光滑假丝酵母菌耐药性分析及对氟康唑耐药机制研究
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摘要
背景
近年来,随着广谱抗生素、免疫抑制剂的广泛应用,器官移植、骨髓移植、介入性诊疗的开展以及艾滋病患者的增多,深部真菌感染的发生率逐年增加,其中以白假丝酵母菌最为常见,其次就是光滑假丝酵母菌。三唑类代表性药物氟康唑因具有良好的生物利用度和安全性,被广泛用于治疗侵袭性假丝酵母菌感染。光滑假丝酵母菌对氟康唑存在固有的低敏感率,在氟康唑的长期治疗下更易发展为氟康唑耐药株。
目前,唑类药物主要的耐药机制在白假丝酵母菌和酿酒酵母中研究的较多的包括:14DM的过表达;唑类药物与靶酶结合位点的改变;麦角固醇生物合成途径中其他基因的突变以及由于ATP-结合盒(ABC)和主要易化扩散载体超家族基因过表达而导致的假丝酵母菌属药物外排的增加。国内尚未见光滑假丝酵母菌对唑类抗真菌药物耐药机制的报道。本课题就2007年7月至2008年7月安徽医科大学第一附属医院临床分离的光滑假丝酵母菌的耐药性及对氟康唑的耐药机制进行研究。
目的了解临床分离的光滑假丝酵母菌的耐药性及其对氟康唑的耐药机制。
方法运用ATB Fungus3药敏板条检测2007年7月至2008年7月安徽医科大学第一附属医院临床分离的190株光滑假丝酵母菌对抗真菌药物的敏感性;从中随机选取氟康唑耐药和敏感各5株,采用微量肉汤稀释法进一步检测对抗真菌药物的最低抑菌浓度;PCR扩增光滑假丝酵母菌靶酶基因CgERG11并测序分析;测定荧光染料罗丹明123在敏感与耐药菌株中的外排情况;RT-PCR检测光滑假丝酵母菌外排泵基因的表达水平。
结果190株光滑假丝酵母菌中有23株对氟康唑耐药,耐药率为12.1%;10株光滑假丝酵母菌的MIC值显示,一株氟康唑耐药株对伊曲康唑剂量依赖性敏感,一株氟康唑敏感株对伊曲康唑耐药;靶酶基因的扩增测序结果显示,一株光滑假丝酵母菌氟康唑耐药株的靶酶基因CgERG11出现了一处点突变,并未引起氨基酸改变;光滑假丝酵母菌氟康唑耐药株与敏感株对荧光染料罗丹明123外排检测结果显示,耐药株和敏感株中外排存在显著的差异;RT-PCR检测氟康唑耐药株与敏感株中外排泵基因结果显示,CgCDR1与CgCDR2基因在耐药株与敏感株之间表达水平有显著性差异。
结论光滑假丝酵母菌对氟康唑的耐药形势严峻;氟康唑和伊曲康唑在光滑假丝酵母菌中存在交叉耐药现象;荧光染料罗丹明123可作为耐药光滑假丝酵母菌外排情况的指示剂;光滑假丝酵母菌对氟康唑耐药的主要机制系外排泵基因的过表达,未发现靶酶基因的有意义突变。
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 fluconazole
The 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.
