应用滚环扩增技术对白念珠菌耐氟康唑相关基因点突变的研究
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摘要
[目的]收集白念珠菌氟康唑耐药株和敏感株,应用滚环扩增技术检测菌株耐药相关基因ERG11和TAC1的点突变,并将所得结果与测序结果进行比较,以期建立一种准确、快速、特异的检测基因单点突变的分子生物学方法,同时进一步了解ERG11和TAC1突变与唑类药物耐药之间的关系。
[方法]从不同临床标本中收集白念珠菌并进行药物敏感性测定,得到白念珠菌氟康唑耐药株25株和敏感株21株；8株美国耐药株已知其ERG11突变位点。根据文献报道的与耐药有关的点突变和挂锁探针设计原则设计挂锁探针,其中ERG11设计24个挂锁探针,TAC1设计4个挂锁探针,每个探针可检测一种点突变。提取DNA、PCR扩增获得目的片段ERG11和TAC1的三个片段,纯化去除多余的缓冲液、引物和dNTP；然后通过挂锁探针的连接、核酸外切酶消化、超分支滚环扩增等过程,用滚环扩增技术分别检测耐药株和敏感株中ERG11的点突变和耐药株中TAC1的点突变。同时将目的片段纯化后送交测序,将滚环扩增所得结果与测序结果进行比较。
[结果]在8株己知突变位点的氟康唑耐药株中,滚环扩增技术准确地检测出与已知突变一致的点突变；应用滚环扩增技术最低可在含有5%目标模板的混合物中检测到滚环扩增信号。对全部临床菌株,应用滚环扩增技术检测到的点突变经测序验证全部正确,滚环扩增技术表现出和DNA测序很好的一致性。对于ERG11,25个氟康唑耐药株的24个中检测出了错义突变导致的20种氨基酸转换(突变率96%),分别是E266D(n=11),V488I(n=8),D116E(n=8),K128T(n=7),G464S(n=4),K143R(n=3),G448E(n=3),G307S(n=3),F145L(n=3),V437I(n=3),F449S(n=2),K108E(n=2),D153E(n=2),G465S(n=1),R467K(n=1),S405F(n=1),Y132H(n=1),F126L(n=1),D278E(n=1),G450V(n=1)。其中G450V以前未见报道,一株菌未检测到任何突变；23个氟康唑敏感株的18个中检测出5种氨基酸置换(突变率78%),分别是E266D(n=15),D116E(n=11),V488I(n=7),K128T(n=3),V437I(n=2)。两种菌株中共有的突变位点是D116E,E266D,K128T,V437I和V488I。对于TAC1,33株耐氟康唑白念珠菌(包括8株美耐药株)中,有5株菌株出现突变,分别为T225A(n=1)和A736V(n=4),其国中4株菌来自美国。
[结论]应用滚环扩增技术检测耐氟康唑白念珠菌耐药基因的点突变,挂锁探针准确地检测出受试菌株中ERG11基因和TAC1基因的突变,其中一些位置较接近的突变,滚环扩增技术均获得了准确的结果,表明滚环扩增技术检测基因单点突变具有良好的特异性和敏感性,是一种准确、快速的检测基因单点突变的分子生物学方法。ERG11和TAC1突变的数目或分布模式与唑类药物的MIC之间未见明显的关联,但是可能随着地理区域的不同而不同。ERG11点突变和耐药的发生密切相关,TAC1点突变与耐药的关系有待于进一步研究。白念珠菌对唑类药物耐药是多种分子机制同时作用的结果,因此今后应进一步探索和完善耐药产生的分子机制,为临床提供理论依据。
[Objective] Clinical Candida albicans strains resistant and susceptible to fluconazole were collected, and were detected point mutation of ERG11 and TAC1 gene by rolling circle amplification (RCA). RCA results were compared with sequencing results, to develop an accurate, rapid and specific assay to detect point mutation; at the same time to better understand the relationship between mutation of ERG 11 or TAC1 and resistance to azoles.
[Methods] C. albicans were collected from Clinical specimens and were determinated drug susceptibility to fluconazole, and 25 fluconazole-resistant strains and 21 fluconazole-susceptible strains were obtained; there were 10 known ERG11 mutations in eight fluconazole-resistant strains from USA. Extracted DNA, and acquired target ERG 11 gene and three fragments of TAC1 gene by PCR amplification, then purified to remove excess buffer, primers and dNTP. After the processes of connection through the padlock probe, exonuclease digestion and hyperbranched rolling circle amplification, RCA assay was used to detect point mutation in ERG 11 of the resistant and susceptible strains and in TAC1 of the resistant strains. At the same time target fragments were sequenced after purification, and the results of RCA were compared with sequencing resuluts.
