Mechanism of Fluconazole Resistance in <i>Candida albicans</i> Biofilms: Phase-Specific Role of Efflux Pumps and Membrane Sterols

TLDR

Candida albicans biofilms develop through three distinct phases and exhibit high fluconazole resistance. The study aimed to determine how deletion of the efflux pumps Cdr1p, Cdr2p, and Mdr1p, together with changes in sterol composition, influence fluconazole resistance during biofilm formation. Isogenic strains lacking one or more of these efflux pumps were used, and sterol profiles were analyzed to assess their contribution to drug resistance. Results showed that all strains formed similar biofilms, but double and triple mutants were more susceptible to fluconazole at 6 h, while by 12–48 h all strains became resistant; early-phase differences in efflux activity and reduced ergosterol levels revealed phase‑specific, multicomponent resistance mechanisms.

Abstract

Candida albicans biofilms are formed through three distinct developmental phases and are associated with high fluconazole (FLU) resistance. In the present study, we used a set of isogenic Candida strains lacking one or more of the drug efflux pumps Cdr1p, Cdr2p, and Mdr1p to determine their role in FLU resistance of biofilms. Additionally, variation in sterol profile as a possible mechanism of drug resistance was investigated. Our results indicate that parent and mutant strains formed similar biofilms. However, biofilms formed by double and triple mutants were more susceptible to FLU at 6 h (MIC = 64 and 16 microg/ml, respectively) than the wild-type strain (MIC > 256 microg/ml). At later time points (12 and 48 h), all the strains became resistant to this azole (MIC > or = 256 microg/ml), indicating lack of involvement of efflux pumps in resistance at late stages of biofilm formation. Northern blot analyses revealed that Candida biofilms expressed CDR and MDR1 genes in all the developmental phases, while planktonic cells expressed these genes only at the 12- and 48-h time points. Functionality of efflux pumps was assayed by rhodamine (Rh123) efflux assays, which revealed significant differences in Rh123 retention between biofilm and planktonic cells at the early phase (P = 0.0006) but not at later stages (12 and 48 h). Sterol analyses showed that ergosterol levels were significantly decreased (P < 0.001) at intermediate and mature phases, compared to those in early-phase biofilms. These studies suggest that multicomponent, phase-specific mechanisms are operative in antifungal resistance of fungal biofilms.