Objectives: To devise an in vitro biofilm model of Aspergillus fumigatus to allow the examination of the ability of antifungal agents to inhibit/kill these biofilms. Methods: Polystyrene microtitre plates were selected to grow biofilms upon, based on previous studies for high throughput analysis of biofilms. Spores were collected from NCPF 7367 and standardized at different densities in RPMI. Biofilm growth kinetics were then observed microscopically over 48 h (1, 2, 4, 6, 24 and 48 h) using a metabolic (XTT) and a biomass assay (crystal violet). Standardized biofilms formed from NCPF 7367 and several clinical isolates were then treated with antifungal agents for 48 h and assayed for viability and biomass. We also examined the effects of biofilm development when antifungals were added at 0, 1, 2 and 4 h postspore inoculation. Following 24 h incubation, the antifungal was removed and replaced with fresh RPMI, and biofilm development reassessed. Results: Spore concentrations of 1 x 106/ml produced the optimal confluent biofilm after 24 h. Biofilm development, assessed by both metabolism and biomass, indicated a slow increase from 0 to 6 h, exponential growth to 24 h, followed by a plateaus. Treatment of mature biofilm structures with amphotericinB, voriconazole and caspofungin was ineffective at clinically achievable concentrations; even at high dosages both metabolism and biomass only decreased minimally. When voriconazole was added during the early stages of adhesion, a dose-dependant effect was observed, and appeared effective at therapeutic concentrations. Replenishment with fresh media allowed biofilm growth in at all concentrations. Conclusions: We have developed a reproducible and robust assay to analyse A. fumigatus biofilms. The results suggest that treatment of mature A. fumigatus biofilms with antifungals is futile. Nevertheless, voriconazole could be used as a prophylactic. The use of this antifungal on early exposure to spores prevented filamentation and subsequent biofilm formation. The continued use of voriconazole would be advocated, as removal of the drug permits biofilm regrowth. Overall, voriconazole appears to offer excellent prophylactic properties against invasive Aspergillosis.
Full conference title:
16th European Congress of Clinical Microbiology and Infectious Diseases
- ECCMID 16th (2006)