Comparative genomics and phenotype microarray of carbapenem-resistant <i>Pseudomonas aeruginosa</i>

Carbapenem-resistant Pseudomonas aeruginosa (CRPA) represents a serious global concern because it combines strong antibiotic evasion strategies with persistence in hospital environments. The combination of intrinsic resistance mechanisms, acquired genetic elements and metabolic adaptability allows CRPA to survive under harsh conditions, contributing to its role in severe nosocomial infections. A comprehensive understanding of the genetic and phenotypic traits of CRPA is essential for developing effective therapeutic and infection control strategies. Whole genome sequencing (WGS) and phenotype microarray (PM) technology were used to investigate the genomic and metabolic characteristics of CRPA strains. Nine CRPA isolates were sequenced and analyzed for genes associated with antimicrobial resistance (AMR), metabolic capabilities and environmental adaptability. Six strains from WGS studies were selected for PM assays to assess carbon source utilization, osmotic stress tolerance, pH adaptability and enzymatic activity, providing a detailed phenotypic profile of CRPA strains. The WGS identified carbapenemase genes ( bla IMP-26 and bla VIM-4 ) embedded within mobile genetic elements, particularly among high-risk ST235 strains. The PM analysis revealed broad metabolic versatility, including tolerance to high osmotic stress, wide pH ranges and utilization of diverse carbon sources, supporting environmental persistence. Across PM1 and PM2A plates (190 carbon sources), the proportion of utilized carbon sources ranged from 72.6% to 88.9% across strains. High-resistance ST235 strains PA153 and PACTR demonstrated the highest utilization, metabolizing 169/190 (88.9%) and 165/190 (86.8%) carbon sources, respectively. In contrast, PA090 and PA109 showed lower utilization levels (138/190; 72.6% and 142/190; 74.7%, respectively), indicating measurable inter-strain variability. Strains PA153, PA232 and PACTR, which clustered closely in the phylogenomic analysis, exhibited the most robust phenotypic profiles and resistance genes. The integration of genomic and phenotypic data reveals that CRPA strains, especially ST235 clones, harbor resistance and stress-response genes that support environmental persistence and pathogenicity. These findings underscore the potential of targeting metabolic vulnerabilities alongside AMR mechanisms in developing therapeutic strategies.