(470g) Vesicle-Mediated Delivery of Antibiotics Increases Their Effectiveness Against Gram-Negative Bacteria

As antibiotic resistance continues to increase and the development of new drugs remains challenging, novel engineering strategies have the potential to improve treatment of bacterial infections. Gram-negative bacteria are intrinsically resistant to many antibiotics because of their dual-membrane cell wall. Small, hydrophilic antibiotics are able to pass through protein channels, called porins to access the bacterial cytosol; however, bacteria can downregulate the expression of these porins, thereby decreasing penetration of these drugs. As a result, there are many fewer drugs available to treat this class of bacteria in comparison to Gram-positive bacteria. To overcome this issue, we have looked toward nature. One means by which bacteria deliver functional molecules to other bacteria is through the use of “outer membrane vesicles” (OMVs). We therefore hypothesized that incorporating antibiotics within the OMV lumen would improve drug uptake and, as a result, increase the effectiveness of these drugs in Gram-negative bacteria. To test this hypothesis, we first investigated the optimal method to load small molecule antibiotics of varying molecular weight and hydrophobicity into OMVs produced by a hypervesiculating strain of Escherichia coli (JC8031). We found that hydrophobic antibiotics, such as moxifloxacin, could be easily loaded within the OMVs using a simple, passive loading process in which purified OMVs are incubated with antibiotic in solution in a high concentration. For more hydrophilic antibiotics, such as imipenem, sonication or electroporation increased the encapsulation efficiency within the OMVs. We next demonstrated that OMV-encapsulated moxifloxacin was as effective as free (unencapsulated) moxifloxacin in killing Pseudomonas aeruginosa strain PAO1, a common laboratory strain. However, in clinical isolates displaying resistance to fluoroquinolones, OMV encapsulation improved the activity of the drugs. OMVs alone had no effect on cell viability, indicating that it was not the mere presence of the OMVs that increased bacterial death, but more likely an improved delivery of the drugs. We hypothesized that by fusing with the bacterial membrane, OMVs enable porin-independent delivery of antibiotics. To this this hypothesis, we compared the delivery of free and OMV-encapsulated drugs in a panel of porin knockouts from the E. coli Keio collection. We observed that in the ΔompF strain (JW0912), imipenem was less effective than in the parent strain (BW25113), demonstrating that this antibiotic is transported through the OmpF porin. When we delivered OMV-encapsulated imipenem to the ΔompD\F knockout, the effectiveness of the antibiotic increased, supporting our model that OMVs deliver their cargo in a porin-independent manner. Together, these results show that OMV-mediated delivery increases the activity of existing antibiotics simply by improving their transport to the bacterial cytosol. Current work is focused on understanding the detailed mechanisms behind this phenomenon to inform the design of a synthetic vehicle with similar properties.