Formation of biofilm is a process in which microbial cells aggregate into clusters, connected with extracellular polymeric substances (EPS). The cells in this survival mode are highly resistant to various stress factors, such as the addition of antibiotics, reduced nutrient concentrations and the formation of anaerobic conditions. In this Master's thesis, we studied the resistance of Bacillus subtilis NCIB 3610 to the antibiotic daptomycin in various conditions, including synthetic biofilms, native biofilms, homogenized native biofilms and cells without added EPS. Additionaly, we investigated the impact of different cell densities and growth phases on the daptomycin resistance of B. subtilis. The results showed that stationary-phase cells in liquid cultures at lower cell densities were less resistant to daptomycin compared to denser cultures, indicating a significant role of cell density in antibiotic sensitivity. We also found that stationary-phase cells were more resistant to daptomycin than pre-stationary-phase cells, which can be attributed to physiological adaptations associated with stress conditions. When comparing the resistance of differently prepared biofilms, native biofilms demonstrated the highest resistance, while synthetic biofilms enriched with EPS showed resistance comparable to homogenized native biofilms. The lowest resistance was observed in cells without added EPS. These findings highlight the essential role of biofilm structure and the extracellular matrix in protecting bacterial cells against antimicrobial agents. They also provide valuable insights into the mechanisms driving the resistance of Bacillus subtilis biofilms to antibiotics.
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