Virulence Determinants of Urinary Pseudomonas aeruginosa Clinical Isolates: A Phenotypic and Genotypic Study

This study aimed to characterize antimicrobial resistance, biofilm formation, and phenotypic and genotypic virulence-associated traits in urinary P. aeruginosa isolates in Iraq. Methods: Twenty P. aeruginosa isolates recovered from 100 urine samples from patients with urinary tract infections at Al-Ramadi Teaching Hospital, Anbar, Iraq, were characterized using conventional biochemical tests and the VITEK® 2 automated system. The expected 16S rRNA gene amplicon was assessed in all isolates; sequencing and phylogenetic analysis were performed on four selected isolates. Biofilm formation was assessed using the crystal violet microtiter plate, tube adherence, and Congo red agar methods. Antimicrobial susceptibility was tested by Kirby–Bauer disk diffusion using CLSI M100, 35th edition (2025), and the multiple antibiotic resistance (MAR) index was calculated using six antimicrobial agents. Phenotypic assays assessed capsule-associated extracellular polysaccharide production, protease, lecithinase, coagulase, hemolytic and pyocin activity, pigment production, and adhesion to human uroepithelial cells. PCR was used to detect algD, lasR, oprD, and mexB. Separately, a retrospective review of 188 urine-culture records from 2025 identified 131 culture-positive specimens. Results: All 20 isolates were identified as P. aeruginosa and met the study’s MDR criterion. MAR indices ranged from 0.33 to 1.00. All isolates formed biofilms; by the crystal violet assay, four (20%) were strong producers and 16 (80%) were moderate producers. Capsule-associated extracellular polysaccharide production, protease activity, β-hemolysis, and pyocin activity were detected in all 20 isolates. Pyocyanin was detected in 16 (80%) isolates, and pyoverdine was detected in seven (35%) isolates. Adhesion indices ranged from 4.3 to 19.5 bacteria per cell, with 11 (55%) isolates classified as high-adherence. PCR detected all four target regions in all isolates, although target detection alone does not establish gene expression or function. In the separate retrospective survey, E. coli was the most frequently reported organism (27.5%), and P. aeruginosa accounted for 7.6% of positive cultures. Conclusions: These findings support local resistance surveillance, susceptibility-guided treatment, and infection-control measures. Larger studies linking molecular, phenotypic, and patient-level outcomes are needed.