Fungicidal synergism of fluconazole and cyclosporine in Candida albicans is not dependent on multidrug efflux transporters encoded by the CDR1, CDR2, CaMDR1, and FLU1 genes

Abstract

The combination of fluconazole (FLC) and cyclosporine (CY) is fungicidal in FLC-susceptible C. albicans (O. Marchetti, P. Moreillon, M. P. Glauser, J. Bille, and D. Sanglard, Antimicrob. Agents Chemother. 44:2373-2381, 2000). The mechanism of this synergism is unknown. CY has several cellular targets including multidrug efflux transporters. The hypothesis that CY might inhibit FLC efflux was investigated by comparing the effect of FLC-CY in FLC-susceptible parent CAF2-1 (FLC MIC, 0.25 mg/liter) and in FLC-hypersusceptible mutant DSY1024 (FLC MIC, 0.03 mg/liter), in which the CDR1, CDR2, CaMDR1, and FLU1 transporter genes have been selectively deleted. We postulated that a loss of the fungicidal effect of FLC-CY in DSY1024 would confirm the roles of these efflux pumps. Time-kill curve studies showed a more potent fungistatic effect of FLC (P = 0.05 at 48 h with an inoculum of 10(3) CFU/ml) and a more rapid fungicidal effect of FLC-CY (P = 0.05 at 24 h with an inoculum of 10(3) CFU/ml) in the FLC-hypersusceptible mutant compared to those in the parent. Rats with experimental endocarditis were treated for 2 or 5 days with high-dose FLC, high-dose CY, or both drugs combined. FLC monotherapy for 5 days was more effective against the hypersusceptible mutant than against the parent. However, the addition of CY to FLC still conferred a therapeutic advantage in animals infected with mutant DSY1024, as indicated by better survival (P = 0.04 versus the results obtained with FLC) and sterilization of valves and kidneys after a very short (2-day) treatment (P = 0.009 and 0.002, respectively, versus the results obtained with FLC). Both in vitro and in vivo experiments consistently showed that the deletion of the four membrane transporters in DSY1024 did not result in loss of the fungicidal effect of FLC-CY. Yet, the accelerated killing in the mutant suggested a "dual-hit" mechanism involving FLC hypersusceptibility due to the efflux pump elimination and fungicidal activity conferred by CY. Thus, inhibition of multidrug efflux transporters encoded by CDR1, CDR2, CaMDR1, and FLU1 genes is not responsible for the fungicidal synergism of FLC-CY. Other cellular targets must be considered.

FIG. 1.

Time-kill curves obtained by using an initial inoculum of 10³ CFU/ml (A) or 10⁵ CFU/ml (B). The effects of FLC, CY, and FLC-CY against C. albicans CAF2-1 (susceptible parent; FLC MIC, 0.25 mg/liter [left panels]) and DSY1024 (hypersusceptible efflux-pump deleted mutant; FLC MIC, 0.03 mg/liter [right panels]) were tested. Mean colony counts and error bars for triplicate experiments are shown.

FIG. 2.

Survival studies in rats with experimental endocarditis due to FLC-susceptible parent CAF2-1 (A) and FLC-hypersusceptible mutant DSY1024 (B). The results for untreated controls and animals receiving CY, FLC, and FLC-CY were compared. The pooled results for duplicate experiments are shown. Day 0 corresponds to the onset of treatment, and day 5 corresponds to the end of treatment. NS, not significant.

FIG. 3.

Fungal densities in aortic valve vegetations (A) and kidneys (B) after 2 days of treatment for experimental endocarditis due to CAF2-1 or DSY1024. The pooled results of duplicate experiments are shown. Each point represents the fungal density observed in a single animal. Horizontal bars indicate the median fungal densities for each group.

FIG. 4.

Fungal densities in aortic valve vegetations (A) and kidneys (B) after 5 days of treatment for experimental endocarditis due to CAF2-1 or DSY1024. The pooled results of duplicate experiments are shown. Each point represents the fungal density observed in a single animal. Horizontal bars indicate the median fungal densities for each group.