ERG11基因与热带念珠菌唑类药物耐药关系的研究

中国真菌学杂志　 2019, Vol. 14 　Issue (6): 332-337.

ERG11基因与热带念珠菌唑类药物耐药关系的研究

王影¹, 项明洁², 刘锦燕³, 叶书来¹, 周馨¹

1. 中国科学技术大学附属第一医院(安徽省立医院)检验科, 合肥 230036;
2. 上海交通大学医学院附属瑞金医院检验科, 上海 200025;
3. 上海交通大学医学院附属瑞金医院卢湾分院放免检验科, 上海 200020

收稿日期:2019-09-05 出版日期:2019-12-28 发布日期:2019-12-28
通讯作者: 周馨,E-mail:zhouxin1806@126.com E-mail:zhouxin1806@126.com
作者简介:王影,女(汉族),硕士,初级检验师.E-mail:sjdwangying@163.com
基金资助:
中央高校基本科研业务费专项资金资助（WK9110000117）

The study on the relationship between ERG11 gene and azole-resistant isolates of Candida tropicalis

WANG Ying¹, XIANG Ming-jie², LIU Jin-yan³, YE Shu-lai¹, ZHOU xin¹

1. Department of Laboratory Medicine, The First Affiliated Hospital of USTC, Division of Life Science and Medicine, University of Science and Technology of China, Hefei 230036, China;
2. Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 20025, China;
3. Department of Laboratory Medicine, Ruijin Hospital Luwan Branch, Shanghai Jiao Tong University School of Medicine, Shanghai 200020, China

Received:2019-09-05 Online:2019-12-28 Published:2019-12-28

摘要/Abstract

摘要：

目的探讨临床唑类药物耐药热带念珠菌菌株ERG11基因突变及表达情况。方法连续收集临床分离的热带念珠菌菌株，采用微量肉汤稀释法检测其对氟康唑、伏立康唑及伊曲康唑的药物敏感性，对唑类药物耐药菌株及部分敏感菌株进行ERG11基因测序，同时采用RT-PCR测定ERG11基因表达量。结果临床共分离92株热带念珠菌菌株，其中有29株为唑类药物耐药菌株，耐药率31.52%。40株热带念珠菌（29株耐药菌株和11株敏感菌株）ERG11基因序列共发现2个错义突变（S154F、Y132F）和5个同义突变，其中24株唑类药物耐药菌株同时出现上述两个错义突变位点。实时荧光定量PCR结果显示，在29株唑类药物耐药菌株中有19株其ERG11基因表达量较敏感菌株增高。分析16株对3种唑类药物全耐药菌株及其余13株仅对一种或两种药物耐药菌株，显示前者ERG11基因表达水平高于后者，差异有统计学意义。结论临床热带念珠菌唑类药物耐药与ERG11基因突变及过表达有关，有关热带念珠菌唑类药物的耐药机制还需进一步研究。

关键词: 热带念珠菌, 唑类药物耐药, ERG11基因

Abstract:

Objective To investigate the mutation and expression of ERG11 gene in clinical azole-resistant Candida tropicalis isolates. Methods The clinical Candida tropicalis isolates were collected and identified, then antifungal susceptibility tests to fluconazole, voriconazole and itraconazole were performed. ERG11 which was the gene coding antifungal drug target enzyme of cytochrome P450 lanosterol 14a-demethylase, was quantified by real-time RT-PCR and sequenced. The sequencing results were compared with the known standard sequence (M23673) in GenBank. Results There were 92 clinical Candida tropicalis isolates were obtained, of which 29 were resistant to at least one of the above three azole antifungals. Two missense mutations (S154F,Y132F) and five synonymous mutations were found in 40 clinical isolates, including 29 azole-resistant isolates and 11 azole-susceptible isolates. It is worth noting that the two missense mutations were existed in 24 of 29 azole-resistant isolates, however, no missense mutations were found in azole-susceptible isolates. The results of real-time PCR showed that the expression of ERG11 gene in azole-resistant clinical isolates was significantly higher than that in azole-susceptible isolates, and 16 isolates resistant to all three of the azole antifungals had a higher level of ERG11 gene expression than 13 isolates that were only resistant to one or two azole antifungals. Conclusions Azole resistance of clinical Candida tropicalis isolates is associated with the mutations and overexpression of ERG11 gene, and the resistance mechanism of Candida tropicalis to azole antifungals needs further study.

Key words: Candida tropicalis, azole resistance, ERG11 gene

中图分类号:

R379.4

王影, 项明洁, 刘锦燕, 叶书来, 周馨. ERG11基因与热带念珠菌唑类药物耐药关系的研究[J]. 中国真菌学杂志, 2019, 14(6): 332-337.

