Relationships between respiration and susceptibility to azole antifungals in Candida glabrata

Abstract

Over the past two decades, the incidence of infections due to Candida glabrata, a yeast with intrinsic low susceptibility to azole antifungals, has increased markedly. Respiratory deficiency due to mutations in mitochondrial DNA (mtDNA) associated with resistance to azoles frequently occurs in vitro in this species. In order to specify the relationships between respiration and azole susceptibility, the effects of respiratory chain inhibitors on a wild-type isolate of C. glabrata were evaluated. Respiration of blastoconidia was immediately blocked after extemporaneous addition of potassium cyanide, whereas a 4-h preincubation was required for sodium azide. Antifungal susceptibility determined by a disk diffusion method on Casitone agar containing sodium azide showed a significant decrease in the susceptibility to azoles. Biweekly subculturing on Casitone agar supplemented with sodium azide was therefore performed. This resulted after 40 passages in the isolation of a respiration-deficient mutant, as suggested by its lack of growth on glycerol-containing agar. This respiratory deficiency was confirmed by flow cytometric analysis of blastoconidia stained with rhodamine 123 and by oxygraphy. Moreover, transmission electron microscopy and restriction endonuclease analysis of the mtDNA of mutant cells demonstrated the mitochondrial origin of the respiratory deficiency. Finally, this mutant exhibited cross-resistance to all the azoles tested. In conclusion, blockage of respiration in C. glabrata induces decreased susceptibility to azoles, culminating in azole resistance due to the deletion of mtDNA. This mechanism could explain the induction of petite mutations by azole antifungals which have been demonstrated to act directly on the mitochondrial respiratory chain.

FIG. 1.

Oxygen consumption by parent blastoconidia. (A) Effect of extemporaneous addition (arrow) of 1 mM potassium cyanide (dashed line) or sodium azide (dotted line). (B) Effect of a 2-h (dashed line) or 4-h (dotted line) preincubation of the cells with 1 mM sodium azide. The continuous line represents oxygen consumption by the cells in the absence of an inhibitor.

FIG. 2.

Flow cytometry of rhodamine 123-stained parent (A) and mutant P40 (B) cells. Yeast cells were preincubated (thick line) or not (grey area) with 1 mM sodium azide before rhodamine 123 staining. The fluorescence of cells incubated without the fluorochrome (black area) is presented as a control.

FIG. 3.

Oxygen consumption by parent (solid line) and mutant P40 (dashed line) blastoconidia. Comparison of the respiration rates showed a marked decrease in oxygen consumption by the mutant cells (1.5 nmol ml⁻¹ min⁻¹ versus 27.3 nmol ml⁻¹ min⁻¹ for the parent cells).

FIG. 4.

Transmission electron micrographs of parent (A) and mutant P40 (B) cells. Note the absence of mitochondria in the respiration-deficient blastoconidia compared to the numerous mitochondrial sections with obvious cristae (arrowheads) in the parent cells. N, nucleus. Bars, 1 μm.

FIG. 5.

Electrophoretic patterns of mtDNA of parent (lane 1) and mutant P40 (lane 2) cells. mtDNA was analyzed by agarose gel electrophoresis after digestion with EcoRV. Lane M, molecular size markers (Marker III; Roche Molecular Biochemicals, Meylan, France). Sizes are shown in base pairs.

FIG. 6.

Antifungal susceptibility of parent (A) and mutant P40 (B) cells. Susceptibility testing was performed by the disk diffusion method on Casitone agar plates with Neosensitab tablets. 1, amphotericin B; 2, nystatin; 3, miconazole; 4, ketoconazole; 5, clotrimazole; 6, fluconazole; and 7, econazole.