Effect of Outer Membrane Permeabilization on Intrinsic Resistance to EIPE-1 in Selected Gram-negative Bacteria

Background: This laboratory has shown that the hydrophobic, melanin-inspired compound EIPE-1 possesses antibacterial properties for aerobic and facultatively anaerobic gram-positive bacteria, while gram-negative bacteria are intrinsically resistant. The intact gram-negative outer membrane is typically impermeable to hydrophobic substances due to the asymmetric localization of lipopolysaccharide in the outer leaflet. Therefore, we hypothesize that the intrinsic resistance of gram-negative bacteria to EIPE-1 is due to the presence of an intact outer membrane that is impermeable to hydrophobic molecules.

Methods: A disk agar diffusion bioassay was used to determine if a strictly anaerobic gram-positive organism is susceptible to EIPE-1, and sensitization to EIPE-1 by compound 48/80, an outer membrane permeabilizer, was employed to determine if EIPE-1 and compound 48/80 synergistically inhibit bacteria growth.

Results: C. difficile 630 and 20291 has slight susceptibility to EIPE-1 at 50 μg, demonstrating that EIPE-1 has some antibacterial properties on an obligate anaerobe. There was no synergistic relationship seen between EIPE-1 and the outer membrane permeabilizer compound 48/80 with regard to sensitization of the three gram-negative test bacteria.

Conclusion: These data confirm that EIPE-1 possesses a gram-positive antibacterial spectrum and extends our work to include at least one strictly anaerobic organism. Despite the hydrophobic nature of EIPE-1, this work also supports the notion that the exclusionary properties of the outer membranes of the gram-negative bacteria examined are not solely responsible for their intrinsic resistance to EIPE-1. The presence of multi-drug efflux pumps and/or enzymes which covalently modify EIPE-1 are suspected. 
Funding: This project was supported by the National Institute of General Medical Sciences of the National Institutes of Health under award P20GM103447. The content in this publication is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Support was also provided by the OSU-CHS Intramural Research Program.