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

Abstract

c Antifungal resistance of Candida species 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 clinical Candida species (Candida albicans JEY355 and Candida tropicalis JEY162) with decreased susceptibility to azoles and polyenes. For JEY355, the fluconazole (FLC) MIC was 8 g/ml. Azole resistance in C. albicans JEY355 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) causing MDR1 overexpression. The C. tropicalis JEY162 isolate demonstrated cross-resistance between FLC (MIC > 128 g/ml), voriconazole (MIC > 16 g/ml), and amphotericin B (MIC > 32 g/ml). Sterol analysis using gas chromatography-mass spectrometry revealed that ergosterol was undetectable in JEY162 and that it accumulated 14␣-methyl fecosterol, thus indicating a perturbation in the function of at least two main ergosterol biosynthesis proteins (Erg11 and Erg3). Sequence analyses of C. tropicalis ERG11 (CtERG11) and CtERG3 from 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 that ERG11 mutants can only survive in combination with other ERG3 mutations. CtERG3 and CtERG11 wild-type alleles were replaced by the defective genes in a wild-type C. tropicalis strain, resulting in a drug resistance phenotype identical to that of JEY162. This genetic evidence demonstrated that CtERG3 and CtERG11 mutations participated in drug resistance. During reconstitution of the drug resistance in C. tropicalis, a strain was obtained harboring only defective Cterg11 allele and containing as a major sterol the toxic metabolite 14␣-methyl-ergosta-8,24(28)-dien-3␣,6␤-diol, suggesting that ERG3 was still functional. This strain therefore challenged the current belief that ERG11 mutations cannot be viable unless accompanied by compensatory mutations. In conclusion, this study, in addition to identifying a novel MRR1 mutation in C. albicans, constitutes the first report on a clinical C. tropicalis with defective activity of sterol 14␣-demethylase and sterol ⌬ 5,6 -desaturase leading to azole-polyene cross-resistance.

Candida tropicalis Antifungal Cross-Resistance Is Related to Different Azole Target (Erg11p) Modifications

“…However, antifungals have been used extensively for the prophylaxis or treatment of candidiasis, and there are increasing reports of azole resistance for this species (13,14,27,54). In fact, the azole-resistant clinical isolates of C. tropicalis studied in this work were recovered from two patients suffering from candidemia and treated with different antifungals (Table 1).…”

For Debate: May the Use of the Polyene Macrolide Natamycin as a Food Additive Foster Emergence of Polyene-Resistance in Candida Species?

“…Other ERGgene defects also confer multi-drug-resistance [98]. Alterations in sterol-and phospholipid composition as well as changes in membrane structure account for different polyene-cross-resistance patterns [130,131] and were found in clinical isolates resistant to Azoles as well as polyenes [98,[132][133][134][135][136][137]. The situation is probably aggravated by the finding that a mutator phenotype caused by a mismatch repair defect is prevalent in C. glabrata [138].…”

“…Amino acid alteration in the target enzyme ERG11 or the replacement of Ergosterol by other sterols in the membrane encoded by ERG3 are examples. As Ergosterol being essential for the mode of action of Azoles as well as polyenes is absent in ERG3 loss-of-function mutants, these strains escape the antifungal effect of both drug classes [91][92][93][94][95][96][97][98]. Other ERGgene defects also confer multi-drug-resistance [98].…”

Mechanisms of Antifungal Drug Resistance

“…Deletion of MRR1 in clinical strains blocked MDR1 up-regulation (Morschhauser et al 2007). Fifteen different MRR1 GOF mutations have been reported (Sanglard 2011;Eddouzi et al 2013), causing constitutive up-regulation of MDR1 (Dunkel et al 2008). Genome-wide transcriptional studies using clinical strains revealed a core set of 14 genes involved in diverse functions that are up-regulated in isolates carrying MRR1 GOF mutations (Morschhauser et al 2007).…”