Synergistic activity of lysozyme and antifungal agents against Candida albicans biofilms on denture acrylic surfaces

Denture related oral candidiasis is a recalcitrant fungal infection not easily resolved by topical antifungals. The antimycotic protein lysozyme, in saliva is an important host defense mechanism although its activity against Candida biofilms on denture acrylic has not been evaluated. Objectives: (i) To establish a clinically relevant denture acrylic assay model to develop standardized Candida albicans biofilms, and (ii) assess the inhibitory effects of lysozyme alone and, the latter combined with antifungals (nystatin, amphotericin B, ketoconazole and 5-fluorocytosine) on sessile Candida cells and, finally (iii) to visualize the accompanying ultrastructural changes. Design: The rotating-disc biofilm reactor was used to develop standardized 48 h Candida biofilms on acrylic discs in YNB/100 mM glucose medium and the biofilm metabolic activity was monitored using a tetrazolium reduction assay. Results: The biofilm metabolic activity was similar in 18 identical denture acrylic discs (p < 0.05) thus validating the rotating-disc biofilm model. Very low concentrations of lysozyme (6.25 mu g/ml) significantly (p < 0.01) inhibited Candida biofilm formation indicating that lysozyme may likely regulate intra-oral Candida biofilm development. Although 100 mu g/ml lysozyme killed 45% of sessile Candida cells, further increasing its concentration (up to 240 mu g/ml) had no such effect. Nystatin, amphotericin B, and ketoconazole in association with 100 mu g/ml lysozyme exhibited effective synergistic killing of biofilm Candida in comparison to drug-free controls. Scanning electron and confocal scanning laser microscopy analysis confirmed the latter trends. Conclusion: Our results indicate that agents found in biological fluids such as lysozyme could be a safe adjunct to antifungals in future treatment strategies for recalcitrant candidal infections. (c) 2008 Elsevier Ltd. All rights reserved.