近年来,随着广谱抗生素、免疫抑制剂的广泛应用,器官移植、骨髓移植、介入性诊疗的开展以及艾滋病患者的增多,深部真菌感染的发生率逐年增加,其中以白假丝酵母菌最为常见,其次就是光滑假丝酵母菌。三唑类代表性药物氟康唑因具有良好的生物利用度和安全性,被广泛用于治疗侵袭性假丝酵母菌感染。光滑假丝酵母菌对氟康唑存在固有的低敏感率,在氟康唑的长期治疗下更易发展为氟康唑耐药株。
目前,唑类药物主要的耐药机制在白假丝酵母菌和酿酒酵母中研究的较多的包括:14DM的过表达;唑类药物与靶酶结合位点的改变;麦角固醇生物合成途径中其他基因的突变以及由于ATP-结合盒(ABC)和主要易化扩散载体超家族基因过表达而导致的假丝酵母菌属药物外排的增加。国内尚未见光滑假丝酵母菌对唑类抗真菌药物耐药机制的报道。本课题就2007年7月至2008年7月安徽医科大学第一附属医院临床分离的光滑假丝酵母菌的耐药性及对氟康唑的耐药机制进行研究。
目的了解临床分离的光滑假丝酵母菌的耐药性及其对氟康唑的耐药机制。
方法运用ATB Fungus3药敏板条检测2007年7月至2008年7月安徽医科大学第一附属医院临床分离的190株光滑假丝酵母菌对抗真菌药物的敏感性;从中随机选取氟康唑耐药和敏感各5株,采用微量肉汤稀释法进一步检测对抗真菌药物的最低抑菌浓度;PCR扩增光滑假丝酵母菌靶酶基因CgERG11并测序分析;测定荧光染料罗丹明123在敏感与耐药菌株中的外排情况;RT-PCR检测光滑假丝酵母菌外排泵基因的表达水平。
结果190株光滑假丝酵母菌中有23株对氟康唑耐药,耐药率为12.1%;10株光滑假丝酵母菌的MIC值显示,一株氟康唑耐药株对伊曲康唑剂量依赖性敏感,一株氟康唑敏感株对伊曲康唑耐药;靶酶基因的扩增测序结果显示,一株光滑假丝酵母菌氟康唑耐药株的靶酶基因CgERG11出现了一处点突变,并未引起氨基酸改变;光滑假丝酵母菌氟康唑耐药株与敏感株对荧光染料罗丹明123外排检测结果显示,耐药株和敏感株中外排存在显著的差异;RT-PCR检测氟康唑耐药株与敏感株中外排泵基因结果显示,CgCDR1与CgCDR2基因在耐药株与敏感株之间表达水平有显著性差异。
结论光滑假丝酵母菌对氟康唑的耐药形势严峻;氟康唑和伊曲康唑在光滑假丝酵母菌中存在交叉耐药现象;荧光染料罗丹明123可作为耐药光滑假丝酵母菌外排情况的指示剂;光滑假丝酵母菌对氟康唑耐药的主要机制系外排泵基因的过表达,未发现靶酶基因的有意义突变。
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 fluconazole
The 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.
引文
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[1]Dominque S,Frank CO.Resistance of candida species to agents : molecular mechanisms and clinical consequences[J].Lancet Infect Dis.2002,2(2):73-85.
[2] Marr KA. Invasive infections: the changing epidemiology [J].On cology (Huntingt). 2004, 18(14):9-14.
[3] Akins R A. An update on antifungal targets and mechanisms of resistance in Candida albicans. MedMycol.2005, 43 (4): 285-318.
[4] Prasad R, Panwar SL, Smriti. Drug resistance in yeasts—an emerging scenario[J]. Adv Microb Physiol.2002, 46: 155-201.
[5] Ji HT,Zhang WN,Zhou YJ,et a1.A Three dimensional Model of Lanosterol 14alpha-demethylase of Candida albicans[J].Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao.1998,30(6):585-592.
[6] Goldman GH, da Silva FerreiraME, dos ReisMarques E, et al. Evaluation of fluconazole resistance mechanisms in Candida albicans clinical isolates from H IV infected patients in Brazil[J]. Diagn Microbiol Infect Dis. 2004, 50 (1): 25-32.
[7] Lamb D C , Kelly D E , Schunck W H , et al. The R467K amino acid substitution in Candida albican sterol 14α-demethylase causes drug resistance through reduced affinity [J]. Antimicrob Agents Chemother . 2000, 44(1): 63-67.
[8] Lee MK, Williams LE, Warnock DW, et al. Drug resistance genes and trailing growth in Cadida albican isolates [J]. J Antimicrob Chemother, 2004, 53 (2): 217-224.
[9] Patrick Vandeputte, Ge′rald Larcher, Thierry Berge,et al.Mechanisms of Azole Resistance in a Clinical Isolate of Candida tropicalis [J]. Antimicrob Agents Chemother.2005, 49(11):4608–4615.
[10] Marichal P, Vanden Bossche H, Odds FC , et al. Molecular biological characterization of an azole-resistant Candida glabrata isolate [J].Antimicrob Agents Chemother.1997, 41(10):2229-2237.
