不同唑类药物体外诱导热带念珠菌耐药特征及其耐药机制研究

中国真菌学杂志　 2018, Vol. 13 　Issue (4): 197-200.

不同唑类药物体外诱导热带念珠菌耐药特征及其耐药机制研究

范欣^1,2,3, 黄晶晶^2,3, 侯欣^2,3, 肖盟^2,3, 张丽^2,3, 徐英春^2,3

1. 首都医科大学附属北京朝阳医院感染和临床微生物科, 北京 100020;
2. 中国医学科学院北京协和医院检验科, 北京 100730;
3. 侵袭性真菌病机制研究与精准诊断北京市重点实验室, 北京 100730

收稿日期:2017-11-23 出版日期:2018-08-28 发布日期:2018-08-28
通讯作者: 徐英春,E-mail:xycpumch@139.com E-mail:xycpumch@139.com
作者简介:范欣,女(汉族),博士,研究实习员.E-mail:fanxin12356@163.com
基金资助:
北京协和医院杰出青年基金项目（JQ201703）

The features and mechanisms of Candida tropicalis resistance in vitro induced by different azoles

FAN Xin^1,2,3, HUANG Jing-jing^2,3, HOU Xin^2,3, XIAO Meng^2,3, ZHANG Li^2,3, XU Ying-chun^2,3

1 Beijing Chao-Yang Hospital, Capital Medical University, Beijing 100020;
2 Department of Clinical Laboratory, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences, Beijng 100730;
3 Beijing Key Laboratory for Mechanisms Research and Precision Diagnosis of Invasive Fungal Diseases, Beijng 100730

Received:2017-11-23 Online:2018-08-28 Published:2018-08-28

摘要/Abstract

摘要：

目的比较体外不同唑类药物诱导热带念珠菌耐药性产生的特点以及耐药机制的不同。方法选取1株临床分离的唑类敏感菌，分别在含16μg/mL氟康唑，2μg/mL伏立康唑，1μg/mL泊沙康唑的液体培养基中进行传代培养。显色微量肉汤稀释法检测每一代菌的抗真菌药物敏感性。对第50代传代菌的唑类作用靶位点基因ERG11进行扩增测序，并对ERG11基因、泵蛋白基因MDR1、CDR1以及线粒体细胞色素b基因CYTb进行荧光定量检测。结果体外氟康唑、伏立康唑暴露的情况下，菌株很快出现耐药性；相比，泊沙康唑并未诱导出耐药菌；氟康唑、伏立康唑诱导耐药性产生的方式呈现不同特征。氟康唑诱导下菌株MIC值逐渐上升，但伏立康唑诱导菌出现了明显的跳跃式升高。耐药机制研究发现，伏立康唑诱导菌ERG11基因出现了与耐药密切相关的G/G1390G/A碱基杂合突变。荧光定量PCR结果显示，仅伏立康唑诱导耐药菌CDR1基因的相对表达量显著升高。结论热带念珠菌在体外氟康唑、伏立康唑暴露情况下会很快出现耐药性，但其出现的特征以及耐药机制有所差别。

关键词: 热带念珠菌, 耐药机制, 诱导唑类耐药

Abstract:

Objective To compare the features of different azoles in vitro induction of azole resistance in Candida tropicalis, and related resistant mechanisms.Methods A clinical azole-susceptible isolate was consecutively subcultured in liquid medium containing 16 μg/mL of fluconazole, 2 μg/mL of voriconazole or 1 μg/mL of posaconazole, respectively, for 50 generations. Antifungal susceptibility testing was performed by colorimetric broth microdilution method. The azoles target gene, ERG11, of the 50^th generation isolates were amplified and sequenced. In addition, the expression level of ERG11, and efflux pump-encoding gene MDR1, CDR1, as well as cytochrome b-encoding gene CYTb, were determined by real-time quantitative PCR.Results Azole resistance was emerged very fast when under in vitro exposure to fluconazole or voriconazole, whilst the isolates induced by posaconazole remained susceptible. However, the resistance features of isolates induced by fluconazole and voriconazole were different. The MICs of isolates induced by fluconazole were gradually increased, while MICs of isolates induced by voriconazole had a jump rise. By resistant mechanism study, a G/G1390G/A heterozygous mutation being responsible for azole resistance was detected in ERG11 gene of voriconazole-induced isolate. The real-time PCR results showed that, only the expression of CDR1 gene in fluconazole-induced isolates increased significantly.Conclusions Azole resistance amongst C.tropicalis isolates emerged rapidly under in vitro exposure to fluconazole or voriconazole, but the resistant features and mechanisms were different.

