Molecular Mechanisms of Fluconazole Resistance in Candida dubliniensis Isolates from Human Immunodeficiency Virus-Infected Patients with Oropharyngeal Candidiasis

Abstract

Candida dubliniensis is a newly identified species of Candida that is phenotypically similar to but genetically distinct from C. albicans. This organism has been recovered with increasing frequency from the oral cavities of human immunodeficiency virus (HIV)-infected and AIDS patients and has been implicated as a causative agent of oral candidiasis and systemic disease. In the present study we characterized the molecular mechanisms of resistance to fluconazole (FLC) in C. dubliniensis clinical isolates from two different HIV-infected patients with oropharyngeal candidiasis. Isolates were identified to the species level by phenotypic and genotypic tests. DNA-typing techniques were used to assess strain identity. Antifungal susceptibility testing was performed by NCCLS techniques. Northern blotting analysis was used to monitor the expression of genes encoding lanosterol demethylase (ERG11) and efflux transporters (CDR and MDR1) in matched sets of C. dubliniensis-susceptible and -resistant isolates by using probes generated from their homologous C. albicans sequences. 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. Decreasing susceptibilities to FLC were detected in C. dubliniensis isolates recovered from both patients during the course of treatment. FLC-resistant C. dubliniensis isolates from one patient demonstrated combined upregulation of the MDR1, CDR1, and ERG11 genes. Among the isolates from the second patient, all isolates showing decreased susceptibility to FLC demonstrated upregulation of MDR1, whereas the levels of mRNA for the ERG11 genes remained constant and the expression of CDR genes was negligible. Fourteen point mutations were found in the ERG11 genes of the isolates with decreased susceptibility to FLC. These data demonstrate that the development of azole resistance in C. dublinensis clinical isolates from HIV-infected patients treated with FLC is mediated by multiple molecular mechanisms of resistance, similar to the observations found in the case of C. albicans.

Fluconazole resistance in Candida glabrata is associated with increased bud formation and metallothionein production

“…Transcriptional upregulation of multidrug transporter genes has frequently been identified in C. glabrata (Vermitsky & Edlind, 2004), other Candida species (Katiyar & Edlind, 2001;Perea et al, 2002) and nonCandida yeasts (Posteraro et al, 2003). In this investigation, we isolated the total proteins, not just the drug-resistant proteins.…”

Azole susceptibility and resistance in Candida dubliniensis

“…Up-regulation of CdCDR1 has been observed in fluconazole-resistant clinical isolates of C. dubliniensis and in-vitrogenerated derivatives [31,38]. However, Moran et al [40] have shown that, while CdCdr1p is important for mediating reduced susceptibility to itraconazole and ketoconazole, it is not required for fluconazole resistance in isolates that exhibit increased CdMDR1 expression.…”

“…While most isolates of C. dubliniensis are susceptible to fluconazole, a number of isolates exhibiting either decreased susceptibility (8 µg/ml MIC 32 µg/ml) or resistance (MIC 64 µg/ml) to fluconazole have been described [12,20,21,[28][29][30][31][32]. These isolates were mostly recovered from HIV-infected patients receiving fluconazole therapy.…”

“…While Cdr1p and Cdr2p can transport a broad range of azole drugs, including itraconazole and ketoconazole, Mdr1p can only transport fluconazole. Homologues of the genes encoding the C. albicans multidrug transporters have been identified in C. dubliniensis (termed CdMDR1, CdCDR1 and CdCDR2 respectively), and their up-regulation has been associated with azole resistance [31]. Overexpression of CdMDR1 has been shown to be involved in mediating a reduced accumulation of drug in fluconazole-resistant clinical isolates and in-vitro-generated derivatives [38].…”

2-Amino-nonyl-6-methoxyl-tetralin muriate inhibits sterol C-14 reductase in the ergosterol biosynthetic pathway

“…Ketoconazole (KCZ) is widely used in the treatment of superficial mycoses. Nevertheless, azole resistance and cross-resistance have been documented in clinical Candida species [3,4] . Despite continuous expansion in the arsenal of antifungal drugs, antifungal drugs available cannot meet the increasing requirements for managing infections in medically complex patients.…”