Johnston, Ella L ORCID: 0000-0003-2880-5719, Zavan, Lauren ORCID: 0000-0003-4700-3033, Bitto, Natalie J ORCID: 0000-0003-3533-9824, Petrovski, Steve, Hill, Andrew F ORCID: 0000-0001-5581-2354 and Kaparakis-Liaskos, Maria ORCID: 0000-0002-8964-4456 (2023) Planktonic and biofilm-derived Pseudomonas aeruginosa outer membrane vesicles facilitate horizontal gene transfer of plasmid DNA. Microbiology Spectrum, 11 (2). ISSN 2165-0497

Abstract

Outer membrane vesicles (OMVs) produced by Gram-negative bacteria package various cargo, including DNA that can be transferred to other bacteria or to host cells. OMV-Associated DNA has been implicated in mediating horizontal gene transfer (HGT) between bacteria, which includes the dissemination of antibiotic resistance genes within and between bacterial species. Despite the known ability of OMVs to mediate HGT, the mechanisms of DNA packaging into OMVs remain poorly characterized, as does the effect of bacterial growth conditions on the DNA cargo composition of OMVs and their subsequent abilities to mediate HGT. In this study, we examined the DNA content of OMVs produced by the opportunistic pathogen Pseudomonas aeruginosa grown in either planktonic or biofilm conditions. Analysis of planktonic growth-derived OMVs revealed their ability to package and protect plasmid DNA from DNase degradation and to transfer plasmid-encoded antibiotic resistance genes to recipient, antibioticsensitive P. aeruginosa bacteria at a greater efficiency than transformation with plasmid alone. Comparisons of planktonic and biofilm-derived P. aeruginosa OMVs demonstrated that biofilm-derived OMVs were smaller but were associated with more plasmid DNA than planktonic-derived OMVs. Additionally, biofilm-derived P. aeruginosa OMVs were more efficient in the transformation of competent P. aeruginosa bacteria, compared to transformations with an equivalent number of planktonic-derived OMVs. The findings of this study highlight the importance of bacterial growth conditions for the packaging of DNA within P. aeruginosa OMVs and their ability to facilitate HGT, thus contributing to the spread of antibiotic resistance genes between P. aeruginosa bacteria. IMPORTANCE Bacterial membrane vesicles (BMVs) mediate interbacterial communication, and their ability to package DNA specifically contributes to biofilm formation, antibiotic resistance, and HGT between bacteria. However, the ability of P. aeruginosa OMVs to mediate HGT has not yet been demonstrated. Here, we reveal that P. aeruginosa planktonic and biofilm-derived OMVs can deliver plasmid-encoded antibiotic resistance to recipient P. aeruginosa. Additionally, we demonstrated that P. aeruginosa biofilmderived OMVs were associated with more plasmid DNA compared to planktonic-derived OMVs and were more efficient in the transfer of plasmid DNA to recipient bacteria. Overall, this demonstrated the ability of P. aeruginosa OMVs to facilitate the dissemination of antibiotic resistance genes, thereby enabling the survival of susceptible bacteria during antibiotic treatment. Investigating the roles of biofilm-derived BMVs may contribute to furthering our understanding of the role of BMVs in HGT and the spread of antibiotic resistance in the environment.

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