Dataset Information

Molecular mechanisms of drug resistance in clinical Candida species isolated from Tunisian hospitals.

ABSTRACT: 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.

SUBMITTER: Eddouzi J

PROVIDER: S-EPMC3697321 | biostudies-literature | 2013 Jul

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Molecular mechanisms of drug resistance in clinical Candida species isolated from Tunisian hospitals.

Eddouzi Jamel J Parker Josie E JE Vale-Silva Luis A LA Coste Alix A Ischer Françoise F Kelly Steve S Manai Mohamed M Sanglard Dominique D

Antimicrobial agents and chemotherapy 20130429 7

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 μg/ml. Azole resistance in C. albicans JEY355 was mainly caused by overexpression of a multidrug ...[more]

PMID: 23629718

Similar Datasets

Project description:Purpose: Candida glabrata has emerged as the second or third most common non-albicans species responsible for an increasing number of systemic infections. Moreover, its high-level of resistance to azole is associated with a high mortality rate. This study aimed to evaluate nosocomial infections and resistance characteristics of C. glabrata and to explore the mechanism of azole resistance in C. glabrata. Patients and methods: Fifty-nine clinical C. glabrata isolates were collected from two hospitals in China. The susceptibility of the strains to antifungal agents was determined by both the ATB Fungus 3 strip and CLSI M27 broth microdilution method. Efflux of rhodamine 6G was examined to evaluate the effects of efflux pumps. The expression levels of CgCDR1, CgCDR2, CgSNQ2, CgERG11, and CgPDR1 were examined by real-time PCR. The sequences of CgERG11 and CgPDR1 were determined by PCR-based DNA sequencing. Results: All 59 isolates of C. glabrata were susceptible to flucytosine and amphotericin B. Twelve (20.3%) isolates were determined to be fluconazole-resistant, whereas 13 (22.0%) and 27 (45.7%) isolates were categorized as non-wild-type for itraconazole and voriconazole, respectively. Efflux pumps in azole-resistant isolates showed stronger effects than those in azole-susceptible-dose dependent isolates, which is consistent with the significant upregulation of CgCDR1 and CgCDR2 (P<0.05), whereas no obvious differences were found for CgSNQ2, CgERG11, and CgPDR1 (P>0.05). Sequencing of CgERG11 showed no alteration favoring the hypothesis that CgERG11 is not involved in the azole resistance of C. glabrata. Four CgPDR1 missense mutations were found in azole-resistant isolates, of which the high frequency of the CgPDR1 mutation, A848V, has not been reported previously. Conclusion: Efflux pump function is the main mechanism of resistance to fluconazole in our collected clinical isolates of C. glabrata, and further studies of the related gene disruption and genome-wide expression are needed to verify the function.

Project description:Resistance to azole antifungals continues to be a significant problem in the common fungal pathogen Candida albicans. Many of the molecular mechanisms of resistance have been defined with matched sets of susceptible and resistant clinical isolates from the same strain. Mechanisms that have been identified include alterations in the gene encoding the target enzyme ERG11 or overexpression of efflux pump genes including CDR1, CDR2, and MDR1. In the present study, a collection of unmatched clinical isolates of C. albicans was analyzed for the known molecular mechanisms of resistance by standard methods. The collection was assembled so that approximately half of the isolates were resistant to azole drugs. Extensive cross-resistance was observed for fluconazole, clotrimazole, itraconazole, and ketoconazole. Northern blotting analyses indicated that overexpression of CDR1 and CDR2 correlates with resistance, suggesting that the two genes may be coregulated. MDR1 overexpression was observed infrequently in some resistant isolates. Overexpression of FLU1, an efflux pump gene related to MDR1, did not correlate with resistance, nor did overexpression of ERG11. Limited analysis of the ERG11 gene sequence identified several point mutations in resistant isolates; these mutations have been described previously. Two of the most common point mutations in ERG11 associated with resistance, D116E and E266D, were tested by restriction fragment length polymorphism analysis of the isolates from this collection. The results indicated that the two mutations occur frequently in different isolates of C. albicans and are not reliably associated with resistance. These analyses emphasize the diversity of mechanisms that result in a phenotype of azole resistance. They suggest that the resistance mechanisms identified in matched sets of susceptible and resistant isolates are not sufficient to explain resistance in a collection of unmatched clinical isolates and that additional mechanisms have yet to be discovered.

Dataset Information

Molecular mechanisms of drug resistance in clinical Candida species isolated from Tunisian hospitals.

Publications

Molecular mechanisms of drug resistance in clinical Candida species isolated from Tunisian hospitals.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets