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The biofilms of filamentous-forming fungi are a novel and still insufficiently understood research topic. We have studied Aspergillus fumigatus, an ubiquitous opportunistic pathogenic fungus, as a representative model for a study of biofilm formation by filamentous fungi and for assessing the potential anti-biofilm activity of natural substances. The activity of antibiotic amphotericin B and selected natural substances: baicalein, chitosan and rhamnolipid was studied. The minimum suspension inhibitory concentrations (MIC) were determined and the biofilm susceptibility was investigated by determining the metabolic activity of sessile cells (XTT assay) and total biofilm biomass (crystal violet staining). Significant time-dependent differences in substances' anti-biofilm activity were observed. Images of A. fumigatus biofilm were obtained by Cellavista automatic light microscope and spinning disc confocal microscopy. Baicalein and rhamnolipid were not found as suitable substances for inhibition of the A. fumigatus biofilm formation, as neither of the substances inhibited the sessile cells metabolic activity or the total biofilm biomass even at tenfold MIC after 48 h. In contrast, chitosan at 10 × MIC (25 µg mL), suppressed the biofilm metabolic activity by 90 % and the total biofilm biomass by 80 % even after 72 h of cultivation. Amphotericin B inhibited only 14 % of total biofilm biomass (crystal violet staining) and 35 % of metabolic activity (XTT assay) of adherent cells under the same conditions. Our results therefore suggest chitosan as potential alternative for treating A. fumigatus biofilm-associated infections.

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Emerging resistance of microbial cells towards antibiotics or disinfectants leads to an increase of nosocomial diseases, often biofilm related, and gives rise to the need for research for new antimicrobial substances. These studies are often focused on substances that are able to support the efficacy of currently used antimicrobials, reduce their required dosage and reduce the probability of resistance development. This work brings new insights into the in vitro interaction of rhamnolipid biosurfactant and amphotericin B against Candida sp. or Trichosporon sp.. The minimum inhibitory concentrations and fractional inhibitory concentration indexes for both planktonic and biofilm cells were determined by the chequerboard microdilution method. Combination effect of rhamnolipid and amphotericin B was observed for both yeasts. Synergy, defined as a fractional inhibitory concentration (FICi) index of 0.50, was observed in both planktonic and biofilm cells of Trichosporon cutaneum. For C. parapsilosis, synergistic effect (FICi 0.5) was observed for planktonic cells, and additive effect (FICi 0.8) for biofilm cells. The influence of concentrations established as FICi on biofilm formation and susceptibility was studied by light microscopy. The highest inhibition (90% colonized area reduction) of initial adhesion of C. parapsilosis was observed when biofilm FIC was applied.