[Results] In eight fluconazole-resistant strains with known mutations, RCA assay correctly detected mutations which were consistent with known point mutations; with application of RCA assay, the RCA signal were detect in a mixture containing a minimum of 5% target template. Of all clinical strains, RCA assay showed excellent concordance with DNA sequencing. For ERG11 gene,24 of 25 fluconazole-resistant strains were detected missense mutations resulting in 20 kinds of amino acids substitutions (prevalence 96%), they were E266D (n=11), V488I (n=8), D116E (n=8), K128T (n=7), G464S(n=4), K143R(n=3), G448E(n=3), G307S(n=3), F145L(n=3), V437I(n=3), F449S(n=2), K108E(n=2), D153E(n=2), G465S(n=1), R467K(n=1), S405F(n=1), Y132H(n=1), F126L(n=1), D278E(n=1), G450V(n=1). Among them, G450V was a new mutation which was not reported before. One strain was not detected any mutation; 18 of 23 fluconazole-susceptible strains were detected 5 kinds of amino acid substitutions (prevalence 78%), they were E266D (n=15), D116E (n=11), V488I (n=7), K128T (n=3), V437I (n=2). Mutations both emerged in resistant and susceptible strains were D116E, E266D, K128T, V437I and V488I. For TAC1 gene, among 33 fluconazole-resistant C. albicans strains (including 8 resistant strains from USA), there were 5 strains containing two mutations, namely T225A (n=1) and A736V (n=4), of which 4 strains from the United States.
[Conclusion] Using RCA assay to detect point mutation in fluconazole-resistant C. albicans, padlock probe correctly detected mutations of ERG11 gene and TAC1 gene in the test strains; accurate results also were obtained by RCA assay with some mutations which were closer in the location. It demonstrated that using RCA assay to detect point mutation has good specificity and sensitivity, RCA is a good assay which can detect point mutation accurately and rapidly. The number of mutations or distribution patterns of ERG11 and TAC1 mutations have no obvious correlation with the MICs to azoles, but may vary with the geographic regions. There were close relation between mutations of ERG11 and resistance to azoles. Further investigation should be carried out to better understanding the relationship between mutations of TAC1 gene and resistance to fluconazole. Multiple molecular mechanisms lead to the resistance to azoles in C. albicans, so in the future further explore should be done to understand molecular mechanisms of resistance to azoles.
[方法]从不同临床标本中收集白念珠菌并进行药物敏感性测定,得到白念珠菌氟康唑耐药株25株和敏感株21株；8株美国耐药株已知其ERG11突变位点。根据文献报道的与耐药有关的点突变和挂锁探针设计原则设计挂锁探针,其中ERG11设计24个挂锁探针,TAC1设计4个挂锁探针,每个探针可检测一种点突变。提取DNA、PCR扩增获得目的片段ERG11和TAC1的三个片段,纯化去除多余的缓冲液、引物和dNTP；然后通过挂锁探针的连接、核酸外切酶消化、超分支滚环扩增等过程,用滚环扩增技术分别检测耐药株和敏感株中ERG11的点突变和耐药株中TAC1的点突变。同时将目的片段纯化后送交测序,将滚环扩增所得结果与测序结果进行比较。
[结果]在8株己知突变位点的氟康唑耐药株中,滚环扩增技术准确地检测出与已知突变一致的点突变；应用滚环扩增技术最低可在含有5%目标模板的混合物中检测到滚环扩增信号。对全部临床菌株,应用滚环扩增技术检测到的点突变经测序验证全部正确,滚环扩增技术表现出和DNA测序很好的一致性。对于ERG11,25个氟康唑耐药株的24个中检测出了错义突变导致的20种氨基酸转换(突变率96%),分别是E266D(n=11),V488I(n=8),D116E(n=8),K128T(n=7),G464S(n=4),K143R(n=3),G448E(n=3),G307S(n=3),F145L(n=3),V437I(n=3),F449S(n=2),K108E(n=2),D153E(n=2),G465S(n=1),R467K(n=1),S405F(n=1),Y132H(n=1),F126L(n=1),D278E(n=1),G450V(n=1)。其中G450V以前未见报道,一株菌未检测到任何突变；23个氟康唑敏感株的18个中检测出5种氨基酸置换(突变率78%),分别是E266D(n=15),D116E(n=11),V488I(n=7),K128T(n=3),V437I(n=2)。两种菌株中共有的突变位点是D116E,E266D,K128T,V437I和V488I。对于TAC1,33株耐氟康唑白念珠菌(包括8株美耐药株)中,有5株菌株出现突变,分别为T225A(n=1)和A736V(n=4),其国中4株菌来自美国。
[结论]应用滚环扩增技术检测耐氟康唑白念珠菌耐药基因的点突变,挂锁探针准确地检测出受试菌株中ERG11基因和TAC1基因的突变,其中一些位置较接近的突变,滚环扩增技术均获得了准确的结果,表明滚环扩增技术检测基因单点突变具有良好的特异性和敏感性,是一种准确、快速的检测基因单点突变的分子生物学方法。ERG11和TAC1突变的数目或分布模式与唑类药物的MIC之间未见明显的关联,但是可能随着地理区域的不同而不同。ERG11点突变和耐药的发生密切相关,TAC1点突变与耐药的关系有待于进一步研究。白念珠菌对唑类药物耐药是多种分子机制同时作用的结果,因此今后应进一步探索和完善耐药产生的分子机制,为临床提供理论依据。
