Genomic, proteolytic, and phenotypic characterization of Pseudomonas aeruginosa isolates causing infective endocarditis.

Infective endocarditis (IE) remains a formidable clinical challenge, characterized by high morbidity and mortality despite advances in antimicrobial therapy and surgery. Pseudomonas aeruginosa is an increasingly relevant IE pathogen due to its intrinsic antibiotic resistance and rising incidence among patients with cardiac implantable electronic devices. However, the specific virulence factors that facilitate P. aeruginosa colonization and persistence in IE are poorly defined. In this study, we analyzed six P. aeruginosa strains isolated from IE patients and compared them genomically, proteomically, and phenotypically to a reference panel of 20 well-characterized clinical isolates from diverse P. aeruginosa infection types. All strains were genome sequenced for genetic comparison. The proteome of five IE isolates was analyzed by mass spectrometry and compared to the proteomes of P. aeruginosa reference strains (PAO1, PA14, and PA_O6) grown in similar conditions. Finally, the entire panel of isolates was analyzed for phenotypes, including motility, biofilm formation, protease activity, serum resistance, pyocyanin production, and cell lysis. While all strains exhibited high genomic similarity, IE isolates lacked a distinct genomic signature. Proteomic profiling revealed consistent upregulation of virulence-associated proteins in IE strains, including components of quorum sensing (e.g., Pseudomonas Quinolone Signal), proteases (e.g., elastase), adhesins (e.g., type IV pili), and toxins (e.g., phenazines), relative to non-IE strains. Phenotypic assays demonstrated that IE isolates displayed traits commonly linked to acute infections, such as O-antigen expression, serum resistance, enhanced motility, and hemolytic activity. Notably, IE strains exhibited significantly elevated proteolytic activity compared to most other clinical isolates, underscoring the potential role of proteases in the pathogenesis and persistence of P. aeruginosa-mediated infective endocarditis.IMPORTANCEPseudomonas aeruginosa infective endocarditis (IE) is a rare but life-threatening condition with limited understanding of its pathogenesis. This study presents the first comprehensive characterization of clinical isolates from patients with P. aeruginosa IE, integrating genomic, proteomic, and phenotypic analyses in comparison with reference strains and a diverse array of clinical isolates. Although no distinct molecular signature was identified, the IE isolates consistently displayed virulence traits and phenotypes commonly associated with acute infections. Proteomic profiling revealed increased expression of prominent virulence factors, including phenazines, proteases, and pili, that likely contribute to disease initiation and persistence. Notably, all IE isolates exhibited markedly elevated proteolytic activity, significantly higher than in isolates from other clinical contexts.