Study of Inter-Species Social Interactions Among Bacterial Cells Using Computer Vision and Zeta Potential Analysis

The interplay between the growth patterns of two or more bacterial species in a co-culture system is often overlooked in traditional microbiology. Analysing the behaviour of pathogens as part of a cohort of bacterial species becomes important because when under a high degree of stress or in large populations, bacterial species can develop mutants. However, the factors affecting the course of such social evolution remain unexplored. In this article, we have attempted to systematically study the social interaction in paired and triplet mixed cultures of Escherichia coli, Salmonella enterica serovar Typhimurium and Staphylococcus aureus. The method is based on computer vision analysis of selective agar plating of both pure and mixed cultures (plated after co-incubation) followed by Zeta potential measurements. Primarily, the social interactions between bacterial species, whether synergetic or antagonistic, are mediated through the exchange of electrical charges. The density of charges which are present on the bacterial surface can be characterised by measuring the Zeta potential. Studying the Zeta potential of co-cultures in various volume ratios aims at probing the effect of mixing of species on the resultant surface charge of the cells in the cohort. Based on the results, we explore how certain species electrically dominate over others in co-cultures, yet they co-exist. Most importantly, the surface charge modifications arising due to the social interactions can severely affect the bactericidal action of antimicrobial agents. To confirm this, the last section of the manuscript is dedicated to the antimicrobial susceptibility tests performed using the disc diffusion method on pure samples and consortia. The results are presented for eleven different antibiotics indicating significant alterations in the efficacy of some of the antimicrobial agents when used against co-cultures.