[Objective] Clinical Candida albicans strains resistant and susceptible to fluconazole were collected, and were detected point mutation of ERG11 and TAC1 gene by rolling circle amplification (RCA). RCA results were compared with sequencing results, to develop an accurate, rapid and specific assay to detect point mutation; at the same time to better understand the relationship between mutation of ERG 11 or TAC1 and resistance to azoles.
[Methods] C. albicans were collected from Clinical specimens and were determinated drug susceptibility to fluconazole, and 25 fluconazole-resistant strains and 21 fluconazole-susceptible strains were obtained; there were 10 known ERG11 mutations in eight fluconazole-resistant strains from USA. Extracted DNA, and acquired target ERG 11 gene and three fragments of TAC1 gene by PCR amplification, then purified to remove excess buffer, primers and dNTP. After the processes of connection through the padlock probe, exonuclease digestion and hyperbranched rolling circle amplification, RCA assay was used to detect point mutation in ERG 11 of the resistant and susceptible strains and in TAC1 of the resistant strains. At the same time target fragments were sequenced after purification, and the results of RCA were compared with sequencing resuluts.
[Results] In eight fluconazole-resistant strains with known mutations, RCA assay correctly detected mutations which were consistent with known point mutations; with application of RCA assay, the RCA signal were detect in a mixture containing a minimum of 5% target template. Of all clinical strains, RCA assay showed excellent concordance with DNA sequencing. For ERG11 gene,24 of 25 fluconazole-resistant strains were detected missense mutations resulting in 20 kinds of amino acids substitutions (prevalence 96%), they were E266D (n=11), V488I (n=8), D116E (n=8), K128T (n=7), G464S(n=4), K143R(n=3), G448E(n=3), G307S(n=3), F145L(n=3), V437I(n=3), F449S(n=2), K108E(n=2), D153E(n=2), G465S(n=1), R467K(n=1), S405F(n=1), Y132H(n=1), F126L(n=1), D278E(n=1), G450V(n=1). Among them, G450V was a new mutation which was not reported before. One strain was not detected any mutation; 18 of 23 fluconazole-susceptible strains were detected 5 kinds of amino acid substitutions (prevalence 78%), they were E266D (n=15), D116E (n=11), V488I (n=7), K128T (n=3), V437I (n=2). Mutations both emerged in resistant and susceptible strains were D116E, E266D, K128T, V437I and V488I. For TAC1 gene, among 33 fluconazole-resistant C. albicans strains (including 8 resistant strains from USA), there were 5 strains containing two mutations, namely T225A (n=1) and A736V (n=4), of which 4 strains from the United States.
[Conclusion] Using RCA assay to detect point mutation in fluconazole-resistant C. albicans, padlock probe correctly detected mutations of ERG11 gene and TAC1 gene in the test strains; accurate results also were obtained by RCA assay with some mutations which were closer in the location. It demonstrated that using RCA assay to detect point mutation has good specificity and sensitivity, RCA is a good assay which can detect point mutation accurately and rapidly. The number of mutations or distribution patterns of ERG11 and TAC1 mutations have no obvious correlation with the MICs to azoles, but may vary with the geographic regions. There were close relation between mutations of ERG11 and resistance to azoles. Further investigation should be carried out to better understanding the relationship between mutations of TAC1 gene and resistance to fluconazole. Multiple molecular mechanisms lead to the resistance to azoles in C. albicans, so in the future further explore should be done to understand molecular mechanisms of resistance to azoles.
