Gram-Negative Bacterial Outer Membrane Vesicles Inhibit Growth of Multi-Drug Resistant Organisms and Induce Wound-Healing Cytokines

Background: Antibiotic resistance is one of the greatest threats to global health. By 2050, it is estimated that 10 million people will die each year from multi-drug resistant organisms (MDROs). Persistent infection, particularly with the MDROs Methicillin Resistant S. aureus (MRSA), Escherichia coli, and Acinetobacter baumannii, is a primary cause of wound dehiscence, highlighting the need for new antimicrobials to treat wound infections. Previous work showed that naturally secreted bacterial outer membrane vesicles (OMVs) are immunostimulatory and contain autolysins capable of degrading the cell wall of competitor bacteria.

Methods: OMVs were purified from the non-pathogenic soil bacterium Burkholderia thailandensis (Bt) and tested for their ability to inhibit the growth of MDROs. MRSA, E. coli, and a highly multi-drug resistant (MDR) clinical isolate of A. baumannii were treated with 20 μg of Bt OMVs or sham control and bacterial growth was assessed every 30 minutes for 6 hours by recording absorbance at 600 nm. We also investigated the ability of Bt OMVs to inhibit biofilm formation by Pseudomonas aeruginosa. P. aeruginosa was seeded in 96 well plates and treated with 10 μg of Bt OMVs or sham control. After a 48 hour incubation, biofilms were stained with crystal violet and absorbance measured at 550 nm to determine biofilm mass. Lastly, we examined the ability of immunogenic Bt OMVs to promote the production of wound-healing cytokines. C57Bl/6 mice were given 10 μg Bt OMVs or sham control by intraperitoneal injection. After 6 hours, a peritoneal lavage was performed and lavage fluid analyzed by Multiplex cytokine assay.

Results: A single dose of OMVs significantly inhibited planktonic growth of MRSA, E.coli and MDR A. baumannii and inhibited biofilm formation by P. aeruginosa compared to control-treated bacteria. OMVs induced the production of cytokines vital for wound healing and repair, including IL-1, TNF-a, GM-CSF, MCP-1, MIP-1, and IL-4.

Conclusion: Bt-derived OMVs inhibit planktonic and biofilm growth of Gram-positive and Gram-negative MDROs in vitro and drive the production of wound-healing cytokines in vivo. These OMV properties may act in synergy to combat infection and promote wound repair. Animal studies are underway to assess OMVs as a novel therapy for MDR bacterial wound infections.