes were detected via PCR, and selected isolates were subjected to WGS using the DNBSEQ platform. Of 361 ESKAPEE isolates, 55 (15.2%) were P. aeruginosa : 30.9% MDR, 54.5% XDR, and 7.3% PDR. Isolates were predominantly from ICU and burn units, and from urine catheter and wound samples. Biofilm formation was found in 86.3% of isolates and correlated strongly with QS genes, particularly LasI , LasR , RhlI , and RhlR in MDR and XDR strains. The WGS of two selected strong biofilm-forming XDR isolates revealed multiple antimicrobial resistance genes (including carbapenemases and efflux pumps), virulence-associated genes (toxins, adhesion, and secretion systems), and CRISPR-Cas regions, highlighting adaptive genomic potential. The study demonstrates a high prevalence of XDR and PDR Pseudomonas aeruginosa , predominantly from ICU and burn units, with wound samples as the main source. Most isolates exhibited strong biofilm formation, frequent quorum-sensing genes ( lasI , lasR ), and extensive multidrug resistance. Whole-genome sequencing identified key resistance determinants, virulence factors, and CRISPR-Cas elements linked to persistence and adaptation.