结核分枝杆菌对环丝氨酸耐药分子特征的初步研究

李超; 李桂莲; 罗巧; 李霜君; 王瑞白; 楼永良; 吕建新; 万康林

Abstract

李超,李桂莲,罗巧,李霜君,王瑞白,楼永良,吕建新,万康林.结核分枝杆菌对环丝氨酸耐药分子特征的初步研究[J].Chinese journal of Epidemiology,2017,38(2):240-243

结核分枝杆菌对环丝氨酸耐药分子特征的初步研究

A preliminary study on the molecular characteristics of D-cycloserine resistance of Mycobacterium tuberculosis

Received:August 20, 2016

DOI：10.3760/cma.j.issn.0254-6450.2017.02.021

KeyWord: 结核分枝杆菌环丝氨酸抗药性基因突变

English Key Word: Mycobacterium tuberculosis Cycloserine Drug resistance Genes mutation

FundProject:国家科技重大专项（2013XZ10003002-001）

Author Name	Affiliation	E-mail
Li Chao	School of Laboratory Medicine and Life Science, Wenzhou Medical University, Wenzhou 325035, China
Li Guilian	Tuberculosis Branch, 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
Luo Qiao	School of Laboratory Medicine and Life Science, Wenzhou Medical University, Wenzhou 325035, China
Li Shuangjun	School of Laboratory Medicine and Life Science, Wenzhou Medical University, Wenzhou 325035, China
Wang Ruibai	Tuberculosis Branch, 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
Lou Yongliang	School of Laboratory Medicine and Life Science, Wenzhou Medical University, Wenzhou 325035, China
Lyu Jianxin	School of Laboratory Medicine and Life Science, Wenzhou Medical University, Wenzhou 325035, China
Wan Kanglin	School of Laboratory Medicine and Life Science, Wenzhou Medical University, Wenzhou 325035, China Tuberculosis Branch, 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	wankanglin@icdc.cn

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Abstract:

目的探讨结核分枝杆菌对环丝氨酸耐药性与alrA、ddlA和cycA基因突变的关系，分析环丝氨酸耐药与基因型的关联性。方法从菌株库中选取145株临床分离株，采用比例法测定菌株对环丝氨酸耐药表型、微孔板刃天青显色法测定最小抑菌浓度，PCR扩增、DNA直接测序法测定目的基因全长，与标准菌株H37Rv比对。间隔区寡核苷酸基因分型（spoligotyping）进行菌株基因型鉴定，分析耐药表型与基因型的关系，采用χ²检验分析差异有无统计学意义。结果 145株临床分离株中，环丝氨酸耐药菌株为24株，敏感株为121株。24株耐药菌株中，3株（12.5%）发生cycA非同义突变，涉及的密码子为188位、318位和508位，1株（4.2%）发生alrA非同义突变，涉及密码子为261位。敏感菌株的目的基因中仅检出同义突变。药敏试验证实，突变株的最小抑菌浓度均有不同程度的升高。北京基因型为88株，环丝氨酸耐药率为20.5%（18/88），非北京基因型为57株，环丝氨酸耐药率为10.5%（6/57），两者耐药率差异无统计学意义（χ²=2.47，P>0.05）。结论 alrA和cycA单核苷酸非同义基因突变可能是环丝氨酸耐药的机制之一。尚不能确定北京基因型或非北京基因型菌株与环丝氨酸耐药有相关性。

English Abstract:

Objective To investigate the relationship between D-cycloserine resistance and the gene mutations of alrA, ddlA and cycA of Mycobacterium (M.) tuberculosis, as well as the association between D-cycloserine resistance and spoligotyping genotyping. Methods A total of 145 M. tuberculosis strains were selected from the strain bank. D-cycloserine resistant phenotypes of the strains were determined by the proportion method and the minimal inhibitory concentration was determined by resazurin microtiter assay. PCR amplification and DNA direct sequencing methods were used for the analysis of gene mutations. Relationship between the resistance phenotype and genotype was analyzed by chi-square test. Results Of the 145 clinically collected strains, 24 (16.6%) of them were D-cycloserine resistant and 121 (83.4%) were sensitive. There were only synonymous mutations noticed on alrA, ddlA and cycA in sensitive strains. Of the 24 D-cycloserine resistant strains, 3 (12.5%) isolates' cycA and 1 (4.2%) isolates' alrA happened to be non-synonymous mutations, in which the codes were 188, 318 and 508 of cycA, and 261 of alrA, respectively. Results on drug sensitivity tests confirmed the minimal inhibitory concentration of the mutant strains were all increased to some degrees. The D-cycloserine resistant rates of 88 Beijing genotype and 57 non-Beijing genotype strains were 20.5% and 10.5%, respectively, but with no statistically significant difference (χ²=2.47, P>0.05). Conclusions The non-synonymous mutations of alrA and cycA might contribute to one of the mechanisms of M. tuberculosis D-cycloserine resistance. M. tuberculosis Beijing genotype or non-Beijing genotype was not considered to be associated with the D-cycloserine resistance.

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