A new method for in vitro Candida biofilm formation

Candida albicans is an opportunistic fungal pathogen capable of high morbidity particularly in immunocompromised patients. An increased prevalence of oral candidiasis that is resistant to the current drugs can be related to the enhanced ability of colonizing yeasts to produce biofilms.

Objective: The aim of this study was to optimize a method for Candida albicans biofilm formation that could be used for testing new antifungal molecules.

Methods: The C. albicans MYA-274 biofilms were formed on acrylic resins discs in batch culture at 37^oC, 5% CO₂ for a total of 7 days. Yeast-based medium containing 1% glucose was changed every 24 h from days 1 through 7. At the end of each day, the biomass, colony formation unit (cfu) and the pH of the media were measured. In addition, the biofilm morphology was evaluated using a combination of fluorescence imaging techniques and Environmental Scanning Electron Microscopy. Fluconazole (250µg/ml) was used as gold standard treatment against mature biofilm.

Results: During the course of the 7 days, the pH of the solution dropped from 6.5 to 4.5. The biomass and cfu reach their plateaus at day 4 of biofilm development. The yeast-to-hypha transition formations were observed from day 2. The treatment with Fluconazole reduces statistically the fungal viability however the structural organization of the biofilm remained intact after the treatment.

Conclusion: In the proposed method, the in vitro Candida biofilm reaches its peak after 4 days, and it is possible to see an exponential growth of the biofilm; more studies are needed to further elucidate the virulence mechanisms. The optimized model can be used to evaluate new antimicrobial agents.

Acknowledgement: Supported by NIH/NCCAM 1K99AT006507-01