Investigation of the expression levels of efflux pumps encoding genes in fluconazole resistant Candida albicans strains by real-time polymerase chain reaction

Investigation of the expression levels of efflux pumps encoding genes in fluconazole resistant Candida albicans strains by real-time polymerase chain reaction ABSTRACT Widespread and repeated use of fluconazole in the prophylaxis and therapy resulted in resistance development in Candida albicans strains. In our study, investigation of the expression of efflux pump encoding genes CDR1, CDR2, and MDR1 in fluconazole resistant and susceptible C. albicans isolates was aimed in order to determine the role of this mechanism in fluconazole resistance. Five fluconazole resistant, six sensitive and four trailing effect showing sensitive C. albicans strains, isolated from various clinical specimens in three hospitals in Turkey were included in our study. MIC values of fluconazole itraconazole, ketoconazole, voriconazole, miconazole, clotrimazole, 5-flucytosine and amphotericin B were determined by microdilution method performed according to CLSI M27-A3 standards. The susceptibilities of fluconazole resistant strains were also studied by fluconazole E-test performed on yeast extract peptone dextrose agar with and without cyclosporin A. The expression of CDR1, CDR2 and MDR1 genes were determined by real-time polymerase chain reaction. The expression of these genes was normalized with their housekeeping gene (ACT1) levels and compared with the drug susceptible C. albicans ATCC 14053 control strain. Gene expression data of the groups were compared by Mann-Whitney U test via using SPSS version 15.0 software. Six out of nine fluconazole resistant/trailing effect showing sensitive strains were resistant to 5-flucytosine, and all except one sensitive strains had found as intermediate to 5-flucytosine. It was seen that all strains were susceptible to amphotericin B and all except one strain were also sensitive to clotrimazole. Seven out of nine fluconazole resistant?trailing isolates were determined as resistant to itraconazole and ketoconazole, and had miconazole MIC values of ?64 µg ml-1. All fluconazole sensitive isolates were found as sensitive to ketoconazole and dose dependent sensitive to itraconazole. Fluconazole resistant and sensitive strains were determined as sensitive to voriconazole. Out of five fluconazole resistant isolates, two strains overexpressed high levels and three strains overexpressed mild levels of CDR1/2; one strain overexpressed high levels and three strains overexpressed low levels of MDR1 according to C. albicans ATCC 14053 control strain. It was observed that CDR1, CDR2 and MDR1 gene expression levels were mild in trailing effect showing strains except one which highly expressed MDR1, while sensitive isolates except three expressed efflux pump genes at low levels. It was determined that the expression levels of CDR1 and CDR2 genes for the same strain were in parallel for all isolates. In our study, although it was observed that the expressions of efflux pump genes were different between fluconazole resistant, trailing and sensitive C. albicans isolates, no statistically significant difference was detected between the groups according to statical analysis (p>0.05). As a result, it can be concluded that overexpression of efflux pumps and ERG11 mutations are important mechanisms responsible for resistance in our fluconazole resistant C. albicans isolates. Key words: C. albicans, fluconazole resistance, efflux pumps