Advancing alternatives to antibiotics: Investigating bacteriocin activity against MRSA and GBS

Introduction/Objectives: Bacteria produce antimicrobial peptides called bacteriocins that inhibit the growth and/or kill other bacteria. Efforts are being made to harness this natural activity to reduce our dependence on antibiotics, thus decreasing the spread of antibiotic resistance and associated human mortality. This study serves as an investigation into bacteriocins against Methicillin Resistant Staphylococcus aureus (MRSA) and Streptococcus agalactiae (Group B Strep/GBS), including improved bioprospecting techniques and antimicrobial activity assays. While this study is still underway, we anticipate zones of inhibition from Bacillus species against our target pathogens MRSA and GBS, with a protective effect against probiotic Lactobacillus spp. Our objective is to use these natural antimicrobial producers with activity against known pathogens to identify peptide structures which may 1) provide a more targeted approach to infections caused by single species, 2) preserve the integrity of our beneficial microbiome, and 3) provide an alternative to antibiotic use in clinical settings.

Methods: Five species of Bacillus, isolated from environmental sources, were used as bacteriocin producers, as this genus has well documented bacteriocin activity. To determine their spectrum of inhibition, Bacillus spp., were tested against growth of pathogenic MRSA and GBS as well as beneficial microbes isolated from the infant probiotic, Flora. The 6-strain probiotic was serially diluted and plated onto BHI and MRS broth for isolation of representative beneficial microbes. An Alto Cerillo microplate reader was used to compare the growth in aerobic and anaerobic conditions. Representative whole genome sequences, obtained from NCBI, were screened through anti-SMASH database, to gather information on homologous bacteriocin genes. Strains were routinely grown in BHI broth media and on agar plates at 37°Cfor 24-48hrs. Gram stain reactivity confirmed the purity of active cultures throughout experimentation.

Results: Temporal spectrophotometric measurements at 600nm using the Alto Cerillo microplate reader showed the growth of GBS and MRSA over 24 hours. Duplicate analysis under anaerobic conditions confirmed the facultative anaerobicity of MRSA. Antimicrobial activity was confirmed using spot plate and well diffusion assays. Spot plates of the Bacillus spp. overlayed with 0.75% BHI and 0.75% MRS agar, imbedded with pathogens and probiotics, respectively, showed zones of inhibition for both MRSA and select probiotic indicator strains. Using the anti-SMASH database, homologous genes to known bacteriocins were confirmed.

Conclusion: This research provides insights into microbial peptides effective against clinical pathogens that will help inform the future clinical options for management of infectious diseases. In order to curb the threat of antibiotic resistance, we must continue to expand our tools, and Bacillus spp. offer an intriguing route for exploration. Future studies will use cell free supernatants to assay purified peptides against additional pathogenic strains to demonstrate further efficacy.