引文
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49. Rustad TR, Stevens DA, Pfaller MA, et al. Homozygosity at the Candida albicans MTL locus associated with azole resistance [J]. Microbiology,2002,148: 1061-1072.
50. Coste A, Karababa M, Ischer F, et al. TAC1, transcriptional activator of CDR genes, is a new transcription gactor involved in the regulation of Candida albicans ABC rransporters CDR1 and CDR2 [J]. Eukaryot Cell,2004,3(6): 1639-1652.
51. De Micheli M, Bille J, Schueller C, et al. A common drug-responsive element mediates the upregulation of the Candida albicans ABC transporters CDR1 and CDR2, two genes involved in antifungal drug resistance[J]. Mol. Microbiol,2002, 43(5):1197-1214.
52. Morschhauser J, Barker KS, Liu TT, et al. The Transcription Factor Mrrlp Controls Expression of the MDR1 Efflux Pump and Mediates Multidrug Resistance in Candida albicans[J]. PLoS Pathog,2007,3(11):1603-1616.
53. Dunkel N, Liu TT, Barker KS, et al. A Gain-of-Function Mutation in the Transcription Factor Upc2p Causes Upregulation of Ergosterol Biosynthesis Genes and Increased Fluconazole Resistance in a Clinical Candida albicans Isolate[J]. Eukaryot Cell,2008,7(7):1180-1190.
54. Heilmann CJ, Schneider S, Barker KS, et al. An A643T mutation in the transcription factor Upc2p causes constitutive ERG 11 upregulation and increased fluconazole resistance in Candida albicans[J]. Antimicrob Agents Chemother, 2010,54(1):353-359.
1. Paulitsch A, Weger W, Ginter-Hanselmayer G, et al. A 5-year (2000-2004) epidemiological survey of Candida and non-Candida yeast species causing vulvovaginal candidiasis in Graz, Austria[J]. Mycoses,2006,49(6):471-475.
2. Lamb DC, Kelly DE, Schunck WH, et al. The mutation T315A in Candida albicans sterol 14alpha-demethylase causes reduced enzyme activity and fluconazole resistance through reduced affinity[J]. The Journal of biological chemistry,1997,272(9):5682-5688.
3. White TC. The presence of an R467K amino acid substitution and loss of allelic variation correlate with an azole-resistant lanosterol 14alpha demethylase in Candida albicans[J]. Antimicro Agents Chemother,1997,41(7):1488-1494.
4. Chau AS, Mendrick CA, Sabatelli FJ, et al. Application of real-time quantitative PCR to molecular analysis of Candida albicans strains exhibiting reduced susceptibility to azoles[J]. Antimicro Agents Chemother,2004,48(6):2124-2131.
5. Perea S, Lopez-Ribot JL, Kirkpatrick WR, et al. Prevalence of molecular mechanisms of resistance to azole antifungal agents in Candida albicans strains displaying high-level fluconazole resistance isolated from human immunodeficiency virus-infected patients[J]. Antimicro Agents Chemother,2001, 45(10):2676-2684.
6. White TC, Holleman S, Dy F, et al. Resistance mechanisms in clinical isolates of Candida albicans[J]. Antimicro Agents Chemother,2002,46(6):1704-1713.
7. Cernicka J, Subik J. Resistance mechanisms in fluconazole-resistant Candida albicans isolates from vaginal candidiasis[J]. Int J Antimicrob Agents,2006, 27(5):403-408.
8. Xu Y, Chen L, Li C. Susceptibility of clinical isolates of Candida species to fluconazole and detection of Candida albicans ERG11 mutations[J]. The Journal of antimicrobial chemotherapy,2008,61(4):798-804.
9. Kelly SL, Lamb DC, Corran AJ, et al. Mode of action and resistance to azole antifungals associated with the formation of 14 alpha-methylergosta-8,24(28)-dien-3 beta,6 alpha-diol[J]. Biochem Biophys Res Commun,1995,207:910-915.
