Outer membrane vesicles mediating horizontal transfer of the epidemic bla_OXA-232 carbapenemase gene among Enterobacterales

OXA-232 is one of the most common OXA-48-like carbapenemase derivatives and is widely disseminated in nosocomial settings across countries. The bla(OXA-232) gene is located on a 6-kb non-conjugative ColKP3-type plasmid, while the dissemination of bla(OXA-232) into different Enterobacterales species and the polyclonal dissemination of OXA-232-producing K. pneumoniae revealed the horizontal transfer of bla(OXA-232). However, it's still unclear how this non-conjugative ColKP3 plasmid could facilitate the mobilization of bla(OXA-232). Here, we observed the in vivo intraspecies transfer of bla(OXA-232 )during a nosocomial outbreak of OXA-232-producing K. pneumoniae. We demonstrated the presence of ColKP3 OXA-232 plasmid in the outer membrane vesicles (OMVs) derived from clinical isolates, and OMVs could facilitate the horizontal transfer of bla(OXA-232) among Enterobacterales. In contrast, for the most prevalent carbapenemase genes, including bla(KPC-2) and bla(NDM-1), though the presence of carbapenemase genes and plasmid backbones in the vesicular lumen was observed, OMVs couldn't promote effective transformation, probably due to the low copy number of plasmids in clinical isolates and the low number of plasmids loaded into vesicles. Conjugation assay revealed that the epidemic IncX3 NDM-1 and IncFII(pHN7A8)/IncR KPC-2 plasmids were conjugative and could be horizontally transferred via independent conjugation or with the help of a co-existent conjugative plasmid. For the large-size and low-copy number conjugative plasmids carrying carbapenemase genes, OMVs-mediated gene exchange may only serve as an alternative pathway for horizontal transfer. In conclusion, diverse mobilization strategies were employed by plasmids harbouring carbapenemase genes, and plasmids display a proper choice of mobility pathway due to their individual properties.