Cell Density and Cell Aging as Factors Modulating Antifungal Resistance ofCandida albicansBiofilms

ABSTRACTBiofilm formation is a major virulence attribute ofCandidapathogenicity 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 ofCandida. A reference and a wild-type strain ofCandida albicanswere 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.Candidabiofilms 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 planktonicCandida. Furthermore,Candidabiofilm-derived planktonic cells (BDPC) were also subjected to antifungal susceptibility testing. The MICs for bothC.albicansstrains in the planktonic mode were low, although on increasing the inoculum concentration (up to 1 × 108cells/ml), a variable MIC was noted. On the contrary, forCandidabiofilms, 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 ofCandidabiofilms is an intrinsic feature possibly related to the biofilm architecture rather than cellular density or cellular aging.