Cell density and cell aging as factors modulating antifungal resistance of Candida albicans biofilms

Biofilm formation is a major virulence attribute of Candida pathogenicity which contributes to higher antifungal resistance. We investigated the roles of cell density and cellular aging on the relative antifungal susceptibility of planktonic, biofilm, and biofilm-derived planktonic modes of Candida. A reference and a wild-type strain of Candida albicans were used to evaluate the MICs of caspofungin (CAS), amphotericin B (AMB), nystatin (NYT), ketoconazole (KTC), and flucytosine (5FC). Standard, NCCLS, and European Committee on Antibiotic Susceptibility Testing methods were used for planktonic MIC determination. Candida biofilms were then developed on polystyrene wells, and MICs were determined with a standard 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-(phenylamino)carbonyl]-2H-tetrazolium hydroxide assay. Subsequently, antifungal susceptibility testing was performed for greater inoculum concentrations and 24- and 48-h-old cultures of planktonic Candida. Furthermore, Candida biofilm-derived planktonic cells (BDPC) were also subjected to antifungal susceptibility testing. The MICs for both C. albicans strains in the planktonic mode were low, although on increasing the inoculum concentration (up to 1 × 10⁸ cells/ml), a variable MIC was noted. On the contrary, for Candida biofilms, the MICs of antifungals were 15- to >1,000-fold higher. Interestingly, the MICs for BDPC were lower and were similar to those for planktonic-mode cells, particularly those of CAS and AMB. Our data indicate that higher antifungal resistance of Candida biofilms is an intrinsic feature possibly related to the biofilm architecture rather than cellular density or cellular aging.