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

Abstract

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 mitochondria1 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.

Disruption of mitochondrial function in Candida albicans leads to reduced cellular ergosterol levels and elevated growth in the presence of amphotericin B

“…Resistance in this case was explained by elevated expression of genes coding for drug efflux pumps in response to the loss of a functional mitochondrion (Sanglard et al 2001). While the respiratory-deficient mutant isolated here demonstrated elevated tolerance to amphotericin B, those isolated by Defontaine et al (1999) and Bouchara et al (2000) did not. This may be attributable to the fact that ethidium bromide mutagenesis can yield a range of different respiratory-deficient mutants in yeast and that, while the majority of the genome may be destroyed, small fragments can be retained and amplified to give a mitochondrial DNA content equivalent in size to that of the wild type (Whittaker and Danks 1978).…”

“…Respiratory-deficient mutants of C. glabrata have been isolated clinically and implicated in the failure of anti-fungal therapy due to elevated resistance to azole anti-fungal drugs (Defontaine et al 1999;Bouchara et al 2000). Resistance in this case was explained by elevated expression of genes coding for drug efflux pumps in response to the loss of a functional mitochondrion (Sanglard et al 2001).…”

“…Respiratory-deficient cells of C. glabrata have previously been shown to manifest an increased tolerance to azole anti-fungal drugs (Defontaine et al 1999;Bouchara et al 2000), and respiratory-deficient mutants of C. albicans show enhanced tolerance to the anti-microbial peptide histatin 5 297 Fig. 1 Cytochrome profiles of Candida albicans strains.…”

“…A number of Candida isolates manifesting anti-fungal drug resistance have been characterised as being respiratorydeficient i.e. incapable of aerobic respiration (Defontaine et al 1999). Respiratory-deficient isolates of Candida glabrata demonstrate tolerance to polyene (Geber et al 1995) and azole anti-fungal drugs (Bouchara et al 2000).…”

Synthesis of new isoxazoles and dihydroisoxazoles and in vitro evaluation of their antifungal activity

“…Evaluation of its antifungal activity pointed out that, contrary to several other analogues of tioconazole [10], the isoxazolinyl ether 3 was much more active against C. glabrata petite mutant (MIC 90 ¼ 16 mg/mL) than on its parent wild-type strain (MIC 90 ¼ 125 mg/mL). With this promising result that clearly demonstrated the sensitivity of the petite mutant to isoxazolinyl antifungal conazole, more hydrophilic compounds 10 and 11 bearing a tertiary hydroxyl group like last generation antifungal azoles (i.e.…”