Characteristics of biofilm formation by Candida albicans.

A variety of manifestations of Candida albicans infections are associated with the formation of biofilms on the surface of biomaterials. Cells in biofilms display phenotypic traits that are dramatically different from their free-floating planktonic counterparts, such as increased resistance to anti-microbial agents and protection form host defenses. Here, we describe the characteristics of C. albicans biofilm development using a 96 well microtitre plate model, microscopic observations and a colorimetric method based on the use of a modified tetrazolium salt (2,3-bis(2-methoxy-4-nitro-5-sulfo-phenyl)-2H-tetrazolium-5-carboxanilide, XTT) to monitor metabolic activities of cells within the biofilm. C. albicans biofilm formation was characterized by initial adherence of yeast cells (0-2 h), followed by germination and micro-colony formation (2-4 h), filamentation (4-6 h), monolayer development (6-8 h), proliferation (8-24 h) and maturation (24-48 h). The XTT-reduction assay showed a linear relationship between cellular density of the biofilm and metabolic activity. Serum and saliva pre-conditioning films increased the initial attachment of C. albicans, but had minimal effect on subsequent biofilm formation. Scanning electron microscopy and confocal scanning laser microscopy were used to visualize C. albicans biofilms. Mature C. albicans biofilms consisted of a dense network of yeasts cells and hyphal elements embedded within exopolymeric material. C. albicans biofilms displayed a complex three dimensional structure which demonstrated spatial heterogeneity and a typical architecture showing microcolonies with ramifying water channels. Antifungal susceptibility testing demonstrated the increased resistance of sessile C. albicans cells against clinically used fluconazole and amphotericin B as compared to their planktonic counterparts.