Outer membrane vesicles (OMVs) are bi-layered structures released by Gram-negative bacteria which contain macromolecules including enzymes, periplasmic proteins, lipids, and DNA. The mechanisms by which DNA is packaged into OMVs are not well characterised. It is suggested that DNA packaging may be increased during stress conditions. Limited studies suggest that OMVs can facilitate the transfer of genes between bacterial cells and protect packaged DNA from degradation. This study examines the ability of OMVs isolated from Pseudomonas aeruginosa and Helicobacter pylori to package and transfer antibiotic resistance genes to other bacteria, which has not been previously demonstrated.
OMVs were isolated from P. aeruginosa and H. pylori harbouring plasmids encoding for antibiotic resistance. Fluorescent staining of OMV-associated DNA revealed DNA was associated with the outer surface in addition to being contained within OMVs, where it was protected from DNase degradation. We confirmed the presence of antibiotic resistance genes within OMVs by PCR. P. aeruginosa OMVs were used to transform antibiotic-sensitive P. aeruginosa, resulting in antibiotic-resistant transformants. We also detected transposon genes encoding for antibiotic resistance associated with P. aeruginosa OMVs, however we are yet to confirm OMV-mediated transfer of transposon DNA. Additionally, we are currently examining the ability of H. pylori OMVs containing a plasmid also encoding for antibiotic resistance to transform antibiotic-sensitive bacteria, as well as the ability of Gram-positive organisms to package and transfer antibiotic-resistance genes via membrane vesicles.
To investigate if OMV DNA packaging is altered in response to stress conditions, we treated P. aeruginosa with sub-lethal doses of antibiotics. Antibiotic stress increased OMV production. We are currently examining the DNA cargo contained within these OMVs and testing the frequency of DNA transfer by OMVs produced under stress conditions. The outcomes of this study suggest that OMV-mediated DNA transfer may contribute to the spread of antibiotic resistance between bacteria.