[11] Franz R, Kelly SL, Lamb DC, et al. Multiple molecular mechanisms contribute to a stepwise development of fluconazole resistance in clinical Candida albicans strains [J]. Antimicrob Agents Chemother, 1998, 42 (12): 3065-3072.
[12] Silver P M, Oliver B G, White T C.Role of Candida albicans transcription factor Upc2p in drug resistance and sterol metabolism [J]. Eukaryot Cell, 2004, 3(6): 1391-1397.
[13] Nico Dunkel, Teresa T. Liu, Katherine S. Barker et al. A Gain-of-Function Mutation in the Transcription Factor Upc2p CausesUpregulation of Ergosterol Biosynthesis Genes and Increased Fluconazole Resistance in a Clinical Candida albicans Isolate[J].EUKARYOTIC CELL.2008,7(7):1180–1190.
[14] Kelly S L , Lamb D C , Kelly D E , et al. Resistance to fluconazole and cross-resistance to amphotericinB in Candida albican from AIDS patients caused by defective sterolΔ5 , 6-desaturation [J] . FEBS Lett .1997, 400 (1): 80-82.
[15]Georgiev VS.Membrane transporters and antifungal drug resistance [J].Curr Drug Target.2000, 1(3):261-284.
[16]Taiga Miyazaki, Yoshitsugu Miyazaki, Koichi Izumikawa. Fluconazole Treatment Is Effective against a Candida albicans erg3/erg3 Mutant in Vivo Despite in Vitro Resistance [J]. Antimirobial Agents and Chemotherapy .2006, 50(2): 580–586.
[17] White TC, Holleman S, Dy F, et al. Resistance mechanisms in clinical isolates of Candida albicans [J]. Antimicrob Agents Chemother.2002, 46 (6):1704-1713.
[18] Kontoyiannis DP, lewis RE.Antifungal drug resistance of pathogenic fungi [J].Lancet 2002, 359:1135-1144.
[19] Jha S, Karnani N, Lynn AM.Covalent modification of cysteine 193 impairs ATPase function of nucleotide binding domain of a Candida drug efflux pump[J].Biochem Biophys Res Commun ,2003 ,310 (3) :869-875.
[20] Wolfger H, Mamnun YM, Kuchler K. Fungal ABC proteins:pleiotropic drug resistance ,stress response and cellular detoxification[J]. Res Microbiol.2001 ,152 (324) :375 -389.
[21] Gauthier C, Weber S, Alarco AM, et al. Functional similarities and differences between Candida albicans Cdr1p and Cdr2p transporters [J]. Antimicrob Agents Chemother.2003,47(5) :1543-1554.
[22] Holmes AR, Tsao S, Ong SW, et al. Heterozygosity and functional allelic variation in the Candida albicans efflux pump genes CDR1 and CDR2[J]. Mol Microbiol.2006,62 (1) :170-186.
[23] Holmes AR, Lin YH, Niimi K,et al. ABC transporter Cdr1p contributes more than Cdr2p does to fluconazole efflux in fluconazole-resistant Candida albicans clinical isolates[J].Antimicrob Agents Chemother. 2008 ,52(11):3851-3862.
[24] Schuetzer muehlbauer M, WilllingerB, EgnerR, et al.Reversal of antifungal resistance mediated by ABC efflux pumps from Candida albicans functionally expressed in yeast [J].Int J Antinicrob Agents.2003,22(3):291-300.
[25] Biswas, K., and J. Morschha¨user. The Mep2p ammonium permease controls nitrogen starvation-induced filamentous growth in Candida albicans[J].Mol. Microbiol. 2005. 56:649–669.
[26] Wirsching S ,Moran GP ,Sullivan D J ,et al .MDR1 mediated drug resistance in Candida dubliniensis [J]. Antimicrob Agents Chemother .2001,45(12) : 3416-3421.
[27] Pasrija R ,Banerjee D , Prasad R. Structure and function analysis of CaMdr1p , a major facilitator superfamily antifungal efflux transporter protein of Candida albicans :identification of amino acid residues critical for drug/ H + transport[J] .Eukaryot Cell ,2007 ,6 (3) :443-453.
