Strain-specific outer membrane vesicle-mediated transfer of a bla OXA-24 -like plasmid in Acinetobacter baumannii restricts cross-species dissemination

ABSTRACT The rapid rise of multidrug-resistant Acinetobacter spp. poses a major global health threat. In Acinetobacter spp., carbapenem resistance is frequently mediated by OXA-type carbapenemases encoded by bla OXA genes, including bla OXA-24 -like genes such as bla OXA-72 . Outer membrane vesicles (OMVs) are nanoscale proteoliposomes secreted by Gram-negative bacteria that can facilitate plasmid transfer, yet the mechanisms limiting their dissemination remain unclear. Nationwide screening in China of 2,880 Acinetobacter clinical isolates identified 10 strains carrying bla OXA-24 and bla OXA-72 plasmids across three species ( A. baumannii , A. pittii , and A. baylyi ). Sequencing revealed nine distinct plasmids, most bearing a Rep_3/OrfX C-module backbone with blaOXA -p dif resistance modules (9/10). Among clinical strains, A2485 was identified as a high-OMV producer (2.74 ± 0.20-fold relative to ATCC 17978, P < 0.001). OMVs from A2485 transferred the bla OXA-72 plasmid to ATCC 17978 at 1.9 × 10⁻⁷ transformants per CFU, exceeding direct supernatant transfer at 3.2 × 10⁻⁸ transformants per CFU, and conferred resistance to meropenem, ceftazidime, and cefoperazone/sulbactam. Transfer was strictly donor-specific, with no transmission observed across 13 non- A . baumannii strains, and recipient strains failed to retransmit the plasmid, indicating a strong cross-species barrier and a non-reciprocal, donor-dependent process. Although all strains produced intact OMVs and encapsulated plasmid DNA, packaging efficiency varied considerably by host (~4.5–28.6 ng/μg). Strains with DNA loading (17978E/17978V: 28.62 and 23.98 ng/μg) equal to or exceeding that of donor A2485 (13.62 ng/μg) remained incapable of transfer. These findings demonstrate that dissemination is constrained by host-specific, post-packaging barriers rather than packaging efficiency, limiting resistance spread to specific lineages while enabling localized adaptation. IMPORTANCE Acinetobacter baumannii is a leading hospital-associated pathogen, and carbapenem resistance mediated by bla OXA-24 -like genes poses a serious clinical challenge. Through nationwide screening of 2,880 clinical Acinetobacter isolates in China, we found that strains carrying bla OXA-24 and bla OXA-72 plasmids are relatively rare, occurring in only 10 isolates across three species. We provide the first evidence that outer membrane vesicle (OMV)-mediated plasmid transfer is subject to strict, host-specific barriers: the bla OXA-72 plasmid was successfully transferred among A. baumannii strains via OMVs, yet no transfer was observed to any of the 13 non- A . baumannii species tested. Transfer was also non-reciprocal—recipient strains that acquired the plasmid were unable to further disseminate it, whether via OMVs or supernatants. Critically, this restriction was not explained by DNA packaging efficiency, as strains with equal or greater plasmid loading than the donor remained incapable of transmission, pointing instead to post-packaging, donor-specific factors, such as membrane composition or vesicle biogenesis machinery. These findings clarify the sporadic distribution of bla OXA-24 -like plasmids in China and challenge the conventional assumption that mobile genetic elements inherently carry broad dissemination potential, revealing that OMV-mediated resistance spread is constrained to specific bacterial lineages.