In vitro biofilms formation of Candida species: Impact of different sugars, its concentrations and effect of two ayurvedic preparations

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Aims: Biofilms are ubiquitous life forms and their pathogenicity is dictated by the constituents of the environment. We evaluated the efficacy of three culture media on biofilm formation of Candida species. Role of sucrose, glucose, saccharine, and inhibitory effect of ayurvedic oral treatments were investigated. Methods: A 96-well plate was inoculated using 106cell/ml of C.albicans, C.tropicalis and 1:1 mixed species and growth rates were determined by measuring the absorbance every 2hrs with the presence of three culture media(Yeast Nitrogen Base (YNB) supplemented with 100 mM glucose, Sabouraud Dextrose Broth (SDB) and RPMI 1640), sweeteners (5 and 10%: glucose, sucrose and saccharin). Adhesion and growth rates were quantified using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyItetrazolium bromide) and Crystal Violet (CV). Inhibitory effect of 0.2% chlohexidine, aqueous extracts of bark of Mimusops elengi and Tripala (mixture of fruits of Emblica officinalis, Terminalia bellirica and Terminalia chebula) were investigated against both sessile and planktonic cells. Scanning electron microscope was performed to assess the biofilm architecture with different treatments. Results: All three biofilms showed maximum adhesion with RPMI 1640. SDB promoted the planktonic cell growth. Glucose and sucrose (5%) had the maximum effect on adhesion of all three biofilms. Planktonic cell growth was highest with 5% glucose while biofilm growth was promoted with 5% sucrose. 0.2% chlorhexidine significantly reduced the biofilm formation within 30 seconds of exposure. Aqueous extract of Triphala (65.0 mg/ml) was effective against planktonic cells while Mimusops elengi had no inhibitory effect. Conclusion: RPMI 1640 effectively facilitate in-vitro biofilm formation of Candida. Our data indicate that researchers should pay more attention on standardization of growth media for cross comparison purposes as sessile cells act differently to the planktonic cells. Further sucrose promotes biofilm formation of Candida compared to glucose and saccharine. Triphala had an inhibitory effect whereas extracts of Mimusops elengi did not show any inhibitory effect. Key words- Candida biofilms, Culture media, Sugars, Herbal mouth rinses, 0.2% chlorhexidine gluconate