10. Sanglard D, Ischer F, Parkinson T, et al. Candida albicans mutations in the ergosterol biosynthetic pathway andresistance to several antifungal agents[J]. Antimicrob Agents Chemother,2003;47:2404-2412.
11.郭慧君,夏忠弟.白色念珠菌耐药蛋白(Cdrlp)表达及调控机制研究进展[J].国外医药抗生素分册,2005,26(6)：244-247.
12. Marr KA, Lyons CN, Ha K, et al. Inducible azole resistance associated with a heterogeneous phenotype in Candida albicans[J]. Antimicrob Agents Chemother, 2001,45(1):52-59.
13. Sanglard D, Ischer F, Monod M, et al. Cloning of Candida albicans genes conferring resistance to azole antifungal agents:characterization of CDR2, a new multidrug ABC transporter gene[J]. Microbiology,1997,143(Pt 2):405-416.
14. Wirsching S, Michel S, Morsch hauser J. Targeted gene disruption in Candida albicans wild-type strains:the role of the MDR1 gene in fluconazole resistance of clinical Candida albicans isolates[J]. Mol Microbiol,2000,36:856-865.
15. Sanglard D, Ischer F, Koymans L, et al. Amino acid substitutions the cytochrome P450 Lanosterol 14ademethylase(CYP51A) from azole resistance Candida albicans clinical isolates contribute to resisitance to azole antifungal agents[J]. Antimicro Agents Chemother,1998,42(2):241-253.
16. Calabrese D, Bille J, Sanglard DA. A novel multidrug efflux transporter gene of the major facilitator superfamily from Candida albicans (FLU1) conferring resistance to fluconazole[J]. Microbiology,2000,146:2743-2754.
17. Alix Coste, Mahir Karababa, Francoise Ischer, et al. TAC1, Transcriptional Activator of CDR Genes, Is a New Transcription Factor Involved in the Regulation of Candida albicans ABC Transporters CDR1 and CDR2[J]. Eukaryotic Cell,2004:1639-1652.
18. Alix Coste, Vincent Turner, Francxoise Ischer, et al. A Mutation in Taclp, a Transcription Factor Regulating CDR1 and CDR2, Is Coupled With Loss of Heterozygosity at Chromosome 5 to Mediate Antifungal Resistance in Candida albicans[J]. Genetics,2006,172:2139-2156.
19. Morschhauser J, Barker KS, Liu TT, et al. The Transcription Factor Mrrlp Controls Expression of the MDR1 Efflux Pump and Mediates Multidrug Resistance in Candida albicans[J]. PLoS Pathog,2007,3(11):1603-1616.
20. Dunkel N, Liu TT, Barker KS, et al. A Gain-of-Function Mutation in the Transcription Factor Upc2p Causes Upregulation of Ergosterol Biosynthesis Genes and Increased Fluconazole Resistance in a Clinical Candida albicans Isolate[J]. Eukaryot Cell,2008,7(7):1180-1190.
21.Donlan RM, Costerton JW. Biofilms:survival mechanisms of clinically relevant microorganisms[J]. Clin Micro boil Rev,2002,15:167-193.
22. Chandra J, Kuhn D M. Mukherjee P K, et al. Biofilm formation by the fungal pathogen Candida albi-ans:developm ent, architecture, and drug resistance[J]. J Bacteriol,2001,183(18):5385 5394.
23. Baillie GS, Douglas LJ. Matrix polymers of Candida biofilms and their possible role in biofilm resistance to antifungal agents[J]. J Antimicrob Chemother,2000, 46:397-403.
24. Mukherjee PK, Chandra J, Kuhn DM, et al. Mechanism of fluconazole resistance in Candida albicans biofilms:phase specific role of efflux pumps and membranesterols[J]. Infect Immun,2003,71:4333-4340.
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15. Sanglard D, Ischer F, Koymans L, et al. Amino acid substitutions in the cytochrome P-450 lanosterol 14alpha-demethylase (CYP51A1) from azole-resistant Candida albicans clinical isolates contribute to resistance to azole antifungal agents[J]. Antimicrob Agents Chemother,1998,42:241-253.
16. White TC:The presence of an R467K amino acid substitution and loss of allelic variation correlate with an azole-resistant lanosterol 14alpha demethylase in Candida albicans[J]. Antimicrob Agents Chemother,1997,41:1488-1494.
17. Favre B, Didmon M, Ryder NS. Multiple amino acid substitutions in lanosterol 14alpha-demethylase contribute to azole resistance in Candida albicans[J]. Microbiology,1999,145:2715-2725.
