The Analyze Of Drug Resistance And Study On Mechanisms Of Fluconazole Resistance In Clinical Isolates Of Candida Glabrata

Background:In recent years, following the widespread use of wide-spectrum antibiotics, immunosuppressive ,the application of organ transplantation,marrow transplantation, interventional therapy and the rise of the acquired immunodeficiency syndrome(AIDS), the incidence of fungous infections had increased year by year. C. albican is the most prevalent opportunistic pathogen of candida species in human, C. glabrata ranks the second. Being one of triazole antifungal agents, fluconazole has becoming the treatment of choice for invasive candidiasis because of its biological availability and safety. C. glabrata exhibits intrinsically low susceptibility to fluconazole, and resistant strains of this species occurs easily after prolonged therapy with fluconazoleThe major mechanisms of azoles resistance described to date have been primarily based on studies done in C. albicans and Saccharomyces cerevisiae include over expression of C14-lanosterol demethylase, alteration of the azole-binding site of C14-lanosterol demethylase, mutation in other ergosterol biosynthetic genes, and increased drug efflux. Increased drug efflux in Candida species is mainly due to increased expression of ATP-binding cassette (ABC) and major facilitator superfamily transporters. There is no report about the mechanisms of C. glabrata to azoles antifungal agents in China. The subject is to analyze the drug resistance of C. glabrata isolated from the First Affiliated Hospital of Anhui Medical University from Jul 2007 to Jul 2008 and to investigate the molecular mechanisms of fluconazole resistance in clinical isolates of C. glabrata. Objective: To analyze the drug resistance of C. glabrata isolated from clinical and to investigate the molecular mechanisms of fluconazole resistance in clinical isolates of C. glabrata.Methods: The ATB Fungus 3 reagents board was performed to determine the drug susceptibilities of antifungal agents to 190 strains of C. glabrata isolated from the First Affiliated Hospital of Anhui Medical University from Jul 2007 to Jul 2008.The broth microdilution method was performed to determine the minimal inhibitory concentration of four sorts of antifungal agents to 5 strains of fluconazole-resistace C. glabrata and 5 strains of fluconazole-susceptible C. glabrata which were selected randomly from 190 strains of C. glabrata isolates. The CgERG11 genes of 5 strains of FCA-susceptible and 5 strains of FCA-resistant isolated C. glabrata were amplified by PCR and were sequenced ; Detected the efflux of fluromchrome Rhodamine 123 between FCA-susceptible and FCA-resistant strains; Reverse transcriptase-poly- -merase chain reaction (RT-PCR) was used to measure the mRNA level of active efflux pump gene CgCDR1 and CgCDR2 .Results: 23 of the 190 isolates were resistant to fluconazole,the rate of resistance is 12.1%;The MIC results of 10 strains of C. glabrata showed that one strain of FCA- resistance C. glabrata was S-DD to itraconazole, one strain of FCA-susceptible isolate was resistance to itraconazole. The CgERG11 genes of all C. glabrata isolates were amplified and sequenced; the results showed that there is a mutation in one strain of FCA-resistant C. glabrata but no varian chang of amino acid. The results of the efflux of fluromchrome Rhodamine 123 between FCA-resistance isolates and FCA-susceptible isolates showed that there were significant difference between FCA-resistance isolates and FCA-susceptible C. glabrata isolates; and the gene expression levels of CgCDR1 and CgCDR2 were also significant difference between FCA-resistance and FCA-susceptible C. glabrata isolates by RT-PCR.Conclusions: The resistance of C. glabrata to fluconazole is severe; There is cross-resistance between fluconazole and itraconazole in C. glabrata; Fluorochrome Rhodamine 123 can use as an indicator of the efflux experiment of FCA- resistance C. glabrata isolates; The main mechanisms of FCA-resistance in clinical isolates of C. glabrata is the over-express of efflux pump genes, no found the missense mutation of the target enzyme genes.