The recent rate of emergence of pathogenic fungi that are resistant to the limited number of commonly used antifungal agents is unprecedented. The azoles, for example, are used not only for human and animal health care and crop protection but also in antifouling coatings and timber preservation. The ubiquity and multiple uses of azoles have hastened the independent evolution of resistance in many environments. One consequence is an increasing risk in human health care from naturally occurring opportunistic fungal pathogens that have acquired resistance to this broad class of chemicals. To avoid a global collapse in our ability to control fungal infections and to avoid critical failures in medicine and food security, we must improve our stewardship of extant chemicals, promote new antifungal discovery, and leverage emerging technologies for alternative solutions.

Azole antifungal agents, and especially fluconazole, have been used widely to treat oropharyngeal candidiasis in patients with AIDS. An increasing number of cases of clinical resistance against fluconazole, often correlating with in vitro resistance, have been reported. To investigate the mechanisms of resistance toward azole antifungal agents at the molecular level in clinical C. albicans isolates, we focused on resistance mechanisms related to the cellular target of azoles, i.e., cytochrome P450 14DM (14DM) and those regulating the transport or accumulation of fluconazole. The analysis of sequential isogenic C. albicans isolates with increasing levels of resistance to fluconazole from five AIDS patients showed that overexpression of the gene encoding 14DM either by gene amplification or by gene deregulation was not the major cause of resistance among these clinical isolates. We found, however, that fluconazole-resistant C. albicans isolates failed to accumulate 3 H-labelled fluconazole. This phenomenon was reversed in resistant cells by inhibiting the cellular energy supply with azide, suggesting that resistance could be mediated by energy-requiring efflux pumps such as those described as ATP-binding cassette (ABC) multidrug transporters. In fact, some but not all fluconazole-resistant clinical C. albicans isolates exhibited up to a 10-fold relative increase in mRNA levels for a recently cloned ABC transporter gene called CDR1. In an azole-resistant C. albicans isolate not overexpressing CDR1, the gene for another efflux pump named BEN r was massively overexpressed. This gene was cloned from C. albicans for conferring benomyl resistance in Saccharomyces cerevisiae. Therefore, at least the overexpression or the deregulation of these two genes potentially mediates resistance to azoles in C. albicans clinical isolates from AIDS patients with oropharyngeal candidiasis. Involvement of ABC transporters in azole resistance was further evidenced with S. cerevisiae mutants lacking specific multidrug transporters which were rendered hypersusceptible to azole derivatives including fluconazole, itraconazole, and ketoconazole.

Resistance to azole antifungal agents in Candida albicans can be mediated by multidrug efflux transporters. In a previous study, w e identified a t least two such transporters, C d r l p and Benp, which belong to the class of ATP-binding -cassette (ABC) transporters and of major facilitators, respectively. To isolate additional factors potentially responsible for resistance to azole antifungal agents in C. albicans, the hypersusceptibility of a Saccharomyces cerevisiae multidrug transporter mutant, Apdr5, to these agents was complemented with a C. albicans genomic library. Several new genes were isolated, one of which was a new ABC transporter gene called CDR2 (Candida drug Eesistance). The protein Cdr2p encoded b y this gene exhibited 84% identity with C d r l p and could confer resistance to azole antif ungal agents, to other antifungals (terbinafine, amorolfine) and to a variety of metabolic inhibitors. The disruption of CDR2 in the C. albicans strain CAM-2 did not render cells more susceptible to these substances. When the disruption of CDR2 was performed in the background of a mutant in which CDR7 was deleted, the resulting double Acdrl Acdr2 mutant was more susceptible to these agents than the single Acdrl mutant. The absence of hypersusceptibility of the single Acdr2 mutant could be explained by the absence of CDR2 mRNA in azole-susceptible C. albicans strains. CDR2 was overexpressed, however, in clinical C. albicans isolates resistant to azole antifungal agents as described previously for CDRI, but to levels exceeding or equal to those reached b y CDRl. Interestingly, CDR2 expression was restored in Acdr7 mutants reverting spontaneously to wildtype levels of susceptibility t o azole antifungal agents. These data demonstrate that CDR2 plays an important role in mediating the resistance of C. albicans to azole antifungal agents.

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Molecular mechanisms of azole resistance in Candida albicans, including alterations in the target enzyme and increased efflux of drug, have been described, but the epidemiology of the resistance mechanisms has not been established. We have investigated the molecular mechanisms of resistance to azoles in C. albicans strains displaying high-level fluconazole resistance (MICs, >64 g/ml) isolated from human immunodeficiency virus (HIV)-infected patients with oropharyngeal candidiasis. The levels of expression of genes encoding lanosterol 14␣-demethylase (ERG11) and efflux transporters (MDR1 and CDR) implicated in azole resistance were monitored in matched sets of susceptible and resistant isolates. In addition, ERG11 genes were amplified by PCR, and their nucleotide sequences were determined in order to detect point mutations with a possible effect in the affinity for azoles. The analysis confirmed the multifactorial nature of azole resistance and the prevalence of these mechanisms of resistance in C. albicans clinical isolates exhibiting frank fluconazole resistance, with a predominance of overexpression of genes encoding efflux pumps, detected in 85% of all resistant isolates, being found. Alterations in the target enzyme, including functional amino acid substitutions and overexpression of the gene that encodes the enzyme, were detected in 65 and 35% of the isolates, respectively. Overall, multiple mechanisms of resistance were combined in 75% of the isolates displaying high-level fluconazole resistance. These results may help in the development of new strategies to overcome the problem of resistance as well as new treatments for this condition.