18. Marichal P, Koymans L, Willemsens S, et al. Contribution of mutations in the cytochrome P450 14alpha-demethylase (Erg11p, Cyp51p) to azole resistance in Candida albicans[J]. Microbiology,1999,145:2701-2713.
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21. Coste A, Turner V, Ischer F, et al. A mutation in Taclp, a transcription factor regulating CDR1 and CDR2, is coupled with loss of heterozygosity at chromosome 5 to mediate antifungal resistance in Candida albicans[J]. Genetics, 2006,172(4):2139-2156.
22. Coste A, Selmecki A, Forche A, et al. Genotypic evolution of azole resistance mechanisms in sequential Candida albicans isolates[J]. Eukaryot Cell,2007, 6(10):1889-1904.
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26. Paulitsch A, Weger W, Ginter-Hanselmayer G, et al. A 5-year (2000-2004) epidemiological survey of Candida and non-Candida yeast species causing vulvovaginal candidiasis in Graz, Austria[J]. Mycoses,2006,49(6):471-475.
27. Lamb DC, Kelly DE, Schunck WH, et al. The mutation T315A in Candida albicans sterol 14alpha-demethylase causes reduced enzyme activity and fluconazole resistance through reduced affinity[J]. J Biol chem,1997,272(9): 5682-5688.
28. White TC, Holleman S, Dy F, et al. Resistance mechanisms in clinical isolates of Candida albicans[J]. Antimicro Agents Chemother,2002,46(6):1704-1713.
29. Kelly SL, Lamb DC, Corran AJ, et al. Mode of action and resistance to azole antifungals associated with the formation of 14 alpha-methylergosta-8,24(28)-dien-3 beta,6 alpha-diol[J]. Biochem Biophys Res Commun,1995,207:910-915.
30. Sanglard D, Ischer F, Parkinson T, et al. Candida albicans mutations in the ergosterol biosynthetic pathway andresistance to several antifungal agents[J]. Antimicrob Agents Chemother,2003;47:2404-2412.
31.郭慧君,夏忠弟.白色念珠菌耐药蛋白(Cdr1p)表达及调控机制研究进展[J].国外医药抗生素分册,2005,26(6)：244-247.
32. Marr KA, Lyons CN, Ha K, et al. Inducible azole resistance associated with a heterogeneous phenotype in Candida albicans[J]. Antimicrob Agents Chemother, 2001,45(1):52-59.
33. Sanglard D, Ischer F, Monod M, et al. Cloning of Candida albicans genes conferring resistance to azole antifungal agents:characterization of CDR2, a new multidrug ABC transporter gene[J]. Microbiology,1997,143(Pt 2):405-416.
34. Wirsching S, Michel S, Morschhauser J. Targeted gene disruption in Candida albicans wild-type strains:the role of the MDR1 gene in fluconazole resistance of clinical Candida albicans isolates[J]. Mol Microbiol,2000,36(4):856-865.
35. Calabrese D, Bille J, Sanglard DA. A novel multidrug efflux transporter gene of the major facilitator superfamily from Candida albicans (FLU1) conferring resistance to fluconazole[J]. Microbiology,2000,146:2743-2754.
36. Donlan RM, Costerton JW. Biofilms:survival mechanisms of clinically relevant microorganisms[J]. Clin Micro boil Rev,2002,15:167-193.
37. Chandra J, Kuhn D M. Mukherjee PK, et al. Biofilm formation by the fungal pathogen Candida albicans:development, architecture, and drug resistance[J]. J Bacteriol,2001,183(18):5385-5394.
38. Baillie GS, Douglas LJ. Matrix polymers of Candida biofilms and their possible role in biofilm resistance to antifungal agents[J]. J Antimicrob Chemother, 2000,46:397-403.
39. Mukherjee PK, Chandra J, Kuhn DM, et al. Mechanism of fluconazole resistance in Candida albicans biofilms:phase specific role of efflux pumps and membranesterols[J]. Infect Immun,2003,71:4333-4340.
40. Cowen LE, Sanglard D, Calabrese D, et al. Evolution of drug resistance in experimental populations of Candida albicans[J]. J Bacteriol,2000,182(6): 1515-1522.
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43. Xu Y, Chen L, Li C. Susceptibility of clinical isolates of Candida species to fluconazole and detection of Candida albicans ERG11 mutations[J]. Antimicrob Chemother,2008;61(4):798-804.
