马西利亚分枝杆菌临床分离株的多位点序列鉴定和药物敏感性试验分析

doi:10.3969/j.issn.1000-6621.2015.03.016

中国防痨杂志 ›› 2015, Vol. 37 ›› Issue (3): 300-306.doi: 10.3969/j.issn.1000-6621.2015.03.016

马西利亚分枝杆菌临床分离株的多位点序列鉴定和药物敏感性试验分析

魏剑浩郭倩李桂莲刘海灿李马超吴移谋楼永良吕建新万康林

325035 温州医科大学检验医学院生命科学学院［魏剑浩、楼永良、吕建新、万康林（特聘教授）］；中国疾病预防控制中心传染病预防控制所传染病预防控制国家重点实验室［魏剑浩（研究生）、郭倩（研究生）、李桂莲、刘海灿、李马超、万康林］；南华大学病原生物学研究所［郭倩、吴移谋、万康林（特聘教授）］；感染性疾病诊治协同创新中心［万康林（特聘研究人员）］

收稿日期:2014-12-01 出版日期:2015-03-10 发布日期:2015-04-03
通信作者: 万康林；吕建新 E-mail:wankanglin@icdc.cn; jxlu313@163.com
基金资助:
“十二五”国家科技重大专项（2013ZX10003006-002-001）；传染病预防控制国家重点实验室重点项目（2014SKLID104）

Species identification with multilocus sequence analysis and drug-sensitivity test in Mycobacterium massiliense clinical isolates

WEI Jian-hao^*, GUO Qian, LI Gui-lian, LIU Hai-can, LI Ma-chao, WU Yi-mou, LOU Yong-liang, LV Jian-xin, WAN Kang-lin

^*School of Laboratory Medicine and Life Science, Wenzhou Medical University, Wenzhou 325035, China (*Now in State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China)

Received:2014-12-01 Online:2015-03-10 Published:2015-04-03
Contact: WAN Kang-lin；LV Jian-xin E-mail:wankanglin@icdc.cn; jxlu313@163.com

摘要/Abstract

摘要： 目的对单基因比对不能区分的马西利亚分枝杆菌（M.massiliense）临床分离株进行菌种鉴定，补充其药物敏感谱，为临床诊治提供依据。方法 2013年中国疾病预防控制中心传染病预防控制所结核病室共收集了47例疑似非结核分枝杆菌（NTM）感染的临床样本，用对硝基苯甲酸、噻吩-2-羧酸肼培养基和多位点PCR方法区分鉴别得到的分枝杆菌，采用基因序列比对进行NTM菌种鉴定，其中未能准确鉴定菌种的6株临床分离株进一步通过多位点序列分析（MLSA）方法进行定种鉴定。采用微孔板阿尔玛蓝测定法（MABA）对马西利亚分枝杆菌菌株开展36种药物的药物敏感性试验（简称“药敏试验“）。结果 47例临床样本中鉴定出34株NTM，其中6株临床分离株综合5个管家基因（16S rRNA、hsp65、rpoB、sodA和recA）的单基因比对结果仍不能鉴定菌种，因此本研究采用MLSA方法构建邻位相加法进化树确认6株均为马西利亚分枝杆菌。药敏试验结果显示M.massiliense对阿米卡星为敏感（16 μg/ml）或中度敏感（32 μg/ml），对利福平、异烟肼、乙胺丁醇、环丙沙星、乙硫异烟胺、卷曲霉素、对氨基水杨酸、氧氟沙星、卡那霉素、环丝氨酸、美罗培南、米诺环素12种药物耐药。结论 MLSA方法能有效鉴定马西利亚分枝杆菌；马西利亚分枝杆菌耐药程度较高。

关键词: 分枝杆菌属, 多位点测序分型, 微生物敏感性试验

Abstract: Objective To confirm the species of the clinical isolates suspected Mycobacterium massiliense (M. massiliense) which could not be identified by the single gene comparison, and test their drug-sensitivity spectrum to provide the basis of clinical diagnosis and treatment. Methods In 2013，we collected 47 clinical samples of the patients suspected with non-tuberculous Mycobacteria (NTM) infection in Tuberculosis Laboratory of National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention. The Mycobacteria isolates were determined with the media containing p-nitrobenzoic acid (PNB) and 2-thiophene carboxy-lic acid hydrazide (TCH) respectively and multilocus PCR. Five house-keeping genes (16S rRNA, hsp65, rpoB, sodaand recA) were analyzed by DNA sequencing, and compared by Basic Local Alignment Search Tool (BLAST) to identifying the species of NTM, in which the species of 6 M. massiliense isolates were confirmed by means of multilocus sequence analysis (MLSA). The drug-sensitivities of M. massiliense isolates to 36 antibiotics were tested with the minimal inhibitory concentrations (MICs) by Microplate AlamarBlue Assay(MABA). Results Of 47 clinical samples from the patients suspected with NTM infection, 34 NTM strains were isolated, in which 6 strains could not be identified by DNA sequencing and BLAST using the single gene sequence comparison with five house-keeping genes. These strains were confirmed as M. massiliense by MLSA with a Neighbor-joining tree. The drug sensitive spectrum to 36 antibiotics showed that M. massiliense isolates were sensitive to Amikacin (16 μg/ml or 32 μg/ml), resistant to 12 kinds of anti-tuberculosis drugs, rifampin, isoniazid, ethambutol, ciprofloxacin, ethionamide, capreomycin, para-aminosalicylic acid, ofloxacin, kanamycin, cycloserine, meropenem, and minocycline; and extremely insensitive to isoniazid aminosalicylate, cefoperazone and thioacetazone. Conclusion The MLSA method is effective in identification of M. massiliense strain. M. massiliense strains had a higher degree of drug resistance.

Key words: Mycobacterium, Multilocus sequence typing, Microbial sensitivity tests

魏剑浩，郭倩，李桂莲，刘海灿，李马超，吴移谋，楼永良，吕建新，万康林. 马西利亚分枝杆菌临床分离株的多位点序列鉴定和药物敏感性试验分析[J]. 中国防痨杂志, 2015, 37(3): 300-306. doi: 10.3969/j.issn.1000-6621.2015.03.016

WEI Jian-hao*, GUO Qian, LI Gui-lian, LIU Hai-can, LI Ma-chao, WU Yi-mou, LOU Yong-liang, LV Jian-xin, WAN Kang-lin. Species identification with multilocus sequence analysis and drug-sensitivity test in Mycobacterium massiliense clinical isolates[J]. Chinese Journal of Antituberculosis, 2015, 37(3): 300-306. doi: 10.3969/j.issn.1000-6621.2015.03.016

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马西利亚分枝杆菌临床分离株的多位点序列鉴定和药物敏感性试验分析

Species identification with multilocus sequence analysis and drug-sensitivity test in Mycobacterium massiliense clinical isolates

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