Doxorubicin selects for fluconazole-resistant petite mutants in Candida glabrata isolates

Candida infections are a permanent threat to immunocompromised individuals such as cancer patients, and Candida glabrata has emerged as a major problem in recent years. Resistance may develop during lengthy antifungal therapies and is often mediated by upregulation of fungal drug efflux pumps. During chemotherapy the yeast cell is also exposed to cytotoxic agents that may affect its drug susceptibility. Four C. glabrata isolates, three susceptible and one resistant to fluconazole (FLU), were incubated with 20 μg/ml of doxorubicin (DOX) for 90 min. In a second experiment, the isolates were cultured with DOX for ten days. Samples were taken on subsequent days to determine the minimal inhibitory concentration (MIC) of FLU and to analyze expression of CgCDR1, CgCDR2, CgSNQ2 and CgPDR1. Samples were also used to assess the petite phenotype. Short-term DOX exposure did not induce efflux pump gene expression, but genes were consistently overexpressed in FLU-susceptible isolates during long-term exposure. An increase in MIC values on day 6 in two of the isolates coincided with the first occurrence of petite mutants in all susceptible isolates. The respiratory deficiency of selected petite mutants was confirmed by culturing mutants on agar containing glycerol as the sole carbon source. FLU MIC values for respiratory-deficient clones were ≥64 μg/ml, and efflux pump gene expression was greatly increased. The resistant isolate did not develop mitochondrial dysfunction. In summary, the cytotoxic agent DOX selects for FLU-resistant respiratory-deficient C. glabrata mutants, which may affect antifungal therapy.