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

Two isolates of Candida glabrata from the same stool sample from a bone mar row transplant recipient treated with fluconazole, and designated 1084-L fo r large colonies on yeast extract-peptone-dextrose-agar and 1084-S for smal l colonies, were analysed. In-vitro susceptibility tests with a commerciall y available disk diffusion procedure showed that isolate 1084-L had a susce ptibility pattern typical of wild-type strains of C, glabrata with sensitiv ity 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 electrophore sis and random amplification of polymorphic DNA to be genetically closely r elated to isolate 1084-L, exhibited cross-resistance to the azole compounds except tioconazole. Determination of MICs by the E-test method confirmed t hese results, showing that isolate 1084-S had greater sensitivity to amphot ericin B and complete resistance to ketoconazole and fluconazole, Growth on agar plates containing glucose or glycerol as the sole carbon source sugge sted that the resistant isolate had a respiratory deficiency, which was fur ther demonstrated by flow cytometric analysis of the fluorescence of rhodam ine 123-stained blastoconidia, Restriction endonuclease analysis of mitocho ndrial DNA (mtDNA) established the mitochondrial origin of the respiratory deficiency. However, PCR amplification of the mtDNA with primers ML1 and ML 6, as well as transmission electron microscopy, suggested a partial deletio n of the mtDNA analogous to that described for rho(-) petite mutants of Sac charomyces cerevisiae, Together, these results provided evidence that the s election of azole-resistant petite mutants of C, glabrata may occur in vivo after fluconazole administration, which might explain, therefore, clinical failure of antifungal therapy.