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The main resistance mechanism for fluconazole in Candida krusei is the diminished sensitivity of the target enzyme cytochrome P450 sterol 14 alpha-demethylase (CYP51) to inhibition by azole agents. An alternative mechanism of resistance, efflux-pump activity, has been proposed. The aim of our study was to find out the possible contribution of efflux-pumps in conferring resistance to fluconazole in 33 C. krusei isolates from different clinical sources. The activity of efflux-pumps was checked using the inhibitor CCCP (carbonyl cyanide 3-chloro-phenylhydrazone), which decreases the minimum inhibitory concentration (MIC) when resistance is attributed. We established a concentration of 0.5 microg/ml of CCCP. The susceptibility patterns of our isolates for five antifungal drugs (amphotericin B, fluconazole, itraconazole, flucytosine and voriconazole) were determined according to an NCCLS M27-A2 protocol modification (Sensititre Yeast One). We tested all the strains before and after adding CCCP to the RPMI medium. The MIC90s and ranges of the drugs were identical before and after addition of CCCP. The MIC for fluconazole was higher than for the other antifungals. The new triazoles were active and the MICs were lower, although this should be interpreted carefully as the drugs showed different cut-offs. Only one isolate showed a two-fold decrease in MIC to fluconazole when CCCP was added. We did not find any multi-resistant strains. According to our study with C. krusei, CCCP-inhibited efflux-pumps do not play a significant role in resistance to fluconazole.

Fluconazole resistance mechanisms inCandida krusei: The contribution of efflux-pumps