44. Cernicka J, Subik J. Resistance mechanisms in fluconazole-resistant Candida albicans isolates from vaginal candidiasis[J]. Int J Antimicrob Agents, 2006;27(5):403-408.
45. Marichal P, Koymans L, Willemsens S, et al. Contribution of mutations in the cytochrome P450 14alpha-demethylase (Ergllp, Cyp51p) to azole resistance in Candida albicans[J]. Microbiology,1999,145:2701-2713.
46. Xiao L, Madison V, Chau AS, et al. Three-dimensional models of wild-type and mutated forms of cytochrome P450 14alpha-sterol demethylases from Aspergillus fumigatus and Candida albicans provide insights into posaconazole binding[J]. Antimicrob Agents Chemother,2004,48:568-574.
47. Lee MK, Williams LE, Warnock DW, et al. Drug resistance genes and trailing growth in Candida albicans isolates[J]. J Antimicrob Chemother,2004,53:217-224.
48.乔建军,刘伟,李若瑜.白念珠菌耐药的分子机制研究进展[J].微生物学通报,2007,34(2)：393-396.
49. Rustad TR, Stevens DA, Pfaller MA, et al. Homozygosity at the Candida albicans MTL locus associated with azole resistance [J]. Microbiology,2002,148: 1061-1072.
50. Coste A, Karababa M, Ischer F, et al. TAC1, transcriptional activator of CDR genes, is a new transcription gactor involved in the regulation of Candida albicans ABC rransporters CDR1 and CDR2 [J]. Eukaryot Cell,2004,3(6): 1639-1652.
51. De Micheli M, Bille J, Schueller C, et al. A common drug-responsive element mediates the upregulation of the Candida albicans ABC transporters CDR1 and CDR2, two genes involved in antifungal drug resistance[J]. Mol. Microbiol,2002, 43(5):1197-1214.
52. Morschhauser J, Barker KS, Liu TT, et al. The Transcription Factor Mrrlp Controls Expression of the MDR1 Efflux Pump and Mediates Multidrug Resistance in Candida albicans[J]. PLoS Pathog,2007,3(11):1603-1616.
53. Dunkel N, Liu TT, Barker KS, et al. A Gain-of-Function Mutation in the Transcription Factor Upc2p Causes Upregulation of Ergosterol Biosynthesis Genes and Increased Fluconazole Resistance in a Clinical Candida albicans Isolate[J]. Eukaryot Cell,2008,7(7):1180-1190.
54. Heilmann CJ, Schneider S, Barker KS, et al. An A643T mutation in the transcription factor Upc2p causes constitutive ERG 11 upregulation and increased fluconazole resistance in Candida albicans[J]. Antimicrob Agents Chemother, 2010,54(1):353-359.
1. Paulitsch A, Weger W, Ginter-Hanselmayer G, et al. A 5-year (2000-2004) epidemiological survey of Candida and non-Candida yeast species causing vulvovaginal candidiasis in Graz, Austria[J]. Mycoses,2006,49(6):471-475.
2. Lamb DC, Kelly DE, Schunck WH, et al. The mutation T315A in Candida albicans sterol 14alpha-demethylase causes reduced enzyme activity and fluconazole resistance through reduced affinity[J]. The Journal of biological chemistry,1997,272(9):5682-5688.
3. White TC. The presence of an R467K amino acid substitution and loss of allelic variation correlate with an azole-resistant lanosterol 14alpha demethylase in Candida albicans[J]. Antimicro Agents Chemother,1997,41(7):1488-1494.
4. Chau AS, Mendrick CA, Sabatelli FJ, et al. Application of real-time quantitative PCR to molecular analysis of Candida albicans strains exhibiting reduced susceptibility to azoles[J]. Antimicro Agents Chemother,2004,48(6):2124-2131.
5. Perea S, Lopez-Ribot JL, Kirkpatrick WR, et al. Prevalence of molecular mechanisms of resistance to azole antifungal agents in Candida albicans strains displaying high-level fluconazole resistance isolated from human immunodeficiency virus-infected patients[J]. Antimicro Agents Chemother,2001, 45(10):2676-2684.
6. White TC, Holleman S, Dy F, et al. Resistance mechanisms in clinical isolates of Candida albicans[J]. Antimicro Agents Chemother,2002,46(6):1704-1713.
