广西壮族自治区结核分枝杆菌异烟肼与利福平耐药基因联合突变特征分析

秦翊翔; 蓝如束; 覃慧芳; 叶婧; 秦小玲; 甘丽; 刘港; 徐若兰; 罗丹

doi:10.3784/jbjc.202112120636

广西壮族自治区结核分枝杆菌异烟肼与利福平耐药基因联合突变特征分析

doi: 10.3784/jbjc.202112120636

秦翊翔^1,,
蓝如束²,
覃慧芳³,
叶婧³,
秦小玲¹,
甘丽¹,
刘港¹,
徐若兰¹,
罗丹^1, ,

1.
广西中医药大学公共卫生与管理学院, 广西南宁 530200
2.
广西江滨医院检验科, 广西南宁 530021
3.
广西壮族自治区疾病预防控制中心结核病防制所, 广西南宁 530021

基金项目: 广西自然科学基金面上项目（No. 2017GXNSFAA198330）；广西中医药大学博士科研启动项目（No. 2017BS004）；广西中医药大学研究生创新项目（No. YCSW2021226）；广西医疗卫生适宜技术开发与推广应用项目（No. S2017066）

详细信息

作者简介:
秦翊翔，男，广西壮族自治区桂林市人，研究生在读，主要从事结核病流行病学，Email:794248760@qq.com

通讯作者:
罗丹，Tel：0771–4928360，Email：Luodan.2005@163.com

中图分类号: R211; R521
计量
- 文章访问数: 107
- HTML全文浏览量: 59
- PDF下载量: 12
- 被引次数: 0
出版历程
- 收稿日期: 2021-12-12
- 网络出版日期: 2022-04-06
- 刊出日期: 2022-07-14

Characterization of combined mutation of isoniazid and rifampicin resistance genes in Mycobacterium tuberculosis in Guangxi

1.
Academy of Public Health and Management of Guangxi University of Traditional Chinese Medicine, Nanning 530200, Guangxi, China
2.
Clinical Laboratory of Guangxi Jiangbin Hospital, Nanning 530021, Guangxi, China
3.
Institute for Tuberculosis Control of Guangxi Zhuang Autonomous Region Center for Disease Control and Prevention, Nanning 530021, Guangxi, China

Funds: This study was supported by the Natural Science Foundation of Guangxi (No. 2017GXNSFAA198330), the Doctoral Research Initiation Program of Guangxi University of Traditional Chinese Medicine (No. 2017BS004), the Graduate Student Innovation Program of Guangxi University of Traditional Chinese Medicine (No. YCSW2021226) and Guangxi Medical Health Suitable Technology Development, Promotion and Application Project (No. S2017066)