Key words: Candida tropicalis, resistant mechanism, induction of azole resistance

中图分类号:

R379.4

范欣, 黄晶晶, 侯欣, 肖盟, 张丽, 徐英春. 不同唑类药物体外诱导热带念珠菌耐药特征及其耐药机制研究[J]. 中国真菌学杂志, 2018, 13(4): 197-200.

FAN Xin, HUANG Jing-jing, HOU Xin, XIAO Meng, ZHANG Li, XU Ying-chun. The features and mechanisms of Candida tropicalis resistance in vitro induced by different azoles[J]. Chinese Journal of Mycology, 2018, 13(4): 197-200.

参考文献

[1] Chai LY, Denning DW, Warn P. Candida tropicalis in human disease[J]. Crit Rev Microbiol, 2010,36(4):282-298.
[2] Fan X, Xiao M, Liao K, et al. Notable increasing trend in azole non-susceptible Candida tropicalis causing invasive candidiasis in China (August 2009 to July 2014):molecular epidemiology and clinical azole consumption[J]. Front Microbiol, 2017, 8:464.
[3] Silva AP, Miranda IM, Guida A, et al. Transcriptional profiling of azole-resistant Candida parapsilosis strains[J]. Antimicrob Agents Chemother, 2011, 55(7):3546-3556.
[4] Fan X, Xiao M, Liu P, et al. Novel polymorphic multilocus microsatellite markers to distinguish Candida tropicalis isolates[J]. PLoS One, 2016, 11(11):e0166156.
[5] Pfaller MA. Antifungal drug resistance:mechanisms, epidemiology, and consequences for treatment[J]. Am J Med, 2012, 125(1 Suppl):S3-13.
[6] de Carvalho Parahym AM, da Silva CM, Leao MP, et al. Invasive infection in an acute myeloblastic leukemia patient due to triazole-resistant Candida tropicalis[J]. Diagn Microbiol Infect Dis, 2011, 71(3):291-293.
[7] Chong Y, Shimoda S, Yakushiji H, et al. Fatal candidemia caused by azole-resistant Candida tropicalis in patients with hematological malignancies[J]. J Infect Chemother, 2012, 18(5):741-746.
[8] Wang H, Xiao M, Chen SC, et al. In vitro susceptibilities of yeast species to fluconazole and voriconazole as determined by the 2010 National China Hospital Invasive Fungal Surveillance Net (CHIF-NET) study[J]. J Clin Microbiol, 2012, 50(12):3952-3959.
[9] Barchiesi F, Calabrese D, Sanglard D, et al. Experimental induction of fluconazole resistance in Candida tropicalis ATCC 750[J]. Antimicrob Agents Chemother, 2000, 44(6):1578-1584.
[10] Tan J, Zhang J, Chen W, et al. The A395T mutation in ERG11 gene confers fluconazole resistance in Candida tropicalis causing candidemia[J]. Mycopathologia, 2015, 179(3-4):213-218.
[11] Jiang C, Dong D, Yu B, et al. Mechanisms of azole resistance in 52 clinical isolates of Candida tropicalis in China[J]. J Antimicrob Chemother, 2013, 68(4):778-785.
[12] Forastiero A, Mesa-Arango AC, Alastruey-Izquierdo A, et al. Candida tropicalis antifungal cross-resistance is related to different azole target (Erg11p) modifications[J]. Antimicrob Agents Chemother, 2013, 57(10):4769-4781.
[13] 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.
[14] 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]. Antimicrob Agents Chemother, 2004, 48(6):2124-2131.

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