Bacterial infections remain a major global health challenge, aggravated by the increasing prevalence of multidrug-resistant (MDR) pathogens. The rapid dissemination of carbapenem resistance mediated by metallo-beta-lactamases, such as VIM, threatens the effective treatment of Pseudomonas aeruginosa infections. Growing evidence indicates that bacterial extracellular vesicles (EVs) contribute to the horizontal transfer of antimicrobial resistance and virulence determinants, although this mechanism remains poorly characterised. This study aimed to analyse and characterise EVs produced by high-risk clones as carriers of resistance and virulence factors.

Two clinical MDR P. aeruginosa strains belonging to high-risk clone ST175 (Ps56 and Ps713) and their respective mutant Δbla_VIM were included. EVs were recovered from culture supernatants by centrifugation and purified by filtration and using the ExoBacteria™ OMV Isolation Kit. Vesicle size and concentration were assessed by Nanoparticle Tracking Analysis and Transmission Electron Microscopy (TEM), protein content was quantified by Bradford assay, and proteomic profiling was performed by data-independent acquisition mass spectrometry. EV-associated DNA (treated with/without DNase) and bacterial genomes were analysed by PCR and whole-genome sequencing (Illumina HiSeq2500 and Nanopore PromethION). Carbapenemase activity was evaluated using NG-Test Carba5.

EVs displayed diameters of 125-145nm and concentrations of 1.52×10⁸-2.66×10⁸ particles/mL, with protein levels of 1436-1592µg/mL. TEM showed vesicles with a single membrane (outer membrane vesicles) and vesicles with a double membrane (outer and inner membrane vesicles). The bla_VIM-2 gene was detected in Ps713 EVs within an In56 integron on a plasmid, while bla_VIM-20 was chromosomally located in Ps56. Both EVs exhibited carbapenemase activity. Proteomic analysis showed enrichment of functional categories related to translation, ribosomal structure, nucleotide and coenzyme transport and metabolism. Multiple virulence factors were detected by proteomic analysis, highlighting toxins such as ExoA and quorum-sensing factors such as Pqs. These findings support a critical role for EVs in P. aeruginosa pathogenicity and dissemination of antibiotic resistance.