In-vivo selection of an azole-resistant petite mutant of Candida glabrata

Abstract

Two isolates of Candida glabrata from the same stool sample from a bone marrow transplant recipient treated with fluconazole, and designated 1084-L for large colonies on yeast extract-peptone-dextrose-agar and 1084-S for small colonies, were analysed. In-vitro susceptibility tests with a commercially available disk diffusion procedure showed that isolate 1084-L had a susceptibility pattern typical of wild-type strains of C. glabrata with sensitivity to polyenes and the presence of resistant colonies randomly distributed within the inhibition zones for all azole compounds except tioconazole. In contrast, isolate 1084-S, which was found by pulsed-field gel electrophoresis and random amplification of polymorphic DNA to be genetically closely related to isolate 1084-L, exhibited cross-resistance to the azole compounds except tioconazole. Determination of MICs by the E-test method confirmed these results, showing that isolate 1084-S had greater sensitivity to amphotericin B and complete resistance to ketoconazole and fluconazole. Growth on agar plates containing glucose or glycerol as the sole carbon source suggested that the resistant isolate had a respiratory deficiency, which was further demonstrated by flow cytometric analysis of the fluorescence of rhodamine 123-stained blastoconidia. Restriction endonuclease analysis of mitochondrial DNA (mtDNA) established the mitochondrial origin of the respiratory deficiency. However, PCR amplification of the mtDNA with primers ML1 and ML6, as well as transmission electron microscopy, suggested a partial deletion of the mtDNA analogous to that described for rho- petite mutants of Saccharomyces cerevisiae. Together, these results provided evidence that the selection of azole-resistant petite mutants of C. glabrata may occur in vivo after fluconazole administration, which might explain, therefore, clinical failure of antifungal therapy.