Phenotypic and Whole-Genome Sequencing-Based Profiling of Antimicrobial Resistance and Virulence in Pseudomonas aeruginosa Isolated from Patients with Ventilator-Associated Pneumonia and Ventilator-Associated Tracheobronchitis in a Croatian Intensive Care Unit.

BACKGROUND/OBJECTIVES: Pseudomonas aeruginosa is one of the leading causes of ventilator-associated pneumonia (VAP) and ventilator-associated tracheobronchitis (VAT), with a worldwide spread of difficult-to-treat high-risk clones. This study aimed to investigate the virulence potential and to characterize phenotypic and genotypic antimicrobial resistance (AMR) in P. aeruginosa causing VAP/VAT in the Intensive Care Unit (ICU), University Hospital of Split, Croatia.

METHODS: The study included P. aeruginosa isolates obtained from ICU patients who met the criteria for VAP or VAT, between January 2023 and January 2024. Isolates were identified using MALDI-TOF MS and tested for antimicrobial susceptibility (AST). A subset of phenotypically multidrug-resistant (MDR) isolates was further analyzed using whole-genome sequencing (WGS) and multilocus sequence typing.

RESULTS: A high rate of resistance was detected to ceftazidime (23.4%), imipenem (39.6%), and meropenem (43.8%). WGS confirmed the presence of multiple AMR genes, including the blaVIM-2 gene, whose genetic environment highlights a complex MDR locus integrating multiple AMR determinants and mobile genetic elements. All tested isolates possessed genes for class C (blaPDC34, blaPDC374 or blaPDC16) and class D (blaOXA-2, blaOXA-10 or blaOXA-50) β-lactamases, fosA, aph(3')-IIb and crpP genes. Additionally, WGS analysis revealed the presence of numerous virulence genes including those for adherence (Type IV pili and Fap protein production), motility (such as flgF), biofilm formation (e.g., algE and mucE), quorum sensing (lasI, lasR, rhlI and rhlR), exotoxin (toxA and plcH) and exoenzyme activity (exoU, exoT, exoS, exoY, pcrV, hcp1 and lasA). The isolates belonged to four different sequence types: ST235, ST446, the high-risk ST253 and the widely distributed ST395. Phylogenomic comparison demonstrated that the isolates from this study do not originate from a single clonal source, but instead represent multiple globally distributed high-risk P. aeruginosa lineages introduced into the clinical setting.

CONCLUSIONS: Due to the emergence of high-risk clones with broad AMR and strong virulence potential, ineffectiveness of standard empirical therapy may be anticipated, highlighting the urgent need for new therapeutic approaches (including those targeting major virulence factors).