In-vitro resistance to azoles associated with mitochondrial DNA deficiency in Candida glabrata

A commercially available disk diffusion procedure was used in a large-scale study to evaluate the susceptibility of a wide range of Candida isolates to polyenes and azoles. With almost all isolates of C. glabrata resistant colonies were present within the inhibition zones for the azole compounds fluconazole, ketoconazole and miconazole, and less frequently for isoconazole, econazole and clotrimazole. Ten randomly selected isolates were cloned by limiting dilution and the susceptibility of the resulting strains to polyenes and azoles was determined. All strains presented a similar susceptibility pattern with sensitivity to polyenes and the presence of resistant colonies for all azole compounds except tioconazole. For each strain and each antifungal agent, one of these resistant colonies was subcultured and studied for antifungal susceptibility. All these colonies showed similar properties regardless of which antifungal agent allowed their selection, with increased sensitivity to polyenes and cross-resistance to the azole compounds except tioconazole. Similar results were obtained on Shadomy's modified medium and on synthetic medium. Likewise, determination of MICs by the Etest method confirmed the resistance to fluconazole. Comparative growth studies revealed a respiratory deficiency in the mutants caused by mitochondrial DNA (mtDNA) deletions. In addition, ‘petite’ mutants were obtained from a wild-type strain by exposure to ethidium bromide, and these respiratory mutants were shown to be resistant to azoles. These results demonstrate the relationship between mtDNA deficiency and resistance to azoles, and provide an interesting model to study the mechanisms of action of these antifungal agents.