IS26-mediated circular bla_KPC transposon in ST2483 Pseudomonas aeruginosa

The global spread of carbapenem-resistant Pseudomonas aeruginosa (CRPA) poses a critical clinical threat, with bla_KPC as a key mobile carbapenem resistance gene. Here, we investigated a novel bla_KPC-2 genetic element and its transmission in clinical ST2483 isolate PA100 via whole-genome sequencing, molecular, and phenotypic testing, and vesicle analysis. A unique 4,746-bp IS26-flanked structure inserted at the chromosomal InaA/pncB2 locus was confirmed, which forms a circular transposition intermediate through IS26 recombination. PA100 exhibited ~40-fold higher bla_KPC-2 copy number and ~25-fold higher mRNA expression vs chromosomal bla_KPC-2 strain PA3; meropenem exposure further elevated its transcription by nearly 70-fold, far exceeding controls. PA100 showed an extensively drug-resistant phenotype with high-level carbapenem resistance, while remaining susceptible to ceftazidime-avibactam and cefiderocol. No classical conjugation or transformation was observed, while intact outer membrane vesicles (OMVs) from PA100 carried bla_KPC-2, supporting a non-plasmid, vesicle-associated route for transient delivery of the resistance gene under the tested conditions. The circular transposon remained genetically stable after 10 generations of passaging in PA100; however, stable maintenance in the PAO1 recipient was not detected after passage. In summary, we first report an IS26-driven circular bla_KPC-2 transposon in ST2483 P. aeruginosa, conferring robust overexpression and stable inheritance in the donor strain, with OMVs potentially facilitating its horizontal spread, although stable maintenance in recipient cells was not demonstrated under the current assay conditions. These data advance understanding of bla_KPC dissemination in CRPA and highlight clinical surveillance priorities for rare high-risk sequence types.