More Information

Corresponding author: Luo Dan, Email: Luodan.2005@163.com

摘要

摘要: 目的了解广西壮族自治区（广西）结核分枝杆菌异烟肼与利福平耐药基因联合突变特征，为耐多药结核病的分子诊断和治疗提供依据。方法 2017—2018年从广西30个结核病防治定点机构收集的结核分枝杆菌中选取49株耐多药菌株和459株全敏感菌株进行全基因组测序。结果耐多药表型与基因联合突变的符合率为71.43%。单基因突变率和基因联合突变率在耐多药菌株中均高于全敏感菌株（χ²=5.753，P=0.016; χ²=284.034，P<0.001）。 katG和rpoB的单基因突变率在耐多药菌株中高于全敏感菌株（χ²=7.524，P=0.006; χ²=4.353，P=0.037）。 katG＋rpoB基因联合突变在耐多药菌株中高于全敏感菌株（χ²=279.956，P<0.001）。在基因联合突变的位点分布中，以katG315＋rpoB450和katG315＋rpoB445位点突变为主，占40.82%（20/49），2种形式的基因位点联合突变率在耐多药菌株中均高于全敏感菌株（χ²=144.232，P<0.001; χ²=19.014，P<0.001）。结论对异烟肼和利福平耐药基因联合突变的检测可作为广西耐多药筛查的重要指标。广西结核分枝杆菌异烟肼和利福平耐药基因突变以katG315、rpoB450和rpoB445位点突变为主，基因联合突变以katG＋rpoB形式为主。 katG315＋rpoB450和katG315＋rpoB445位点突变是广西地区耐多药产生的主要分子机制。
- 分枝杆菌 /
- 肺结核 /
- 耐多药 /
- 基因 /
- 联合突变
Abstract: Objective To understand the combined mutation characteristics of isoniazid and rifampicin resistance genes of Mycobacterium tuberculosis in Guangxi and provide a basis for the molecular diagnosis and treatment of multidrug-resistant tuberculosis (MDR-TB). Methods A total of 49 MDR strains and 459 fully sensitive strains of M. tuberculosis collected from 30 designated TB treatment institutions in Guangxi during 2017–2018 were selected for whole genome sequencing. Results The consistent rate between the MDR phenotype and the combined mutation of genes was 71.43%. The single gene mutation rate and combined gene mutation rate were higher in the MDR strains than in the fully sensitive strains (χ²=5.753, P=0.016; χ²=284.034, P<0.001). The single gene mutation rates of katG and rpoB were higher in the MDR strains than in the fully sensitive strains (χ²=7.524, P=0.006; χ²=4.353, P=0.037). Combined mutations in katG＋rpoB genes were higher in the MDR strains than in the fully sensitive strains (χ²=279.956, P<0.001). Among the distribution of combined gene mutations, mutations at the katG315＋rpoB450 and katG315＋rpoB445 loci accounted for 40.82% (20/49), and the combined mutation rate of gene loci in both forms was higher in the MDR strains than in the fully sensitive strains (χ²=144.232, P<0.001; χ²=19.014, P<0.001). Conclusion Detection of combined mutations of resistance genes to isoniazid and rifampicin can be used as an important method for MDR screening in Guangxi. Isoniazid and rifampicin resistance gene mutations of M. tuberculosis in Guangxi were mainly caused by mutations at the loci of katG315, rpoB450 and rpoB445, while the gene combined mutations were mainly in the form of katG＋rpoB. Mutations at the katG315＋rpoB450 and katG315＋rpoB445 loci are the main molecular mechanisms of MDR in Guangxi.
- Mycobacteria /
- Tuberculosis /
- Multidrug resistance /
- Gene /
- Combined mutation

HTML全文

表 1 基因测序结果与比例法药敏结果的比较

Table 1. Comparison of gene sequencing results with drug susceptibility results by ratio method

基因测序	比例法药敏（株）		总计（株）
基因测序	耐多药	全敏感	总计（株）
INH＋RFP基因联合突变	35	6	41
未发生联合突变	14	453	467
合计	49	459	508
注：INH. 异烟肼；RFP. 利福平

下载: 导出CSV

表 2 耐多药和全敏感菌株中异烟肼与利福平耐药基因突变分布情况

Table 2. Distribution of mutations between isoniazid and rifampicin resistance genes in MDR and fully sensitive strains

基因	耐多药（n=49）		全敏感（n=459）		χ²值	P值
基因	突变（株）	突变率（%）	突变（株）	突变率（%）	χ²值	P值
单基因突变	7	14.29	22	4.79	5.753	0.016^a
katG	4	8.16	6	1.31	7.524	0.006^a
kasA	0	0.00	3	0.65	0.000	1.000^a
fabG1	0	0.00	8	1.74	0.108	0.743^a
rpoB	3	6.12	5	1.09	4.353	0.037^a
基因联合突变	35	71.43	6	1.31	284.034	<0.001^a
katG＋rpoB	33	67.35	4	0.87	279.956	<0.001^a
inhA＋rpoB	1	2.04	0	0.00	1.872	0.171^a
ahpC＋rpoB	1	2.04	1	0.00	0.543	0.461^a
kasA＋rpoB	0	0.00	1	0.22	−	1.000^b
注：a. 矫正χ²值，b. Fisher精确概率，−. 无数据

下载: 导出CSV

表 3 异烟肼与利福平耐药基因联合突变位点分布

Table 3. Distribution of combined mutation sites of isoniazid and rifampicin resistance genes

