Genetic and virulence factors behind the success of high-risk <i>Pseudomonas aeruginosa</i> clones: insights from comparative genomics and an experimental infection model
Abstract
<h4>Introduction</h4><i>Pseudomonas aeruginosa</i> causes severe healthcare-associated infections. High-risk clones are defined by global dissemination and multidrug resistance, yet virulence is heterogeneous. We sought to map accessory-genome determinants associated with high-risk clones by integrating whole-genome sequencing (WGS) with a <i>Caenorhabditis elegans</i> infection model.<h4>Methods</h4>We analyzed 84 clinical isolates plus publicly available genomes using WGS, phylogenomics, and resistome/virulome profiling. Virulence was measured by <i>C. elegans</i> slow-killing (SK). A GWAS of accessory-genome subelements (AGEs) identified <i>loci</i> with high- (HVA) or low-virulence association (LVA). Coding sequences were annotated with Prokka and InterPro.<h4>Results</h4>Although high-risk and sporadic clones carried a similar total number of antimicrobial-resistance genes, 15/67 (22.38%) genes/variants were enriched in high-risk clones, producing class-level enrichment (<i>p</i> < 0.002) for aminoglycosides, phenicols, trimethoprim, sulfonamides, and tetracyclines, but not <i>β</i>-lactams or fosfomycin. Many resistance determinants are recognized mobile-element cargo such as integron cassettes or plasmid/ICE-borne genes (e.g., <i>aadA</i>, <i>dfrB</i>, <i>bla</i> <sub>VIM-2</sub>, <i>crpP</i>, <i>cmlA</i>, <i>floR</i>), indicating a mobility-linked resistome in high-risk clones. GWAS identified 113 AGEs linked to SK virulence (42 HVA, 71 LVA). HVA regions were enriched for pyoverdine (<i>fpvA</i>, <i>pvdE</i>, <i>pvdD</i>) and LPS O-antigen (<i>wbpA/B/D</i>) <i>loci</i>, whereas LVA regions were enriched for ICE/conjugation/integrase motifs. <i>cdsA</i> and <i>clpP</i> were newly associated with <i>P. aeruginosa</i> virulence. Phenotypically, high-risk clones were more often strong biofilm producers and none were non-producers. High-risk clones were not consistently more virulent in SK, suggesting success reflects persistence traits (mobile DNA and biofilm under antibiotic pressure).<h4>Conclusion</h4>Accessory-genome GWAS revealed two risk dimensions: acute-virulence programs (HVA) versus mobility functions (LVA) favoring persistence and spread. Because SK measures acute virulence, readouts did not align with high-risk designations. Genomic reports should combine high-risk assignment with accessory-genome and effector profiling to support earlier containment and mechanism-aware, biofilm-focused care.