[28] Mukherjee PK, Chandra J, Kuhn DM, et a . Mechanism of fluconazoleresistance in Candida albicans biofilms: phase specific role of efflux pumps and membrane sterols [J]. Infect Immun.2003,71(8) :4333-4340.
[29]Davina Hiller, Dominique Sanglard and Joachim Morschha¨user .Over expression of the MDR1 Gene Is Sufficient To Confer Increased Resistance to Toxic Compounds in Candida albicans Antimirobial Agents and Chemotherapy. 2006, 5(4):1365–1371.
[30] Manjistha S,Asis D.Two membrane proteins located in the Nag regulom of candida albicans confer multidrug resistance [J].Biochem Biophys Res commtm.2003,30(1):1099-1108.
[31] Sanglard D,Odds FC.Resistance of Candida species to antifungal agents: molecular mechanisms an d clinical consequences[J].kmcet Infect Dis 2002,2:73-85.
[32] Lyons CN, White TC. Transcrip tional analyses of antifungal drug resistance in Candida albicans [J].Antimicrob Agents Chemother. 2000,44: 2296-2303.
[33] Coste, A.T., Karababa, M., Ischer, F., et al.TAC1, transcriptional activator of CDR genes, is a new transcription factor involved in the regulation of Candida albicans ABC transporters CDR1 and CDR 2[J].Eukaryot Cell .2007, 3: 1639–1652.
[34] Morschh?user J, Barker K.S., Liu T.T, et al. Transcription factor Mrr1p controls expression of the MDR1 efflux pump and mediates multidrug resistance in Candida albicans[J].PLoS Pathog .2008 ,3:1603-1616.
[35] Nico Dunkel, Julia Bla?, P. David Rogers et al. Mutations in the multi-drug resistance regulator MRR1,followed by loss of heterozygosity, are the main cause of MDR1 overexpression in fluconazole-resistant Candida albicans strains[J]. Molecular Microbiology 2008, 69(4):827–840.
[36]DuglasL J.Candida biofilms and their role in infection [J].Trends in microbiobgy.2003, 11 (1):30-36.
[37] Andes D, Nett J, Oschel P, et al. Development and characterization of an In vivo central venous catheter candida albicans biofilm model [J]. Infect Immun, 2004, 72(10): 6023-6031.
[38] Vlahou A , Schellhammer PF , Mendrinos S , et al. Development of a novelproteomic approach for the detection of transitional cell carcinoma of the bladder in urine[J].Am J Pathol. 2001,158 (4):1491-1502.
[39] Sanglard D, Ischer F, Parkinson T, et al. Candida albicans mutations in the ergosterol biosynthetic pathway and resistance to several antifungal agents[J]. Antimicrob Agents Chemother.2003, 47 (8):2404-2412.
[40]Patrick Vandeputte, Guy Tronchin, Thierry Berge`s, et al. Reduced Susceptibility to Polyenes Associated with a Missense Mutation in the ERG6 Gene in a Clinical Isolate of Candida glabrata with Pseudohyphal Growth. Antimirob Agents Chemother [J] .2007, 51 (3):982–990.
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48. Maesaki S,Marichal P, Vanden Bossche H, et al. Rhodamine 6G efflux for the detection of CDR1 -overexpressing azole-resistant Candida albicans strains [J]. J Antimicrob Chemother.1999, 44(1): 27-31.
[1]Dominque S,Frank CO.Resistance of candida species to agents : molecular mechanisms and clinical consequences[J].Lancet Infect Dis.2002,2(2):73-85.
[2] Marr KA. Invasive infections: the changing epidemiology [J].On cology (Huntingt). 2004, 18(14):9-14.
[3] Akins R A. An update on antifungal targets and mechanisms of resistance in Candida albicans. MedMycol.2005, 43 (4): 285-318.
[4] Prasad R, Panwar SL, Smriti. Drug resistance in yeasts—an emerging scenario[J]. Adv Microb Physiol.2002, 46: 155-201.
[5] Ji HT,Zhang WN,Zhou YJ,et a1.A Three dimensional Model of Lanosterol 14alpha-demethylase of Candida albicans[J].Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao.1998,30(6):585-592.
