Fluconazole resistance due to energy-dependent drug efflux in Candida glabrata

Abstract

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...

In Vitro Resistance of Staphylococcus aureus to Thrombin-Induced Platelet Microbicidal Protein Is Associated with Alterations in Cytoplasmic Membrane Fluidity

“…F-74, 1996). Such Candida strains have been shown to possess an energy-dependent drug efflux mechanism, putatively analogous to the multidrug resistance efflux pumps of mammalian tumor cells (15,16,24,30). Likewise, there are at least two examples of energydependent systems conferring a cationic peptide-resistant phenotype in bacterial species.…”

Multiple amino acid substitutions in lanosterol 14α-demethylase contribute to azole resistance in Candida albicans

“…Drug susceptibility testing of C. albicans isolated from these patients revealed in many cases that the organism had acquired resistance to fluconazole (Denning et al, 1997 ;Law et al, 1994 ;Rex et al, 1995 ;Tumbarello et al, 1996 ;. Several mechanisms, independent of 14DM, have been reported which could contribute at least partially to fluconazole resistance in Candida species, including increased ergosterol biosynthesis (Vanden Bossche et al, 1992), a lesion in ∆5,6 sterol desaturase , and lower intracellular accumulation of the drug (Marichal et al, 1995 ;Parkinson et al, 1995 ;Sanglard et al, 1995 ;Vanden Bossche et al, 1992 ;Venkateswarlu et al, 1996Venkateswarlu et al, , 1997. This last mechanism seems to be the most common and is associated with the overexpression of two types of multidrug transporters, the ATP-binding cassette (ABC) transporters (Albertson et al, 1996 ;Miyazaki et al, 1998 ;Moran et al, 1998 ;Prasad et al, 1995 ;Sanglard et al, 1995Sanglard et al, , 1996Sanglard et al, , 1997van Veen & Konings, 1998 ;White, 1997a) and the major facilitators (Albertson et al, 1996 ;Goldway et al, 1995 ;Moran et al, 1998 ;Sanglard et al, 1995Sanglard et al, , 1996White, 1997a).…”

The Candida albicans CDR3 gene codes for an opaque-phase ABC transporter

“…Azole derivatives such as fluconazole (FCZ) are often used for clinical treatment, but resistance to this class of drugs is becoming increasingly common in patients undergoing long-term or prophylactic treatment (36). A number of studies investigating the mechanisms of FCZ resistance in C. albicans and other Candida species have previously shown that resistant strains fail to accumulate FCZ due to an increased drug efflux, suggesting the participation of transporter-mediated drug resistance mechanisms in these strains (33,41). Two C. albicans genes of the ABC family, CDR1 and CDR2, have been isolated by functional complementation of an S. cerevisiae pdr5 null mutant strain, screening for cycloheximide and FCZ resis-tance, respectively (34,40).…”