High-risk lineages shape the resistome and virulome of multidrug-resistant Pseudomonas aeruginosa.

Multidrug-resistant (MDR) and extensively drug-resistant (XDR) Pseudomonas aeruginosa strains remain a major threat in clinical settings, yet no contemporary genomic data are available from the Olomouc region, Czech Republic. We performed whole-genome sequencing of 157 MDR isolates obtained from 133 patients hospitalized at two tertiary-care hospitals in Olomouc between April 2020 and April 2024. The aim of this study was to delineate the clonal structure, resistance and virulence repertoires shaping the local epidemiology. Of the 157 isolates, 81 (51.6%) met XDR criteria, most commonly exhibiting susceptibility to colistin (37/81, 45.7%). Four dominant high-risk sequence types (STs) were identified: ST175 (n = 53), ST357 (n = 49), ST233 (n = 25), and ST235 (n = 11). ST357 comprised two phylogenetically and functionally distinct sublineages: a major cluster (43/49) carrying bla IMP-7 and bla OXA-2, and a smaller bla VIM-2-positive cluster (5/49) lacking bla OXA-2, oprD, soxR, and the pvc operon, correlating with an XDR phenotype and reduced biofilm-associated virulence potential. All ST357 isolates carried the cytotoxic exoU gene. Both bla IMP-7 and bla VIM-2 were chromosomally encoded. ST175 isolates lacked acquired carbapenemase genes, except for a single bla VIM-2-positive isolate. This lineage carried the highest number of virulence genes, dominated by adherence- and biofilm-associated determinants together with the characteristic exoS +/exoT +/exoY + type III secretion system profile. All ST233 isolates carried bla VIM-2 and exhibited an XDR phenotype, consistent with globally disseminated VIM-2 lineages. ST235 displayed the greatest diversity of carbapenemase genes, including bla GES-14 (5/11), bla GES-29 (1/11), and bla VIM-1 (1/11). This represented the first identification of a bla VIM-1-positive ST235 isolate in Olomouc University Hospital, and, to our knowledge, the first documented occurrence of a VIM-1-producing ST235 strain in the Czech Republic. All ST235 isolates were exoU-positive. This study provides a recent whole-genome analysis of MDR/XDR P. aeruginosa in the Czech Republic, integrating resistome, virulome and molecular epidemiology. Our findings demonstrate that the local population is shaped by multiple high-risk lineages with distinct resistance-virulence profiles, underscoring their clinical relevance and the necessity of genomic surveillance to guide therapy and prevent hospital transmission.