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

Abstract

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 ؋ 10 8 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.

Transcriptional regulation of drug-resistance genes in Candida albicans biofilms in response to antifungals

“…However, all cultures, including stationary-phase, high-density planktonic cultures, were resistant to ketoconazole. This is in agreement with previous studies on the dose-dependent response of fungistatic drugs (Seneviratne et al, 2008b;Beggs, 1989).…”

“…Biofilm C. albicans cultures were generally more resistant to antifungals than recently adhered cells and stationary-phase, high-density planktonic cultures (Table 2), as reported in previous studies (Baillie & Douglas, 1999;Chandra et al, 2001;Seneviratne et al, 2008b). However, all cultures, including stationary-phase, high-density planktonic cultures, were resistant to ketoconazole.…”

“…This phenomenon has been Regulation of drug-resistance genes in Candida biofilms observed by various research groups, including ourselves (Mateus et al, 2004;Seneviratne et al, 2008b). However, levels of expression of the drug efflux pump-related genes CDR1, CDR2 and MDR1 were significantly lower in adhered cells than in planktonic cultures.…”

“…The biofilms were first washed with 200 ml PBS, and then 200 ml XTT solution (PBS : XTT : menadione at 158 : 40 : 2) was added to each of the pre-washed wells and the control wells. High-density planktonic cultures exposed to antifungals in microtitre plates were mixed directly with 200 ml XTT solution, as described in previous studies (Seneviratne et al, 2008b). Microtitre plates with biofilm and high-density planktonic cultures were then incubated in the dark for 3 h at 37 uC.…”

“…In brief, stock solutions of each drug were serially diluted with RPMI 1640 (Life Technologies) supplemented with 2 % glucose to obtain drug concentrations ranging from 480 to 0.225 mg ml 21 for amphotericin B, 100 to 0.1 mg ml 21 for caspofungin and 64 to 0.125 mg ml 21 for ketoconazole. Antifungal susceptibility testing was performed using an XTT reduction assay, as described previously (Seneviratne et al, 2008b). In brief, 24 h Candida biofilms were washed with 100 ml PBS to remove non-adherent cells.…”