Synergistic Inhibition of Candida biofilms by Lysozyme and Common Antifungals

Candida-associated denture stomatitis (CADS) is a recalcitrant fungal infection managed using topical antifungals and, the major agent of the disease is biofilm or sessile Candida residing on acrylic dentures. Although lysozyme - a glycol-protein present in saliva - is a important host defense mechanism, the combined effect of lysozyme and common antifungals against biofilm Candida has not been evaluated. Objectives: a) To assess the combined antifungal effect of lysozyme plus, nystatin (Ny), amphotericin B (AmB), ketoconazole (Keto) and 5-fluorocytosine (5FC) on Candida albicans by determining the percentage viability of biofilm/sessile cells b) to visualize the accompanying ultrastructural changes. Methods: The rotating-disc biofilm reactor system was used to develop standardized 2-day old Candida biofilms on denture acrylic discs in YNB/100mM glucose medium, incubated at 37C under standard atmospheric and growth conditions. Candida biofilm metabolic activity with and without antimycotics and/or lysozyme was monitored using a conventional tetrazolium (XTT) reduction assay. SEM and CSLM were used to visualize morphological changes and, also to calculate the percentage viability of the drug exposed cells. Results: When compared with the inhibition by 100ug/ml lysozyme (control), Ny (Range: 6-12 Minimum inhibitory concentrations MIC), AmB (X 6-12 MIC ) and Keto (x12 MIC) in tandem with 100ug/ml lysozyme demonstrated a highly synergistic inhibitory activity of sessile Candida cells (p<0.001). When the antifungal activity of x12 MIC concentrations of Ny, Keto and 5FC were compared, the most effective antifungal agent for the inhibition of biofilm growth of Candida was Ny, whereas Candida biofilm cells appeared to be intrinsically resistant against even the highest concentrations of 5FC. Scanning and confocal scanning laser microscopy also revealed the latter trend of antifungal activity.Conclusion: Salivary lysozyme appear to potentiate commonly used antifungals in the management of CADS.

Supported by the RGC and, CRCG (Grant No/ 10204061) of HKU