Implication of efflux pumps and genes in resistance of clinical isolates to fluconazole.

has been recognized as an opportunistic agent having a limited sensitivity to antifungal treatment. Molecular mechanisms of azole resistance have been rarely reported for . Similar to other fungi, we hypothesized that both gene mutation and efflux pumps genes hyper-expression were implicated. The current work aimed to study the sensitivity of clinical isolates to different antifungal agents and to explore their resistance mechanisms by molecular methods including real-time PCR and gene sequencing. The sensitivity of isolates to fluconazole, amphotericin B and voriconazole was estimated by the Etest method. Real-time PCR was used to measure the relative expression of and genes via the housekeeping gene. Three pairs of primers were also chosen to sequence the gene. This exploration was followed by statistical study including the receiver operating characteristic (ROC) curve analysis to identify a relationship between gene mean expression and the sensitivity of isolates. In 31 clinical isolates, the resistance frequencies were 87, 16.1 and 3.2 %, respectively, for amphotericin B, fluconazole and voriconazole. Quantitative real-time PCR demonstrated that only over-expression was significantly associated with FCZ resistance confirmed by univariate statistical study and the ROC curve analysis ( <0.05). The sequencing revealed two mutations H380G and S381A in TN325U11 (MIC FCZ=8 µg ml) and H437R in TN114U09 (MIC FCZ=256 µg ml) in highly conserved regions (close to the haem-binding domain) but their involvement in the resistance mechanism has not yet been assigned. FCZ resistance mechanisms are proven to be much more complex and gene alteration sequence and/or expression can be involved. Only gene over-expression was significantly associated with FCZ resistance and no good correlation was observed between FCZ and VCZ MIC values and relative gene expression. sequence alteration seems to play a major role in FCZ resistance mechanism but their involvement needs further confirmation.