Molecular Mechanisms of Drug Resistance in Clinical Candida Species Isolated from Tunisian Hospitals

Abstract

ABSTRACTAntifungal resistance ofCandidaspecies is a clinical problem in the management of diseases caused by these pathogens. In this study we identified from a collection of 423 clinical samples taken from Tunisian hospitals two clinicalCandidaspecies (Candida albicansJEY355 andCandida tropicalisJEY162) with decreased susceptibility to azoles and polyenes. For JEY355, the fluconazole (FLC) MIC was 8 μg/ml. Azole resistance inC. albicansJEY355 was mainly caused by overexpression of a multidrug efflux pump of the major facilitator superfamily, Mdr1. The regulator of Mdr1,MRR1, contained a yet-unknown gain-of-function mutation (V877F) causingMDR1overexpression. TheC. tropicalisJEY162 isolate demonstrated cross-resistance between FLC (MIC > 128 μg/ml), voriconazole (MIC > 16 μg/ml), and amphotericin B (MIC > 32 μg/ml). Sterol analysis using gas chromatography-mass spectrometry revealed that ergosterol was undetectable in JEY162 and that it accumulated 14α-methyl fecosterol, thus indicating a perturbation in the function of at least two main ergosterol biosynthesis proteins (Erg11 and Erg3). Sequence analyses ofC. tropicalis ERG11(CtERG11) andCtERG3from JEY162 revealed a deletion of 132 nucleotides and a single amino acid substitution (S258F), respectively. These two alleles were demonstrated to be nonfunctional and thus are consistent with previous studies showing thatERG11mutants can only survive in combination with otherERG3mutations.CtERG3andCtERG11wild-type alleles were replaced by the defective genes in a wild-typeC. tropicalisstrain, resulting in a drug resistance phenotype identical to that of JEY162. This genetic evidence demonstrated thatCtERG3andCtERG11mutations participated in drug resistance. During reconstitution of the drug resistance inC. tropicalis, a strain was obtained harboring only defectiveCterg11allele and containing as a major sterol the toxic metabolite 14α-methyl-ergosta-8,24(28)-dien-3α,6β-diol, suggesting thatERG3was still functional. This strain therefore challenged the current belief thatERG11mutations cannot be viable unless accompanied by compensatory mutations. In conclusion, this study, in addition to identifying a novelMRR1mutation inC. albicans, constitutes the first report on a clinicalC. tropicaliswith defective activity of sterol 14α-demethylase and sterol Δ5,6-desaturase leading to azole-polyene cross-resistance.