| Background:Candida species are the opportunities fungus that reside in the skin and membrana mucosa. Candida species are major human pathogens responsible for a wide spectrum of diseases ranging from superficial to life-threatening invasive infections,and.serious harm to human health. In recent years, Candida species infections is increasing with the application of broad spectrum antibiotics and corticosteroids, invasive treatment, organ transplantation, as well as in immunocompromised patients more. Currently, the clinical treatment of C. albicans to azole antifungals mainly based. Due to the impact of non-standard clinical treatment and many other factors, led to growing drug resistance of Candida clinical treatment often fails. Mortality due to systemic C. albicans infection has reached 50% [ 7 ], has become one of the clinical seriously endanger the life and health of the major diseases of patients, and therefore resistance mechanisms of C. albicans is particularly urgent.Objective:To investigate the relationship of overexpression and mutation of ERG4 with drug resistance of C. albicans using clinical isolates from patients.Methods C. albicans clinical isolates were collect from fungal laboratory of Department of Dermatology of the Second Hospital of Shanxi Medical University in December 2012 to May 2013,including 19 from respiratory secretions,genitourinary tract secretions 10( both sexually transmitted diseases),blood one,A crumbs 1,3 scaly skin.Standard strains of C.albicans ATCC11006, purchased from Peking fungi and fungal disease research center.this project will collect clinical isolates of C. Albicans strains resistant and susceptible strains of obtaining the required follow-up by the Institute of in vitro drug sensitivity test,ERG4 gene in clinical isolates of C. albicans were cloned and sequenced,bioinformatic analysis find mutations;then by the expression level of real-time quantitative PCR Erg4p;and investigate ERG4 gene mutation, overexpression C. albicans azole antifungal resistance.Results:(1) The in vitro drug susceptibility test of 34 isolates showed that all isolates were susceptible to Flucytosine. Out of 34 isolates, one isolate was resistant to Amphotericine B,18 isolates were resistant to Fluconuzole(FCA), 24 isolates were resistant to Itraconazole(ITR), 17 isolates were resistant to Virionazole(VRC) and with a cross resistance of44.12% and multi-drug resistance of 2.94%.(2) The gene expression of ERG4 in FCA-resistant isolates(3.44±4.03) were significantly higher when compared to FCA-susceptible isolates(1.48±1.51)(p < 0.05).Same trend were detected in ITR-resistant isolates and VRC-resistant isolates, that the gene expression of ERG4 were significantly higher in ITR-resistant(2.46±3.88) and VRC-resistant isolates(3.99 ± 2.72) when compared to ITR-susceptible(1.25±2.27) and VRC-susceptible isolates(1.85±1.41), respectively(p < 0.05). The gene expression of ERG4 in isolates that showed cross resistance also followed the same trend.(3) There were two mutations of ERG4 identified from the 34 isolates. The mutation C711 T is sensitive to FCA, ITR and VRC, whereas the mutation C435 T showed opposite response. When comparing both mutations, the gene expression of ERG4 is much higher in C435T(2.06±0.20) compared to C711T(0.01±0.00).Conclusion(1) Of 5-FC experimental strains were sensitive to 2.1 susceptibility test results 34 clinical isolates of C. albicans; for AMB has high sensitivity,only one(CA22 strain) their resistance; for FCA, ITR and VRC exist different levels of resistance;which one(CA22secretions from the glans) for AMB,FCA,ITR while resistance,and VRC intermediary;15for FCA, ITR, VRC while resistance;two pairs of FCA,ITR while resistance;two pairs of ITR,VRC while resistance;the existence of cross-resistance and multidrug resistance phenomenon.Standard strain ATCC11006 and nine of them on five clinical isolates were sensitive to antifungal drugs.It is thus clear that drug susceptivity test could contribute to therapy by antifungal drug established under objective basis and to choose drug reasonable.Through these, drug-resistant strains were prevented created as far as possible.(2) Clinical isolates of C. albicans resistant strains and sensitive strains are present ERG4 gene point mutations were detected in two experimental clinical isolates the presence of two point mutations,C711 T and C 435 T,but the encoded amino acid not changed,are synonymous mutations,not found azole resistant C. albicans strains exist missense mutations ERG4 gene or frameshift mutations.Therefore, in this study,C. albicans azole drug resistance could not because ERG4 mutation causes an amino acid change in the enzyme encoded by the formation, there may be other mechanisms involved in the formation of resistance.The presence of C. albicans sensitive strains CA133 synonymous mutation C711 T,the presence of drug-resistant strains CA137 synonymous mutation C 435 T, which to some extent reflects the clinical isolates of C. albicans polymorphic gene sequences ERG4 Features.(3) In the present study,C. albicans resistant to FCA,IRT and the expression of genes ERG4 VRC strain group were significantly higher than the sensitive strain group;and for FCA,IRT and VRC resistant strains at the same time the expression of the gene group was also significantly higher than ERG4 At the same time sensitive strains groups.The experimental results proved the ERG4 lead to increased gene expression of C. albicans resistant and can cause cross-resistance.(4) The strain CA22( from glans secretions) on FCA,IRT and AMB while resistance of VRC intermediary,but ERG4 gene expression levels(1.70 ± 0.14) is not too high,nor point mutations, this prompted us to AMB resistance mechanisms of C. albicans may not significantly correlated with changes in ERG4 genes or gene expression ERG4 may be unrelated to multidrug resistance phenomenon,which still need to expand the sample sizefor further study. |
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