对唑类药物交叉耐药的烟曲霉临床分离株耐药机制的初步探讨

中华皮肤科杂志 ›› 2011, Vol. 44 ›› Issue (4): 244-248.

对唑类药物交叉耐药的烟曲霉临床分离株耐药机制的初步探讨

孙毅¹,刘伟²,陈伟³,万喆²,李若瑜²

1. 北京大学第一医院
2. 北京大学第一医院皮肤科
3.

收稿日期:2010-08-30 修回日期:2010-10-20 出版日期:2011-04-15 发布日期:2011-04-01
通讯作者: 刘伟 E-mail:liuw90@yahoo.com
基金资助:
国家自然科学基金;北京市自然科学基金

Molecular mechanisms of cross-resistance to azole antifungal agents in a clinical isolate of Aspergillus fumigatus: a preliminary study

Received:2010-08-30 Revised:2010-10-20 Online:2011-04-15 Published:2011-04-01

摘要/Abstract

摘要：

目的研究对唑类药物交叉耐药的烟曲霉临床分离株的耐药机制。方法自1例侵袭性曲霉病患者体内分离得到1株烟曲霉，分别利用美国临床实验室标准化研究所（CLSI）M38-A2中的微量液基稀释法和E-test法测定伊曲康唑、伏立康唑、两性霉素B、泊沙康唑和卡泊芬净对该烟曲霉的最低抑菌浓度（MIC）/最低有效浓度（MEC）；并对该菌株中唑类药物靶酶基因cyp51A进行克隆和序列测定分析。结果微量液基稀释法显示，该菌株伊曲康唑、伏立康唑、两性霉素B的MIC分别为≥16、8、1 mg/L，卡泊芬净的MEC为0.5 mg/L。E-test法显示，伊曲康唑、伏立康唑、两性霉素B和泊沙康唑的MIC分别为≥32、≥32、12和≥32 mg/L，卡泊芬净的MIC为0.047 mg/L。对cyp51A基因进行测序并分析后发现，该菌株的cyp51A序列中存在启动子区-288到-322位间34 bp的串联序列的插入，以及该基因编码区364位碱基的点突变（T364A），导致了编码区98位亮氨酸的置换（L98H）；该菌株的cyp51A基因编码区还存在137位碱基（A137T）、585位碱基（G585A）、814位碱基（C814A）、836位碱基（G836C）、991位碱基（T991C）、1350位碱基（A1350G）的突变，并分别导致编码区相应氨基酸的置换。结论分离到对唑类药物交叉耐药的烟曲霉临床株，该菌株的cyp51A基因存在启动子区34 bp的串联序列插入和编码区364位的突变（T364A），这种变化参与了其对伊曲康唑、伏立康唑和泊沙康唑交叉耐药；该菌株cyp51A基因编码区还存在一些其他的点突变，并可导致相应部位的氨基酸置换。

关键词: cyp51A

Abstract:

Objective To investigate the molecular mechanisms of cross-resistance to azoles in a clinical isolate of Aspergillus fumigatus. Methods A. fumigatus was isolated from a patient with invasive aspergillosis. Clinical Laboratory Standard Institute M38-A2 broth microdilution method and E-test method were used to determine the minimum inhibitory concentrations （MICs） or minimum effective concentration （MEC） of itraconazole, voriconazole, amphotericin B, posaconazole and caspofungin for the A. fumigatus isolate. DNA was extracted from the isolate and subjected to the amplification of cyp51A gene encoding the target enzyme of azole antifungal agents followed by sequence analysis. Results The broth microdilution test showed that the MEC of caspofungin was 0.5 mg/L, and MICs of itraconazole, voriconazole and amphotericin B were ≥16 mg/L, 8 mg/L and 1 mg/L, respectively, for this isolate; while E-test assay revealed that the MICs of caspofungin, itraconazole, voriconazole, amphotericin B and posaconazole were 0.047 mg/L, ≥32 mg/L, ≥32 mg/L, 12 mg/L and ≥32 mg/L, respectively. Sequence analysis showed an insertion of a 34-bp tandem sequence in the promoter region of the cyp51A gene as well as a T364A point mutation causing the substitution of leucine 98 （L98H）. In addition, there were some other mutations in the cyp51A gene of this isolate, such as A137T, G585A, C814A, G836C, T991C and A1350G, which could result in corresponding amino acid substitutions. Conclusions An A. fumigatus strain with cross-resistance to azole antifungal agents is isolated. There is an insertion of a 34-bp tandem sequence into the promoter region as well as a T364A point mutation in the cyp51A gene, which contribute to the cross resistance to azole antifungal agents including itraconazole, voriconazole, and posaconazole. In addition, other mutations causing amino acid substitutions have also been detected in the cyp51A gene of this isolate.

