Introduction: Pseudomonas aeruginosa is a ubiquitous opportunistic pathogen responsible for significant healthcare-associated infections, particularly among immunocompromised patients and healthcare workers. Its metabolic adaptability and resistance gene acquisition contribute to its survival in hospital environments, especially in high-risk areas such as burn units and intensive care units. The rise of carbapenem-resistant P. aeruginosa (CRPA) poses a significant public health challenge due to the limited treatment options and the potential for rapid dissemination. Objectives: This study aimed to assess the prevalence of P. aeruginosa in clinical and environmental healthcare settings, characterize its antimicrobial resistance patterns particularly carbapenem resistance, and identify key resistance genes, including metallo-β-lactamases (MBLs). Additionally, the study evaluated major virulence factors and explored the relationship between multidrug resistance and virulence traits. Methods: A total of 80 clinical and environmental samples were collected from critical healthcare settings. Phenotypic identification of P. aeruginosa was performed, followed by molecular confirmation using OprL-targeted PCR. Antimicrobial susceptibility testing was conducted using the Kirby-Bauer disk diffusion method. The presence of MBL genes (blaNDM, blaVIM, and blaIMP) was detected via molecular assays. Virulence factor analysis included assessments of pyocyanin production, elastase activity, biofilm formation, and hemolytic activity. Correlations between biofilm formation and multidrug resistance (MDR) were statistically evaluated. Results: From 80 analyzed samples, 22 (27.5%) yielded P. aeruginosa, predominantly from burn wounds (63.6%), with 9.1% recovered from environmental surfaces, suggesting potential reservoirs. All isolates were PCR-confirmed. Antimicrobial testing revealed 27.3% multidrug resistance (MDR), 13.6% carbapenem resistance, and 73% susceptibility to colistin. Molecular analysis showed blaNDM and blaVIM in all tested isolates, blaIMP in 80%, and co-occurrence of blaNDM and blaVIM in 40%, indicating a high risk for gene dissemination. Virulence factor profiling demonstrated strong expression of pyocyanin, elastase, hemolysin, and biofilms, with a significant positive correlation (r = 0.62) between biofilm formation and MDR. Conclusion: This study highlights the alarming presence of CRPA in both clinical and environmental settings, particularly in burn units. The widespread occurrence of MBL genes, including co-expression of blaNDM and blaVIM, suggests a high potential for resistance gene dissemination. The observed association between virulence and resistance underlines the threat posed by these strains. Immediate measures—including molecular surveillance, robust antimicrobial stewardship, and stringent infection control—are urgently needed. Further multicenter studies and genomic investigations are recommended to better understand resistance mechanisms and guide effective containment strategies.