7. Cernicka J, Subik J. Resistance mechanisms in fluconazole-resistant Candida albicans isolates from vaginal candidiasis[J]. Int J Antimicrob Agents,2006, 27(5):403-408.
8. Xu Y, Chen L, Li C. Susceptibility of clinical isolates of Candida species to fluconazole and detection of Candida albicans ERG11 mutations[J]. The Journal of antimicrobial chemotherapy,2008,61(4):798-804.
9. Kelly SL, Lamb DC, Corran AJ, et al. Mode of action and resistance to azole antifungals associated with the formation of 14 alpha-methylergosta-8,24(28)-dien-3 beta,6 alpha-diol[J]. Biochem Biophys Res Commun,1995,207:910-915.
10. Sanglard D, Ischer F, Parkinson T, et al. Candida albicans mutations in the ergosterol biosynthetic pathway andresistance to several antifungal agents[J]. Antimicrob Agents Chemother,2003;47:2404-2412.
11.郭慧君,夏忠弟.白色念珠菌耐药蛋白(Cdrlp)表达及调控机制研究进展[J].国外医药抗生素分册,2005,26(6)：244-247.
12. Marr KA, Lyons CN, Ha K, et al. Inducible azole resistance associated with a heterogeneous phenotype in Candida albicans[J]. Antimicrob Agents Chemother, 2001,45(1):52-59.
13. Sanglard D, Ischer F, Monod M, et al. Cloning of Candida albicans genes conferring resistance to azole antifungal agents:characterization of CDR2, a new multidrug ABC transporter gene[J]. Microbiology,1997,143(Pt 2):405-416.
14. Wirsching S, Michel S, Morsch hauser J. Targeted gene disruption in Candida albicans wild-type strains:the role of the MDR1 gene in fluconazole resistance of clinical Candida albicans isolates[J]. Mol Microbiol,2000,36:856-865.
15. Sanglard D, Ischer F, Koymans L, et al. Amino acid substitutions the cytochrome P450 Lanosterol 14ademethylase(CYP51A) from azole resistance Candida albicans clinical isolates contribute to resisitance to azole antifungal agents[J]. Antimicro Agents Chemother,1998,42(2):241-253.
16. Calabrese D, Bille J, Sanglard DA. A novel multidrug efflux transporter gene of the major facilitator superfamily from Candida albicans (FLU1) conferring resistance to fluconazole[J]. Microbiology,2000,146:2743-2754.
17. Alix Coste, Mahir Karababa, Francoise Ischer, et al. TAC1, Transcriptional Activator of CDR Genes, Is a New Transcription Factor Involved in the Regulation of Candida albicans ABC Transporters CDR1 and CDR2[J]. Eukaryotic Cell,2004:1639-1652.
18. Alix Coste, Vincent Turner, Francxoise Ischer, et al. A Mutation in Taclp, a Transcription Factor Regulating CDR1 and CDR2, Is Coupled With Loss of Heterozygosity at Chromosome 5 to Mediate Antifungal Resistance in Candida albicans[J]. Genetics,2006,172:2139-2156.
19. Morschhauser J, Barker KS, Liu TT, et al. The Transcription Factor Mrrlp Controls Expression of the MDR1 Efflux Pump and Mediates Multidrug Resistance in Candida albicans[J]. PLoS Pathog,2007,3(11):1603-1616.
20. Dunkel N, Liu TT, Barker KS, et al. A Gain-of-Function Mutation in the Transcription Factor Upc2p Causes Upregulation of Ergosterol Biosynthesis Genes and Increased Fluconazole Resistance in a Clinical Candida albicans Isolate[J]. Eukaryot Cell,2008,7(7):1180-1190.
21.Donlan RM, Costerton JW. Biofilms:survival mechanisms of clinically relevant microorganisms[J]. Clin Micro boil Rev,2002,15:167-193.
22. Chandra J, Kuhn D M. Mukherjee P K, et al. Biofilm formation by the fungal pathogen Candida albi-ans:developm ent, architecture, and drug resistance[J]. J Bacteriol,2001,183(18):5385 5394.
23. Baillie GS, Douglas LJ. Matrix polymers of Candida biofilms and their possible role in biofilm resistance to antifungal agents[J]. J Antimicrob Chemother,2000, 46:397-403.
24. Mukherjee PK, Chandra J, Kuhn DM, et al. Mechanism of fluconazole resistance in Candida albicans biofilms:phase specific role of efflux pumps and membranesterols[J]. Infect Immun,2003,71:4333-4340.