Antifungal Efficacy of Citrus Fruit Flavonoids Against Candida albicans Biofilms

Candidal infections have gained considerable attention in recent years due to a growing immunocompromised population. Current antifungal agents in combination with prescribed medications often contribute to systemic toxicity in medically compromised patients. Additionally, the increased prevalence of antifungal drug resistance necessitates the development of new antimicrobial agents with better antifungal activity with the goal of improving current therapeutic strategies. Natural products, especially plant extracts are being used in medicine and are often a good alternative to synthetic chemicals. Objectives: To investigate the antifungal efficacy of bioflavonoids derived from citrus fruits against Candida albicans biofilms. Methods: C. albicans biofilms (wild-type strain and Nystatin resistant human isolate) were developed on polystyrene microtiter plates. Early (1-6 h), intermediate (16-24 h), and mature (48-72 h) biofilms were treated for 60 sec with saline (control), Biosecur c320c (bioflavonoids), mycostatin (Nystatin), or Biosecur c320c/Nystatin combination. Cell survival was determined by CFU counting and drug interaction assessed via checkerboard assay. Results: Cell viability of C. albicans biofilms treated with 10% Biosecur c320c was significantly reduced by 90 to 99% depending on biofilm age (versus control). A combination of 10% Biosecur c320c and 90,000 U/ml Nystatin demonstrated greater efficacy at killing biofilm cells from both wild-type and Nystatin resistant strains; yet synergy was not detected in the checkerboard assay using planktonic cells. After 14 passages with planktonic C. albicans cells, resistance to Biosecur c320c did not develop while exposure to Nystatin led rapidly to resistance (10-fold increase in Nystatin MIC). Scanning electron microscopy of C. albicans biofilms revealed the presence of a large amount of cellular debris after treatment with a combination of Biosecur c320c and Nystatin. Conclusions: Bioflavonoids are effective at rapidly killing C. albicans biofilms. They could represent a new therapeutic approach for the treatment of oral candidiasis particularly in those refractory to Nystatin therapy.