Development of high-throughput techniques to assess bacteriocin activity for targeted pathogen management

Introduction/Objectives: As antibiotic resistance in bacterial pathogens is on the rise, antimicrobial peptides (AMPs) offer an effective solution against potential global pandemics. These AMPs, termed ‘bacteriocins’ are naturally produced by many microbial strains. AMPs may provide an alternative strategy to clinical antibiotic use due to their diverse mechanisms of action and diminished imposition of resistance. In an effort to develop high-throughput methods to assay bacteriocin activity against known pathogens, we employed spot plate, well diffusion, and microplate reader techniques to analyze the growth of bacteriocin-producing microbes on Streptococcus agalactiae (Group B Strep) and Staphylococcus aureus (MRSA). In order to assess the collateral damage to beneficial microbes such as Lactic Acid Bacteria, we also considered the bacteriocin activity against Lactobacillus spp. Our objective was to determine prejudicial effects of various species within the Bacillus genus (Bacillus cereus, Bacillus pumilus, Bacillus thuringiensis, and Bacillus subtilis) on Group B Strep, MRSA, and Lactobacillus. By developing these high-throughput methods, we will facilitate the screening of bacteriocin activity from different clinical and environmental samples against the World Health Organization’s Top Priority Pathogen List.

Methods: Isolation of Lactobacillus strains was performed from a commercial toddler probiotic via serial dilution and plating onto MRS plates. Pathogenic organisms were acquired from ATCC (GBS NCTC_8532 and Staphylococcus aureus NCTC_8181) and cultured in BHI broth at 37°C aerobically. Freshly grown strains were transferred into a 96-well plate, placed into the Alto microplate reader, and conditionally assayed to establish a working SOP. The Alto device was assessed under ambient conditions using endpoint modality, followed by aerobically at 37°C using end-point and kinetic data collection, and lastly within the anaerobic chamber in kinetic mode for 24 hours. Results from the microplate reader were transformed into growth curves. Separately, Bacillus species were isolated and stamped onto BHI agar plates. After 24 hours, spot plates were overlayed with sloppy agar (0.75% BHI and MRS agar) containing active cultures of Lactobacillus, GBS, and MRSA strains. Zones of inhibition were measured after overnight incubation. A well diffusion assay (WDA) was also performed using the cell free supernatant of Bacillus strains overlayed with MRSA and GBS in 0.75% BHI broth to determine whether bacteriocin activity is associated with cells or is released into the environment.

Results: Growth curves were observed for Streptococcus agalactiae and Staphylococcus aureus in both the incubator and anaerobic chamber. Antimicrobial activity was confirmed using spot plate and WDAs. The stamped Bacillus plates overlayed with 0.75% BHI and 0.75% MRS agar with probiotics revealed zones of inhibition.

Conclusion: In this study, known bacteriocin producing Bacillus species were used to determine their inhibitory effects on clinical pathogens by using augmented techniques. Further development of this high-throughput methodology will facilitate our ability to identify unknown bacteriocin-producing organisms and add to our arsenal of antimicrobial compounds.