WANG Ying, XIANG Ming-jie, LIU Jin-yan, YE Shu-lai, ZHOU xin. The study on the relationship between ERG11 gene and azole-resistant isolates of Candida tropicalis[J]. Chinese Journal of Mycology, 2019, 14(6): 332-337.

参考文献

[1] Kolaczkowska A, Kolaczkowski M. Drug resistance mechanisms and their regulation in non-albicans Candida species[J]. J Antimicrob Chemother, 2016,71(6):1438-1450.
[2] Kothavade RJ, Kura MM, Valand AG, et al. Candida tropicalis:its prevalence, pathogenicity and increasing resistance to fluconazole[J]. J Med Microbiol, 2010,59(Pt 8):873-880.
[3] Quindós G. Epidemiology of candidaemia and invasive candidiasis. A changing face[J]. Rev Iberoam Micol, 2014,31(1):42-48.
[4] Chakrabarti A, Chatterjee SS, Rao KLN, et al. Recent experience with fungaemia:change in species distribution and azole resistance[J]. Scand J Infect Dis, 2009, 41(4):275-284.
[5] Negri M, Silva S, Henriques M, et al. Insights into Candida tropicalis nosocomial infections and virulence factors[J]. Eur J Clin Microbiol Infect Dis, 2012,31(7):1399-1412.
[6] Pappas PG, Kauffman CA, Andes DR, et al. Clinical practice guideline for the management of candidiasis:2016 update by the Infectious Diseases Society of America[J]. Clin Infect Dis, 2016, 62(4):e1-50.
[7] Cornely OA, Bassetti M, Calandra T, et al. ESCMID guideline for the diagnosis and management of Candida diseases 2012:Non-neutropenic adult patients[J]. Clin Microbiol Infect, 2012, 18(s7):19-37.
[8] Castanheira M, Messer SA, Rhomberg PR, et al. Antifungal susceptibility patterns of a global collection of fungal isolates:results of the SENTRY Antifungal Surveillance Program (2013)[J]. Diagn Microbiol Infect Dis, 2016,85(2):200-204.
[9] Flowers SA, Brendan C, Whaley SG, et al. Contribution of clinically derived mutations in ERG11 to azole resistance in Candida albicans[J]. Antimicrob Agents Chemother, 2015, 59(1):450-460.
[10] Vandeputte P, Larcher G, Berges T, et al. Mechanisms of azole resistance in a clinical isolate of Candida tropicalis[J]. Antimicrob Agents Chemother, 2005, 49(11):4608-4615.
[11] Jiang C, Dong DF, 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] Heilmann CJ, Schneider S, Barker KS, et al. An A643T mutation in the transcription factor Upc2p causes constitutive ERG11 upregulation and increased fluconazole resistance in Candida albicans[J]. Antimicrob Agents Chemother, 2010, 54(1):353-359.
[13] Jin LY, Cao ZR, Wang Q, et al. MDR1 overexpression combined with ERG11 mutations induce high-level fluconazole resistance in Candida tropicalis clinical isolates[J]. BMC Infect Dis, 2018,18(1):162.
[14] Xiang MJ, Liu JY, Ni PH, et al. Erg11 mutations associated with azole resistance in clinical isolates of Candida albicans[J]. FEMS Yeast Res, 2013,13(4):386-393.
[15] Choi YJ, Kim YJ, Yong D, et al. Fluconazole-resistant Candida parapsilosis bloodstream isolates with Y132F mutation in ERG11 gene, South Korea[J]. Emerg Infect Dis, 2018,24(9):1768-1770.
[16] Selb R, Fuchs V, Graf B, et al. Molecular typing and in vitro resistance of Cryptococcus neoformans clinical isolates obtained in Germany between 2011 and 2017[J]. Int J Med Microbiol, 2019,309(6):151336.
[17] Castanheira M, Deshpande LM, Messer SA, et al. Analysis of global antifungal surveillance results reveals predominance of Erg11 Y132F alteration among azole-resistant Candida parapsilosis and Candida tropicalis and country-specific isolate dissemination[J]. Int J Antimicrob Agents, 2019, S0924-8579(19):30246-30248.
[18] Xisto MI, Caramalho RD, Rocha DA, et al. Pan-azole-resistant Candida tropicalis carrying homozygous erg11 mutations at position K143R:a new emerging superbug[J]? J Antimicrob Chemother, 2017,72(4):988-992.
[19] Fan X, Xiao M, Zhang D, et al. Molecular mechanisms of azole resistance in Candida tropicalis isolates causing invasive candidiasis in China[J]. Clin Microbiol Infect, 2019,25(7):885-891.