Identification of Outer Membrane Vesicles as a New Vehicle Mediating Antibiotic Resistance Gene Transfer in <i>Campylobacter</i>

The emergence and worldwide dissemination of antibiotic resistance genes (ARGs) compromise antibiotic therapy and are a major public health crisis. Horizontal gene transfer (HGT) plays a major role in the spread of ARGs among bacterial pathogens. Outer membrane vesicles (OMVs), which are membrane-bound particles and naturally released by Gram-negative bacteria, have been reported to carry a variety of cargos such as DNA, proteins and lipids. However, it remains unknown whether OMVs mediate transfer of ARGs in Campylobacter, an important foodborne pathogen whose resistance to antibiotics poses a serious threat to public health. To close this knowledge gap, we determined the role of OMVs in ARG transfer. Using a non-conjugative plasmid (pRY112), we demonstrated that OMVs successfully transferred the plasmid from Campylobacter coli to Campylobacter jejuni. Additionally, OMVs transferred chromosomally encoded florfenicol resistance from a clinical C. coli isolate (SH89) to C. jejuni. The OMV-mediated transfer is independent of natural transformation as both DNase I treatment (for digestion of external-free DNA) and use of a strain deficient of natural transformation as the recipient strain did not affect OMV-mediated ARG transfer. Transmission electron microscopy revealed direct fusion between OMVs and recipient bacterial membranes, suggesting membrane fusion as the mechanism for OMV-mediated DNA transfer. Furthermore, we showed that OMVs derived from strains expressing a functionally-enhanced CmeB (FE-CmeB) transiently protect florfenicol-susceptible C. jejuni against selection by the antibiotic. Together, these findings indicate that OMVs mediate the transfer of both plasmid- and chromosome-encoded ARGs in Campylobacter and define OMVs as a novel pathway for Campylobacter to acquire antibiotic resistance via HGT.