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.

Routine use of CHROMagar Candida medium for presumptive identification of Candida yeast species and detection of mixed fungal populations.

OBJECTIVE: To assess the value of the new differential culture medium CHROMagar Candida for routine investigation of clinical specimens. METHODS: During a whole year, 6150 clinical samples were plated on CHROMagar Candida medium. After incubation, the green colonies were considered to be Candida albicans. The colonies of other colors were identified using Bichrolatex-krusei, or by their assimilation pattern on ID 32C test strips and their morphology on rice cream-agar-Tween. RESULTS: Among the 6150 clinical samples, 1643 were positive for fungi. Aspergillus fumigatus and Geotrichum sp. were the predominant filamentous fungi isolated. Candida albicans was the most common species isolated (1274 of the positive samples; 77.5%), and Candida glabrata was the second most common yeast isolated (174 positive samples; 10.6%). Other yeast species were detected at lower frequencies, mainly Candida tropicalis (3.8%), Candida krusei (2.7%), Saccharomyces cerevisiae (2.7%) and Candida kefyr (2.3%), and 16 samples revealed a lipophilic species, Malassezia furfur. Mixed fungal populations accounted for 14.7% of the positive samples. Two or more yeast species were detected in 206 of the 242 specimens containing mixed fungal populations, and five yeast species were detected in one sample. Additionally, we did not observe significant differences in the isolation of yeasts or filamentous fungi from the 366 samples simultaneously plated on CHROMagar Candida and Sabouraud dextrose agar. Close agreement between the two culture media was observed for 89.9% of these samples. CONCLUSIONS: CHROMagar Candida medium was shown to be extremely helpful in a routine clinical mycology service, facilitating the detection of mixed cultures of yeasts and allowing direct identification of C. albicans, as well as rapid presumptive identification of the other yeasts: C. glabrata, C. tropicalis, C. krusei and S. cerevisiae. This chromogenic medium thus appears to be suitable as a primary culture medium, particularly for the mycologic surveillance of immunocompromised patients.