[6] Goldman GH, da Silva FerreiraME, dos ReisMarques E, et al. Evaluation of fluconazole resistance mechanisms in Candida albicans clinical isolates from H IV infected patients in Brazil[J]. Diagn Microbiol Infect Dis. 2004, 50 (1): 25-32.
[7] Lamb D C , Kelly D E , Schunck W H , et al. The R467K amino acid substitution in Candida albican sterol 14α-demethylase causes drug resistance through reduced affinity [J]. Antimicrob Agents Chemother . 2000, 44(1): 63-67.
[8] Lee MK, Williams LE, Warnock DW, et al. Drug resistance genes and trailing growth in Cadida albican isolates [J]. J Antimicrob Chemother, 2004, 53 (2): 217-224.
[9] Patrick Vandeputte, Ge′rald Larcher, Thierry Berge,et al.Mechanisms of Azole Resistance in a Clinical Isolate of Candida tropicalis [J]. Antimicrob Agents Chemother.2005, 49(11):4608–4615.
[10] Marichal P, Vanden Bossche H, Odds FC , et al. Molecular biological characterization of an azole-resistant Candida glabrata isolate [J].Antimicrob Agents Chemother.1997, 41(10):2229-2237.
[11] Franz R, Kelly SL, Lamb DC, et al. Multiple molecular mechanisms contribute to a stepwise development of fluconazole resistance in clinical Candida albicans strains [J]. Antimicrob Agents Chemother, 1998, 42 (12): 3065-3072.
[12] Silver P M, Oliver B G, White T C.Role of Candida albicans transcription factor Upc2p in drug resistance and sterol metabolism [J]. Eukaryot Cell, 2004, 3(6): 1391-1397.
[13] Nico Dunkel, Teresa T. Liu, Katherine S. Barker et al. A Gain-of-Function Mutation in the Transcription Factor Upc2p CausesUpregulation of Ergosterol Biosynthesis Genes and Increased Fluconazole Resistance in a Clinical Candida albicans Isolate[J].EUKARYOTIC CELL.2008,7(7):1180–1190.
[14] Kelly S L , Lamb D C , Kelly D E , et al. Resistance to fluconazole and cross-resistance to amphotericinB in Candida albican from AIDS patients caused by defective sterolΔ5 , 6-desaturation [J] . FEBS Lett .1997, 400 (1): 80-82.
[15]Georgiev VS.Membrane transporters and antifungal drug resistance [J].Curr Drug Target.2000, 1(3):261-284.
[16]Taiga Miyazaki, Yoshitsugu Miyazaki, Koichi Izumikawa. Fluconazole Treatment Is Effective against a Candida albicans erg3/erg3 Mutant in Vivo Despite in Vitro Resistance [J]. Antimirobial Agents and Chemotherapy .2006, 50(2): 580–586.
[17] White TC, Holleman S, Dy F, et al. Resistance mechanisms in clinical isolates of Candida albicans [J]. Antimicrob Agents Chemother.2002, 46 (6):1704-1713.
[18] Kontoyiannis DP, lewis RE.Antifungal drug resistance of pathogenic fungi [J].Lancet 2002, 359:1135-1144.
[19] Jha S, Karnani N, Lynn AM.Covalent modification of cysteine 193 impairs ATPase function of nucleotide binding domain of a Candida drug efflux pump[J].Biochem Biophys Res Commun ,2003 ,310 (3) :869-875.
[20] Wolfger H, Mamnun YM, Kuchler K. Fungal ABC proteins:pleiotropic drug resistance ,stress response and cellular detoxification[J]. Res Microbiol.2001 ,152 (324) :375 -389.
[21] Gauthier C, Weber S, Alarco AM, et al. Functional similarities and differences between Candida albicans Cdr1p and Cdr2p transporters [J]. Antimicrob Agents Chemother.2003,47(5) :1543-1554.
[22] Holmes AR, Tsao S, Ong SW, et al. Heterozygosity and functional allelic variation in the Candida albicans efflux pump genes CDR1 and CDR2[J]. Mol Microbiol.2006,62 (1) :170-186.
[23] Holmes AR, Lin YH, Niimi K,et al. ABC transporter Cdr1p contributes more than Cdr2p does to fluconazole efflux in fluconazole-resistant Candida albicans clinical isolates[J].Antimicrob Agents Chemother. 2008 ,52(11):3851-3862.
