ERG11 mutations and expression of resistance genes in fluconazole-resistant Candida albicans isolates
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ORIGINAL PAPER
ERG11 mutations and expression of resistance genes in fluconazole‑resistant Candida albicans isolates Yonghao Xu1 · Fang Sheng2 · Jie Zhao1 · Lamei Chen3 · Chunyang Li1
Received: 9 January 2015 / Revised: 5 August 2015 / Accepted: 31 August 2015 © Springer-Verlag Berlin Heidelberg 2015
Abstract Azole resistance in the pathogenic yeast Candida albicans poses significant challenges for its antibiotic treatment. The conformational change of the target enzyme 14 alpha-demethylase (Erg11p) due to ERG11 gene mutations is one of the mechanisms resulting in the azole resistance. ERG11 of 23 isolates (8 susceptible and 15 resistant) and 6 standard strains of Candida albicans were amplified and sequenced. Nineteen missense mutations were detected. Two mutations, G487T (A114S) and T916C (Y257H), coexisted exclusively in 14 fluconazoleresistant isolates. To identify the resistance mechanisms in the isolates with G487T and T916C mutations, we compared the expression of 5 resistance-related genes in the 14 azole-resistant isolates with those in the susceptible type strain ATCC 10231, Saccharomyces cerevisiae AD/ CDR1 and AD/CDR2. The tested values of mRNA transcription of CDR1 and CDR2 were higher than that of control strain, while the semi-quantified Cdr1p values were not higher in all of the 14 resistant isolates. And the data analyzed with t test suggest that both of the differences are significant (P 64 μg ml−1 were obtained from vaginal secreta, or urine, or secreta of raw surface of nonHIV patients in Ji′nan (Nos. 592, 4263, and 4266) or in Guangzhou (No. GZ03, GZ04, GZ09, GZ15, GZ16, GZ17, GZ18, GZ23, GZ29, GZ34, GZ51, and GZ58) of China. The fluconazole-susceptible isolates were from patients in Ji′nan (Nos. C261, C307, and C522) or in Beijing (Nos. 0461, 0920, 2827, 2855, and 2928). Candida krusei ATCC 6258, Candida parapsilosis ATCC 22019, and the following type strains of C. albicans, including fluconazole-susceptible C1b, C1c, C1d, C1e, and fluconazole-resistant ATCC 7661519, were obtained from Chinese Cultural Collection Commission for Microbiology (CCCCM, Nanjing, China). C. albicans ATCC 10231 was purchased from Anhui Provincial Center for Disease Prevention and Control (Hefei, China). C. albicans Darlington strain was provided by Professor John E. Bennett of National Institutes of Health, Bethesda, Maryland. Saccharomyces cerevisiae AD/CDR1 (MATα PDR1-3 ura3 his1Δyor1::hisGΔsnq2::hisGΔpdr10::hisGΔpdr11:: hisGΔycf1::hisGΔpdr3::hisGΔpdr15::hisGΔpdr5::CaCD R1A-URA3) and AD/CDR2 (MATα PDR1-3 ura3 his1Δyor1 ::hisGΔsnq2::hisGΔpdr10::hisGΔpdr11::hisGΔycf1::hisG Δpdr3::hisGΔpdr15::hisGΔpdr5::CaCDR2A-URA3) were gifts from Professor Ann R. Holmes of Otago University, New Zealand. All isolates and standard strains were reserved on Sabouraud dextrose agar (SDA) at 4 °C.
Arch Microbiol
Agents and media SDA and yeast extract peptone dextrose (YEPD) broth were prepared according to conventional laboratory methods. The ingredients of SDA include dextrose 20 g, peptone 10 g, agar 20 g, chloromycin 50 mg, a
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