The role of sterol mutations in the resistance of Candida albicans to antifungal agents has not been thoroughly investigated. Previous work reported that clinical C. albicans strains resistant to both azole antifungals and amphotericin B were defective in ERG3, a gene encoding sterol ⌬ 5,6 -desaturase. It is also believed that a deletion of the lanosterol 14␣-demethylase gene, ERG11, is possible only under aerobic conditions when ERG3 is not functional. We tested these hypotheses by creating mutants by targeted deletion of the ERG3 and ERG11 genes and subjecting those mutants to antifungal susceptibility testing and sterol analysis. The homozygous erg3/erg3 mutant created, DSY1751, was resistant to azole derivatives, as expected. This mutant was, however, slightly more susceptible to amphotericin B than the parent wild type. It was possible to generate erg11/erg11 mutants in the DSY1751 background but also, surprisingly, in the background of a wild-type isolate with functional ERG3 alleles under aerobic conditions. This mutant (DSY1769) was obtained by exposure of an ERG11/erg11 heterozygous strain in a medium containing 10 g of amphotericin B per ml. Amphotericin B-resistant strains were obtained only from ERG11/erg11 heterozygotes at a frequency of approximately 5 ؋ 10 ؊5 to 7 ؋ 10 ؊5 , which was consistent with mitotic recombination between the first disrupted erg11 allele and the other remaining functional ERG11 allele. DSY1769 was also resistant to azole derivatives. The main sterol fraction in DSY1769 contained lanosterol and eburicol. These studies showed that erg11/erg11 mutants of a C. albicans strain harboring a defective erg11 allele can be obtained in vitro in the presence of amphotericin B. Amphotericin B-resistant strains could therefore be selected by similar mechanisms during antifungal therapy.

The mechanisms of fluconazole resistance in three clinical isolates of Candida krusei were investigated. Analysis of sterols of organisms grown in the absence and presence of fluconazole demonstrated that the predominant sterol of C. krusei is ergosterol and that fluconazole inhibits 14α-demethylase in this organism. The 14α-demethylase activity in cell extracts of C. kruseiwas 16- to 46-fold more resistant to inhibition by fluconazole than was 14α-demethylase activity in cell extracts of two fluconazole-susceptible strains of Candida albicans. Comparing the carbon monoxide difference spectra of microsomes fromC. krusei with those of microsomes from C. albicans indicated that the total cytochrome P-450 content ofC. krusei is similar to that of C. albicans. The Soret absorption maximum in these spectra was located at 448 nm forC. krusei and at 450 nm for C. albicans. Finally, the fluconazole accumulation of two of the C. krusei isolates was similar to if not greater than that ofC. albicans. Thus, there are significant qualitative differences between the 14α-demethylase of C. albicansand C. krusei. In addition, fluconazole resistance in these strains of C. krusei appears to be mediated predominantly by a reduced susceptibility of 14α-demethylase to inhibition by this drug.

Sequential Candida glabrata isolates were obtained from the mouth of a patient infected with human immunodeficiency virus type 1 who was receiving high doses of fluconazole for oropharyngeal thrush. Fluconazole-susceptible colonies were replaced by resistant colonies that exhibited both increased fluconazole efflux and increased transcripts of a gene which codes for a protein with 72.5% identity to Pdr5p, an ABC multidrug transporter in Saccharomyces cerevisiae. The deduced protein had a molecular mass of 175 kDa and was composed of two homologous halves, each with six putative transmembrane domains and highly conserved sequences of ATP-binding domains. When the earliest and most azole-susceptible isolate of C. glabrata from this patient was exposed to fluconazole, increased transcripts of thePDR5 homolog appeared, linking azole exposure to regulation of this gene.

We report on the mechanism of fluconazole resistance in Candida glabrata from a case of infection in which pre-and posttreatment isolates were available for comparison. The resistant, posttreatment isolate was cross-resistant to ketoconazole and itraconazole, in common with other azole-resistant yeasts. Resistance was due to reduced levels of accumulation of [ 3 H]fluconazole rather than to changes at the level of ergosterol biosynthesis. Studies with metabolic or respiratory inhibitors showed that this phenomenon was a consequence of energy-dependent drug efflux, as opposed to a barrier to influx. Since energy-dependent efflux is a characteristic of multidrug resistance in bacteria, yeasts, and mammalian cells, we investigated the possibility that fluconazole resistance is mediated by a multidrug resistance-type mechanism. Benomyl, a substrate for the Candida albicans multidrug resistance protein, showed competition with fluconazole for efflux from resistant C. glabrata isolates, consistent with a common efflux mechanism for these compounds. By contrast, other standard substrates or inhibitors of multidrug resistance proteins had no effect on fluconazole efflux. In conclusion, we have identified energy-dependent efflux of fluconazole, possibly via a multidrug resistance-type transporter, as the mechanism of resistance to fluconazole in C. glabrata.A growing number of debilitated and immunocompromised patients are at risk of serious fungal infections. These include patients receiving cancer therapies and organ transplants and those infected with the virus that causes AIDS. The latter are particularly susceptible to fungal infections because they are permanently immunocompromised, unlike other groups, in whom immunosuppression is transient. Amphotericin B has been the mainstay of therapy for patients with life-threatening mycoses, although nephrotoxicity and administration by slow intravenous infusion are frequent complications (2). The newer azole class of antifungal agents (fluconazole, itraconazole, and ketoconazole) has also proven to be effective in treating invasive mycoses and represents an important alternative to amphotericin B for some indications (for a review, see reference 28).The azole antifungal agents work by inhibiting cytochrome P-450-dependent 14␣-sterol demethylase of ergosterol biosynthesis (P-450 DM ) (for a review, see reference 10). Azoletreated fungi are depleted of ergosterol and accumulate 14␣-methylated sterols which inhibit fungal growth. All of the azoles are fungistatic, as opposed to fungicidal, against Candida spp., underlining the importance of the host's immune system for eradicating the infecting organism and achieving a clinical cure. A corollary of this situation is that AIDS patients require indefinite suppressive therapy, and given the widespread use of fluconazole in end-stage AIDS (including patients who have failed ketoconazole or itraconazole therapy), it is not unexpected that fluconazole-resistant Candida strains have been isolated from this group of patients (22).Stu...