Mechanisms Of Fluconazole Resistance In Candida Glabtata

Objective:Because of broad-spectrum antibiotics was used in clinic, and increased number of immunocompromised patients, the infections of Fungus is increasing quickly. Candida is the popular opportunistic pathogen in clinic, Candida albicans remains is the most common conditional pathogenic fungi isolated from clinical samples, but Candida glabrata is now emerging as an important agent infections. Candida glabrata currency ranks as the second most frequency isolated Candida species form reported cases of candidasis in some area, only to Candida albicans. Because the sensitivity of Candida glabrata is low for the azoles, which were used in clinic, a growing percentage of Candida glabrata are resistant for fluconazole. So research mechanisms of drug resistance of Candida glabrata is an important problem.We gather the Candida glabrata of isolate in clinic, in order to research the drug-resistance for fluconazole, and we study the target gene ERG11 gene and effuse pump gene CDR1, CDR2 and MDR1 gene for fluconazole-resistance, to realize the molecular mechanism formative of drug-resistance, and give high attention to clinical doctors.Methods:1 Candida glabrata which 98 strains were isolated form patients of the clinic. In order to investigate the prevalence of antifungal resistance of Candida glabrata, used M44-P tablet assay and M27-4 micro dilution.2 We used the PCR technology amplificated the ERG11 geng of fluconazole-resistance and fluconazole-sensitive Candida glabrata, then we sequenced the gene of bidirectional, the sequences were compared to the published sequence, we evaluated the mutation of ERG11 gene, in order to study the relate of gene mutation and drug-resistance.3 We used semi quantitative RT-PCR technology to analysis ERG11 gene and the effuse pump gene CDR1, CDR2 and MDR1 gene, fluconazole-resistance and fluconazole-sensitive Candida glabrata, to evaluate the expression level of these genes, in order to study the relate of expression level different of these genes between fluconazole-resistance and fluconazole-sensitive.Results:1 In the 98 strains of Candida glabrata, resistant to fluconazole are 8 strains, proportion 8.16%, fluconazole S-DD (sensitive dose dependent) are 15 strains, proportion 15.31%, sensitive to fluconazole are 75 strains, proportion 76.53%. We discovered the Candida glabrata are all sensitive to amphotericin B and 5-flucytosine, but the 8 strains fluconazole-resistance are all resistant to itraconazole, and 3 strains resistant to voficonazole at one time.2 We sequenced the ERG11 gene of Candida glabrata, and compared to the published sequence, we discovered 8 points mutation, there are T866A, C1103T, T1280C, A1325G, C1640T, G1661A, T1931A and A2042G, but all the points are all the same sense mutation, which do not lead to the amino acid changes. Among the 8 points, 5 points exist in both fluconazole-resistance and fluconazole-sensitive, there are C1103T, T1280C, A1325G, C1640T and G1661A, 2 points only exist in fluconazole-resistance there are T866A and A2042G, and 1 point exists in fluconazole-sensitive, there is T1931A. But we do not know the signification of there mutation.3 When we compared to the mRNA expression level of ERG11 CDR1 CDR2 and MDR1 in Candida glabrata, we discovered fluconazole-resistance expression ERG11, CDR1 and CDR2 genes at higher level than fluconazole-sensitive, but we did not discover the difference of MDR1 gene in fluconazole-resistance and fluconazole-sensitive, so we provide that the over-expression level of ERG11, CDR1 and CDR2 genes are associate with fluconazole-resistance Candida glabrata. The function of MDR1 gene in fluconazole-resistance is advance to study. Conclusion:1 The study discovered Candida glabrata have low sensitivity and have cross-resistance to the azoles agents, but have high sensitivity to amphotericin B and 5-flucytosine.2 We did not discover ERG11 gene mutation involved in development of fluconazole resistant in Candida glabrata. And we also do not know the signification of there mutation. We must to study advance.3 We discovered the over-expression level of ERG11,CDR1 and CDR2 genes were associated with fluconazole-resistance Candida glabrata. But MDR1 gene was not had this function. We also must to study advance.