Key words: cyp51A

孙毅刘伟陈伟万喆李若瑜. 对唑类药物交叉耐药的烟曲霉临床分离株耐药机制的初步探讨[J]. 中华皮肤科杂志, 2011, 44(4): 244-248.

参考文献

参考文献 [1] Pagano L, Caira M, Candoni A, et al. The epidemiology of fungal infections in patients with hematologic malignancies: the SEIFEM-2004 study. Haematologica,2006,91(8):1068-1075. [2] Denning DW, Hope WW. Therapy for fungal diseases: opportunities and priorities. Trends Microbiol,2010,18(5):195-204. [3] Herbrecht R, Denning DW, Patterson TF, et al. Voriconazole versus amphotericin B for primary therapy of invasive aspergillosis. N Engl J Med,2002,347(6):408-415. [4] Snelders E, van derLeeHA, Kuijpers J, et al. Emergence of azole resistance in Aspergillus fumigatus and spread of a single resistance mechanism. PLoS Med,2008,5(11):e219. [5] Samson RA, Varga J. What is a species in Aspergillus? Med Mycol,2009,47 Suppl 1:S13-S20. [6] 齐祖同. 中国真菌志第五卷.1版北京：科学出版社,1997；27-33. [7] Hong SB, Shin HD, Hong J, et al. New taxa of Neosartorya and Aspergillus in Aspergillus section Fumigati. Antonie Van Leeuwenhoek,2008,93(1-2):87-98. [8] Samson RA, Hong S, Peterson SW, et al. Polyphasic taxonomy of Aspergillus section Fumigati and its teleomorph Neosartorya. Stud Mycol,2007,59:147-203. [9] Varga J, Frisvad JC, Samson RA. Polyphasic taxonomy of Aspergillus section Candidi based on molecular, morphological and physiological data. Stud Mycol,2007,59:75-88. [10] 卢圣栋. 现代分子生物学实验技术. 北京:高等教育出版社,1993. [11] Hong SB, Go SJ, Shin HD, et al. Polyphasic taxonomy of Aspergillus fumigatus and related species. Mycologia,2005,97(6):1316-1329. [12] Glass NL, Donaldson GC. Development of primer sets designed for use with the PCR to amplify conserved genes from filamentous ascomycetes. Appl Environ Microbiol,1995,61(4):1323-1330. [13] Clinical and Laboratory Standards Institute. Reference method for broth dilution antifungal susceptibility testing of filamentous fungi; Approved standard ---- M38-A2. CLSI, Wayne, PA, USA, 2008. [14] Kurtz MB, Heath IB, Marrinan J, et al. Morphological effects of lipopeptides against Aspergillus fumigatus correlate with activities against (1,3)-beta-D-glucan synthase. Antimicrob Agents Chemother,1994,38(7):1480-1489. [15] Walsh TJ, Anaissie EJ, Denning DW, et al. Treatment of Aspergillosis: Clinical Practice Guidelines of the Infectious Diseases Society of America. Clinical Infectious Diseases,2008,46:327-360. [16] Chamilos G, Kontoyiannis DP. Update on antifungal drug resistance mechanisms of Aspergillus fumigatus. Drug Resist Updat,2005,8(6):344-358. [17] Mellado E, Garcia-Effron G, Alcazar-Fuoli L, et al. A new Aspergillus fumigatus resistance mechanism conferring in vitro cross-resistance to azole antifungals involves a combination of cyp51A alterations. Antimicrob Agents Chemother,2007,51(6):1897-1904. [18] Chen J, Li H, Li R, et al. Mutations in the cyp51A gene and susceptibility to itraconazole in Aspergillus fumigatus serially isolated from a patient with lung aspergilloma. J Antimicrob Chemother,2005,55(1):31-37. [19] Howard SJ, Cerar D, Anderson MJ, et al. Frequency and evolution of Azole resistance in Aspergillus fumigatus associated with treatment failure. Emerg Infect Dis,2009,15(7):1068-1076. [20] Rodriguez-Tudela JL, Alcazar-Fuoli L, Mellado E, et al. Epidemiological cutoffs and cross-resistance to azole drugs in Aspergillus fumigatus. Antimicrob Agents Chemother,2008,52(7):2468-2472.