Probiotic AntimicrobialEvaluation Via Real-Time Profilingof Bacterial Cell Proliferation Using Stochastic Kinetics

Probiotic metabolites are gaining attention as potential antibiotic candidates against antibiotic-resistant bacteria. The disk diffusion test, by measuring bacterial aggregate responses, faces challenges in accurately evaluating antimicrobial efficacy when these responses to different probiotic strains are indistinguishable at a macroscopic level. Here, this study presents an analytical method for accurately evaluating antimicrobial activity by analyzing bacterial cell proliferation suppression at a microscopic level. This assay can be used in a coculture system, designed to continuously expose pathogenic bacteria growing on the bottom surface of the culture plate to probiotic metabolites, selectively released from porous capsules positioned above. Bacterial proliferation is optically monitored in real-time and tracked via a computer vision algorithm. Specifically, bacterial proliferation is quantified as their doubling time, calculated using a proposed stochastic kinetic model. This method identifies the most potent antimicrobial strains by determining which probiotic candidates most effectively extend the bacterial doubling time. In comparative experiments using the same strains, this proposed method demonstrated clear distinctions in the antimicrobial efficacy of each strain, unlike the disk diffusion test. Therefore, this approach provides a reliable solution for identifying superior probiotic strains, with potential for widespread use in discovering new antimicrobial agents.