Characterization of Cryptococcus neoformans biofilms in vitro

Cryptococcus neoformans is a pathogenic fungus responsible for over fifty diseases including biofilm-type infections. Biofilms are composed of individual microbial cells that aggregate to form layered cellular masses attached to a surface, surrounded by a matrix coating. Cryptococcus neoformans biofilms were characterized using 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT), confocal laser scanning microscopy, Hoffman modulation microscopy, phase contrast microscopy and drug susceptibility testing. Capsule size and growth in liquid culture correlated with the ability to form biofilms, while colony morphology did not. The ability of 32 strains of C. neoformans to form biofilms on polystyrene, glass, and silicone elastomer (SE) at 30°C and 35°C was quantified using XTT and microscopy. Biofilms formed on polystyrene, SE, and glass at 35°C and 30°C, but relative biofilm tenacity was substrate and temperature dependent. The experimental compound Auristatin PHE (AUR PHE) is specific primarily for Cryptococcus neoformans. The possibility that AUR PHE's spectrum could be broadened when in combination with membrane damaging drugs was investigated. In vitro planktonic interactions of the approved antifungals amphotericin B (AMB), fluconazole (FLC), and 5-fluorouracil (5FC) with AUR PHE and 1-(3',4',5'-trimethoxyphenyl)-2-nitro-ethylene (TMPN) against the fungal genera Candida and Aspergillus were evaluated. For Candida, AUR PHE-AMB was synergistic for 22% of the isolates and TMPN-FLC was synergistic for 100% of the isolates. For Aspergillus, AUR-5FC was synergistic for 38% of the isolates. Synergistic planktonic combinations were applied to biofilms using a visual endpoint, but interactions were indifferent. There is no standardized method for evaluating fungal biofilm drug susceptibility. The alamar blue (AB) biofilm drug susceptibility assay has been successfully used with Staphylococcus epidermidis; its application to Candida albicans and Cryptococcus neoformans was evaluated. For all Candida strains, AB results had excellent correlation with XTT (r=0.88-0.98) and CFU/ml (r=0.93-0.99). The AB assay also allowed quantitation of C. neoformans biofilm susceptibility; however, it required longer incubation than the XTT method. The demonstration that C. neoformans forms temperature and substrate dependent biofilms in vitro will contribute to the development of disease-specific treatments.