[24] Schuetzer muehlbauer M, WilllingerB, EgnerR, et al.Reversal of antifungal resistance mediated by ABC efflux pumps from Candida albicans functionally expressed in yeast [J].Int J Antinicrob Agents.2003,22(3):291-300.
[25] Biswas, K., and J. Morschha¨user. The Mep2p ammonium permease controls nitrogen starvation-induced filamentous growth in Candida albicans[J].Mol. Microbiol. 2005. 56:649–669.
[26] Wirsching S ,Moran GP ,Sullivan D J ,et al .MDR1 mediated drug resistance in Candida dubliniensis [J]. Antimicrob Agents Chemother .2001,45(12) : 3416-3421.
[27] Pasrija R ,Banerjee D , Prasad R. Structure and function analysis of CaMdr1p , a major facilitator superfamily antifungal efflux transporter protein of Candida albicans :identification of amino acid residues critical for drug/ H + transport[J] .Eukaryot Cell ,2007 ,6 (3) :443-453.
[28] Mukherjee PK, Chandra J, Kuhn DM, et a . Mechanism of fluconazoleresistance in Candida albicans biofilms: phase specific role of efflux pumps and membrane sterols [J]. Infect Immun.2003,71(8) :4333-4340.
[29]Davina Hiller, Dominique Sanglard and Joachim Morschha¨user .Over expression of the MDR1 Gene Is Sufficient To Confer Increased Resistance to Toxic Compounds in Candida albicans Antimirobial Agents and Chemotherapy. 2006, 5(4):1365–1371.
[30] Manjistha S,Asis D.Two membrane proteins located in the Nag regulom of candida albicans confer multidrug resistance [J].Biochem Biophys Res commtm.2003,30(1):1099-1108.
[31] Sanglard D,Odds FC.Resistance of Candida species to antifungal agents: molecular mechanisms an d clinical consequences[J].kmcet Infect Dis 2002,2:73-85.
[32] Lyons CN, White TC. Transcrip tional analyses of antifungal drug resistance in Candida albicans [J].Antimicrob Agents Chemother. 2000,44: 2296-2303.
[33] Coste, A.T., Karababa, M., Ischer, F., et al.TAC1, transcriptional activator of CDR genes, is a new transcription factor involved in the regulation of Candida albicans ABC transporters CDR1 and CDR 2[J].Eukaryot Cell .2007, 3: 1639–1652.
[34] Morschh?user J, Barker K.S., Liu T.T, et al. Transcription factor Mrr1p controls expression of the MDR1 efflux pump and mediates multidrug resistance in Candida albicans[J].PLoS Pathog .2008 ,3:1603-1616.
[35] Nico Dunkel, Julia Bla?, P. David Rogers et al. Mutations in the multi-drug resistance regulator MRR1,followed by loss of heterozygosity, are the main cause of MDR1 overexpression in fluconazole-resistant Candida albicans strains[J]. Molecular Microbiology 2008, 69(4):827–840.
[36]DuglasL J.Candida biofilms and their role in infection [J].Trends in microbiobgy.2003, 11 (1):30-36.
[37] Andes D, Nett J, Oschel P, et al. Development and characterization of an In vivo central venous catheter candida albicans biofilm model [J]. Infect Immun, 2004, 72(10): 6023-6031.
[38] Vlahou A , Schellhammer PF , Mendrinos S , et al. Development of a novelproteomic approach for the detection of transitional cell carcinoma of the bladder in urine[J].Am J Pathol. 2001,158 (4):1491-1502.
[39] Sanglard D, Ischer F, Parkinson T, et al. Candida albicans mutations in the ergosterol biosynthetic pathway and resistance to several antifungal agents[J]. Antimicrob Agents Chemother.2003, 47 (8):2404-2412.
[40]Patrick Vandeputte, Guy Tronchin, Thierry Berge`s, et al. Reduced Susceptibility to Polyenes Associated with a Missense Mutation in the ERG6 Gene in a Clinical Isolate of Candida glabrata with Pseudohyphal Growth. Antimirob Agents Chemother [J] .2007, 51 (3):982–990.