The cytochrome P450 14α-demethylase, encoded by the ERG11 (CYP51) gene, is the primary target for the azole class of antifungals. Changes in the azole affinity of this enzyme caused by amino acid substitutions have been reported as a resistance mechanism. Nine Candida albicans strains were used in this study. The ERG11 base sequence of seven isolates, of which only two were azole-sensitive, were determined. The ERG11 base sequences of the other two strains have been published previously. In these seven isolates, 12 different amino acid substitutions were identified, of which six have not been described previously (A149V, D153E, E165Y, S279F, V452A and G465S). In addition, 16 silent mutations were found. Two different biochemical assays, subcellular sterol biosynthesis and CO binding to reduced microsomal fractions, were used to evaluate the sensitivity of the cytochromes for fluconazole and itraconazole. Enzyme preparations from four isolates showed reduced itraconazole susceptibility, whereas more pronounced resistance to fluconazole was observed in five isolates. A three-dimensional model of C. albicans Cyp51p was used to position all 29 reported substitutions, 98 in total identified in 53 sequences. These 29 substitutions were not randomly distributed over the sequence but clustered in three regions from amino acids 105 to 165, from 266 to 287 and from 405 to 488, suggesting the existence of hotspot regions. Of the mutations found in the two N-terminal regions only Y132H was demonstrated to be of importance for azole resistance. In the Cterminal region three mutations are associated with resistance, suggesting that the non-characterized substitutions found in this region should be prioritized for further analysis.

A Candida (Torulopsis) glabrata strain (B57149) became resistant to fluconazole after a patient carrying the organism was treated with the drug at 400 mg once daily for 9 days. Growth of the pretreatment isolate (B57148) was inhibited by 50%v with 0.67 FM ketoconazole, 1.0 pM itraconazole, and 43 pM fluconazole, whereas growth of B57149 was inhibited slightly by 10 FM ketoconazole but was unaffected by 10 FM itraconazole or 100 pM fluconazole. This indicates cross-resistance to all three azole antifungal agents. The cellular fluconazole content of B57149 was from 1.5-to 3-fold lower than that of B57148, suggesting a difference in drug uptake between the strains. However, this difference was smaller than the measured difference in susceptibility and, therefore, cannot fully explain the fluconazole resistance of B57149. Moreover, the intracellular contents of ketoconazole and itraconazole differed by less than twofold between the strains, so that uptake differences did not account for the azole cross-resistance of B57149. The microsomal cytochrome P-450 content of B57149 was about twice that of B57148, a difference quantitatively similar to the increased subcellular ergosterol synthesis from mevalonate or lanosterol. These results indicate that the level of P450-dependent 14u-demethylation of lanosterol is higher in B57149. Increased ergosterol synthesis was also seen in intact B57149 cells, and this coincided with a decreased susceptibility of B57149 toward all three azoles and amphotericin B. B57149 also had higher squalene epoxidase activity, and thus, more terbinafine was needed to inhibit the synthesis of 2,3-oxidosqualene from squalene. P-450 content and ergosterol synthesis both decreased when isolate B57149 was subcultured repeatedly on drug-free medium. This repeated subculture also fiuly restored the strain's itraconazole susceptibility, but only partly increased its susceptibility to fluconazole.The results suggest that both lower fluconazole uptake and increased P-450-dependent ergosterol synthesis are involved in the mechanism of fluconazole resistance but that only the increased ergosterol synthesis contributes to itraconazole cross-resistance.

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We investigated the drug efflux mechanism in azole-resistant strains of Candida albicans using rhodamine 6G (R6G). No significant differences in R6G uptake were observed between azole-sensitive B2630 (9.02 +/- 0.02 nmol/10(8) cells) and azole-resistant B67081 (8.86 +/- 0.03 nmol/10(8) cells) strains incubated in glucose-free phosphate buffered saline. A significantly higher R6G efflux (2.0 +/- 0.21 nmol/10(8) cells) was noted in the azole-resistant strain (B67081) when glucose was added, compared with that in the sensitive strain B2630 (0.23 < or = 0.14 nmol/10(8) cells). A fluconazole-resistant strain C40 that expressed the benomyl resistance gene (CaMDR) also showed a low R6G efflux (0.16 +/- 0.06 nmol/10(8) cells) as did the sensitive strains. Accumulation of R6G in growing C. albicans cells was inversely correlated with the level of CDR1 mRNA expression. Our data also suggest that measurement of intracellular accumulation of R6G is a useful method for identification of azole-resistant strains due to CDR1-expressed drug efflux pump.