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

A commercially available disk diffusion procedure was used in a large-scale study to evaluate the susceptibility of a nide range of Candida isolates t o polyenes and azoles. With almost all isolates of C. glabrata resistant co lonies were present within the inhibition zones for the azole compounds flu conazole, 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 polyen es and azoles was determined. All strains presented a similar susceptibilit y pattern with sensitivity to polyenes and the presence of resistant coloni es for all azole compounds except tioconazole. For each strain and each ant ifungal 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 increas ed sensitivity to polyenes and cross-resistance to the azole compounds exce pt tioconazole. Similar results were obtained on Shadomy's modified medium and on synthetic medium. Likewise, determination of MICs by the Etest metho d confirmed the resistance to fluconazole. Comparative growth studies revea led a respiratory deficiency in the mutants caused by mitochondrial DNA (mt DNA) deletions. In addition, 'petite' mutants were obtained from a wild-typ e strain by exposure to ethidium bromide, and these respiratory mutants wer e shown to be resistant to azoles. These results demonstrate the relationsh ip between mtDNA deficiency and resistance to azoles, and provide an intere sting model to study the mechanisms of action of these antifungal agents.