ERG11-Overexpression Contributes to azole Resistance in Candida tropicalis.

J. LOEFFLER, M. KAPP, M. KOEBERLE, K. SCHMIDT, H. EINSELE, H. HEBART1, U. SCHUMACHER

Author address: 

Medizinische Klinik, Tuebingen, Germany, Institut fuer Medizinische Mikrobiologie, Tuebingen, Germany.

Abstract: 

Background: Candida species belong to the most abundant nosocomial pathogens and especially Candida tropicalis is characterized by both a high pathogenicity and invasivity. Point mutations in the lanosteroldemethylase gene ERG11, overexpression of ERG11 and an increased expression of the efflux pump gene MDR1 are known to cause azole resistance in Candida albicans. For Candida tropicalis, data about resistance mechanisms are limited. The aim of this study was to investigate whether similar resistance mechanisms exist in C. tropicalis. Methods: azole resistance was induced by culturing C. tropicalis strains (n=4) in RPMI 1640 medium supplemented with 128 µg/ml fluconazole for 3 weeks. De-induction was performed by culturing in RPMI 1640 without fluconazole for an additional 3 weeks. Resistance was measured by the YeastOne microbouillon dilution test. Total RNA was prepared using the TRIZOL LS Reagent and 1 µg total RNA was subsequently reverse transcribed with Oligo-dT-priming. Gene expression analysis was performed for both the ERG11 and the MDR1 gene using the LightCycler instrument and specific oligonucleotide probe sets. For mutation analysis, the heme binding region of ERG11 was characterized by both melting curve analysis (LightCycler) and cycle sequencing. Results: All strains analyzed showed Minimum Inhibitory Concentrations of >256 µg/ml after 3 weeks of culturing with fluconazole. In contrast to C. albicans, no evidence of point mutations contributing to azole resistance was found. Furthermore, there was no increase in the expression of the efflux system ctMDR1. However, ERG11 expression was up-regulated about 64-fold corresponding to an increased resistance to fluconazole in the YeastOne Test. Conclusions: azole resistance in C. tropicalis might be influenced by an increased expression of genes involved in the ergosterol synthesis pathway.
2003

abstract No: 

M-395

Full conference title: 

43rd Interscience Conference on Antimicrobial Agents
    • ICAAC 43rd