Genomic Insights into Carbapenem-Resistant Pseudomonas aeruginosa (CRPA): Resistome and Virulome Analysis Beyond Carbapenemases

<b>Background:</b> Carbapenem-resistant <i>Pseudomonas aeruginosa</i> (CRPA) has been added to the World Health Organization's list as a high-priority pathogen for which new antibiotics are urgently needed. Herein, we investigated the association between resistance/virulence genes and high-risk CRPA clinical isolates by whole genome sequencing (WGS). <b>Methods</b>: Between 2019 and 2025, twenty-six CRPA strains from patients hospitalized in the "Renato Dulbecco" University Hospital were characterized. WGS analysis was performed using the next generation sequencing (NGS) technique. Multi-locus sequence typing (MLST) prediction was performed. Antibiotic resistance genes were detected using Antibiotic Resistance Gene-ANNOTation, Comprehensive Antibiotic Resistance Database, and ResFinder. Virulence genes were identified by the Virulence Factor Database. <b>Results</b>: The MLST analysis detected 14 different sequence types (ST). The 26 strains exhibited the same resistome profile: aac(3)-Ic, aphA15, catB7, catB10, cmlA, blaCARB, blaVIM-1, and tetG genes. The genes encoding enzymes involved in resistance to chloramphenicol and beta-lactams were found in all isolates using the three databases. Biofilm formation genes, metalloproteinase, chemotaxis, fimbriae, and pyoverdine were identified in all strains. Genes of the type III secretion system exoS, exoT, exoU, and exoY were found in 46.15%, 84.61%, 53.84%, and 84.61% of the strains, respectively. <b>Conclusions</b>: The analysis of the 26 clinical isolates showed high clonal heterogeneity, with a predominance of ST235, a high-risk clone associated with multiple resistances. Interestingly, cefiderocol resistance was carried by 4/8 isolates belonging to the ST235 strain. The surveillance based on resistome and virulome analysis could monitor the dynamic evolution of high priorityhigh-priority pathogens to guide clinical treatment and to adapt healthcare control measures, limiting their spread in the near future.