A-053. Characterization of Lactosporin, a Novel Membrane-Active Antimicrobial from Bacillus coagulans, for Control of Gardnerella vaginalis
Bacterial vaginosis (BV) is a condition that results from the replacement of healthy vaginal microflora mostly with anaerobic pathogens such as Gardnerella vaginalis. BV increases susceptibility to HIV infection and causes complications in pregnant women and fetuses. Current treatment of BV often results in re-occurring infection and development of antibiotic-resistant bacteria. We hypothesized that Bacillus coagulans (Lactobacillus sporogenes) produces a novel and non-cytotoxic antimicrobial substance that is active against G. vaginalis, including antibiotic-resistant mutants by targeting their cytoplasmic membrane. This antimicrobial was produced by growing B. coagulans cells in MRS medium at 37°C aerobically. The cell free supernatant was concentrated 10 fold (named Lactosporin Preparation, LP) and was tested for antimicrobial activity in well-diffusion assay against G. vaginalis, and 9 healthy vaginal Lactobacillus isolates. To characterize the nature of LP, it was treated with various enzymes. The activity of LP was tested at a broad range of pH and temperatures. The isoelectric point(pI) of LP was determined by isoelectric focusing. The LP sample was subjected to SDS-PAGE and native-PAGE to visualize the antimicrobial activity. The cytotoxicity of LP was tested in Epi-Vaginal™ model. The mechanism of action of the LP was investigated by measuring the dissipation of Proton Motive Force (PMF) components, the release of K+ and the ATP from the cells. LP inhibited G. vaginalis but not the healthy vaginal lactobacilli. The activity of LP was inhibited after exposure to proteinase K, protease, trypsin and α-chymotrypsin and lipase, LP was stable at 30-100°C and active at pH < 7 with a pI of approx. 3.5. The antimicrobial activity of LP was visualized by native-PAGE but not SDS-PAGE. The Epi-Vaginal™ model study showed no toxicity of the LP for 48h. The LP caused dissipation of PMF components, release of K+ and ATP from the target cells. In conclusion, B. coagulans produces a novel antimicrobial that is active against G. vaginalis. This substance is an anionic protein with a possible lipoprotein structure which is not toxic for human application that targets the membrane of sensitive cells.