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 bla_VIM-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 (bla_PDC34, bla_PDC374 or bla_PDC16) and class D (bla_OXA-2, bla_OXA-10 or bla_OXA-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).

Background/Objectives: Acinetobacter baumannii is a major cause of infections in Intensive Care Units (ICUs), driving mortality through high-level antimicrobial resistance. This study utilized whole-genome sequencing (WGS) to evaluate the phenotypic, genotypic and virulence features of carbapenem-resistant A. baumannii (CRAB), which has caused ventilator-associated pneumonia/tracheobronchitis (VAP/VAT) in the ICU of the University Hospital of Split, Croatia. Methods: Over 1 year, lower respiratory tract specimens from 79 VAP/VAT patients were analyzed. CRAB isolates were identified via MALDI-TOF MS and evaluated for antimicrobial susceptibility, and a representative subset underwent WGS and multilocus sequence typing (MLST). Results: Out of 106 specimens, 18 non-duplicate CRAB strains were isolated. Five isolates underwent genomic analysis, identifying two globally distributed, high-risk Pasteur lineages: ST2 and ST492. These lineages displayed distinct resistomes: ST2 carried bla_OXA-23 and bla_ADC-73, while ST492 harbored plasmid-borne bla_OXA-72 (Rep3-T1/AB082 cluster) and bla_ADC-30. All isolates shared aminoglycoside/macrolide-resistance genes, conserved efflux pumps, and virulence determinants (bau, bas, ent, bar) crucial for acinetobactin synthesis and respiratory colonization. Phylogenetic analysis confirmed regional circulation and genetic links to neighboring countries. Conclusions: This study highlights the evolutionary dynamics of endemic CRAB lineages in a Croatian ICU and their global dissemination, which poses a critical threat, demanding strict infection control and novel therapeutics.