Bacterium Bacillus subtilis adapts to the stressful conditions by binding cells into biofilms, synthesizing antimicrobial substances and developing endospores. These properties are important for applying probiotics of the Bacillus genus, including to control antibiotic-resistant pathogenic bacteria. However, our understanding regarding the impact of long-term pathogen-probiotic co-cultivation is limited. As part of the Master's Thesis, we cultivated a probiotic (B. subtilis PS-216 mKate) and an enteropathogenic bacterium (S. Typhimurium SL1344 GFP) at different initial cell ratios (1:1, 10:1, 100:1), assessed their survival rate by determining their number of colony-forming units (CFU/mL) and estimated S. Typhimurium biofilms' thickness with the help of fluorescence microscopy. The results showed a significant drop in growth of S. Typhimurium in co-cultures. The same goes for its biofilm's thickness, but at initial cell ratios of 10:1 and 100:1. Next, we co-evolved B. subtilis and 100-fold diluted S. Typhimurium, following their number of CFU/mL and changes in colony morphology over the course of 20 cycles. We observed pathogen's eventual adaptation to the probiotic whose number of spores gradually decreased. Finally, we co-cultured isolates from certain trajectories of the 20th cycle with the ancestral strain of the opponent species. We found that evolved B. subtilis with a low spore count had a much lower impact on the survival of ancestral S. Typhimurium in comparison to the strains that maintained a high level of sporulation, while the evolved pathogen was not resistant to the ancestral probiotic.
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