Biofilms are complex microbial communities in which cells are surrounded by extracellular polymeric substances (EPS), which are thought to limit the transport of antimicrobial agents within biofilms. In this master’s thesis, we investigated the diffusion properties of a mature Bacillus subtilis biofilm, focusing on the effect of the extracellular polysaccharide EpsA-O on the diffusion, transport, and availability of the fluorescently labeled antibiotic daptomycin. We also aimed to evaluate the influence of molecular size and biofilm structure on diffusion and to elucidate the role of EpsA-O in the reduced efficacy, distribution, and localization of daptomycin within the biofilm. Diffusion was studied using the method with fluorescently labeled dextrans of various molecular weights in aqueous solutions and in the biofilm of wild type B. subtilis and Δeps mutants, which do not produce EpsA-O. The results showed that diffusion decreases with increasing molecular weight and is significantly slowed in biofilms compared to aqueous solutions. Using confocal microscopy, we revealed that daptomycin accumulation in Δeps biofilm cells is higher compared to wild type biofilm. Colocalization analysis revealed that daptomycin coincided with the cell membrane in the Δeps mutant, whereas in the wild type strain, it was displaced relative to the membrane, suggesting interactions with EPS. Despite the low concentration and fluorescent labeling of daptomycin, we observed greater resistance of the wild type biofilm to daptomycin than the Δeps mutant. In addition, a faster decrease in the local concentration of daptomycin was observed in the wild type biofilm, suggesting additional mechanisms of antibiotic redistribution and dilution. The results indicate that the EpsA-O polysaccharide significantly influences the transport, mobility, distribution, localization, and efficacy of daptomycin and contributes to biofilm resistance.
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