基因	位点	突变碱基	氨基酸	耐多药（n=49）		全敏感（n=459）		χ²值	P值
基因	位点	突变碱基	氨基酸	菌株数（株）	突变率（%）	菌株数（株）	突变率（%）	χ²值	P值
katG＋rpoB	315＋450	AGC-ACC＋TCG-TTG	丝氨酸-苏氨酸＋丝氨酸-亮氨酸	16	32.65	0	0.00	144.232	0.000^a
katG＋rpoB	315＋445	AGC-ACC＋CAC-TAC	丝氨酸-苏氨酸＋组氨酸-酪氨酸	4	8.16	0	0.00	19.014	0.000^a
katG＋rpoB	315＋450	AGC-AAC＋TCG-TTG	丝氨酸-天冬酰胺＋丝氨酸-亮氨酸	1	2.04	0	0.00	−	0.171^b
katG＋rpoB	315＋452	AGC-ACC＋CTG-CCG	丝氨酸-苏氨酸＋亮氨酸-脯氨酸	1	2.04	0	0.00	−	0.171^b
katG＋rpoB	315＋445	AGC-ACC＋CAC-AAC	丝氨酸-苏氨酸＋组氨酸-天冬酰胺	1	2.04	0	0.00	−	0.171^b
katG＋rpoB	315＋445	AGC-ACC＋CAC-CCC	丝氨酸-苏氨酸＋组氨酸-脯氨酸	1	2.04	0	0.00	−	0.171^b
katG＋rpoB	315＋445	AGC-AAC＋CAC-GAC	丝氨酸-苏氨酸＋组氨酸-天冬氨酸	1	2.04	0	0.00	−	0.171^b
katG＋rpoB	315＋435	AGC-AAC＋CAC-CTC	丝氨酸-天冬酰胺＋天冬氨酸-缬氨酸	1	2.04	0	0.00	−	0.171^b
katG＋rpoB	315＋444	AGC-ACC＋ACC-ATC	丝氨酸-苏氨酸＋苏氨酸-异亮氨酸	1	2.04	0	0.00	−	0.171^b
katG＋rpoB	315＋437	AGC-ACC＋AAC-GAC	丝氨酸-苏氨酸＋天冬酰胺-天冬氨酸	1	2.04	0	0.00	−	0.171^b
katG＋rpoB	315＋435	AGC-AAC＋GAC-GTC	丝氨酸-天冬酰胺＋天冬氨酸-缬氨酸	1	2.04	0	0.00	−	0.171^b
katG＋rpoB	315＋435	AGC-ACC＋GAC-GTC	丝氨酸-苏氨酸＋天冬氨酸-缬氨酸	1	2.04	0	0.00	−	0.171^b
katG＋rpoB	315＋430	AGC-ACC＋CTG-CCG	丝氨酸-苏氨酸＋亮氨酸-脯氨酸	1	2.04	0	0.00	−	0.171^b
katG＋rpoB	315＋400	AGC-ACC＋ACC-GCC	丝氨酸-苏氨酸＋苏氨酸-丙氨酸	1	2.04	0	0.00	−	0.171^b
katG＋rpoB	461＋430	CAG-CCG＋CTG-CCG	谷氨酰胺-脯氨酸＋亮氨酸-脯氨酸	1	2.04	0	0.00	−	0.171^b
inhA＋rpoB	194＋450	ATG-ACC＋TCG-TTG	蛋氨酸-苏氨酸＋丝氨酸-亮氨酸	1	2.04	0	0.00	−	0.171^b
ahpC＋rpoB	-81＋450	C-T＋TCG-TTG	−＋丝氨酸-亮氨酸	1	2.04	1	0.22	0.543	0.461^a
katG＋rpoB	315＋445	AGC-ACC＋CAC-AAC	丝氨酸-苏氨酸＋组氨酸-天冬酰胺	0	0.00	1	0.22	−	0.904^b
katG＋rpoB	315＋445	AGC-ACC＋CAC-CGC	丝氨酸-苏氨酸＋组氨酸-精氨酸	0	0.00	1	0.22	−	0.904^b
katG＋rpoB	315＋435	AGC-AAC＋GAC-GGC	丝氨酸-天冬酰胺＋天冬氨酸-甘氨酸	0	0.00	1	0.22	−	0.904^b
katG＋rpoB	335＋431	ATC-ACC＋AGC-AGG	异亮氨酸-苏氨酸＋丝氨酸-精氨酸	0	0.00	1	0.22	−	0.904^b
katG＋rpoB	315＋445	AGC-ACC＋CAC-CGC	丝氨酸-苏氨酸＋组氨酸-精氨酸	0	0.00	1	0.22	−	0.904^b
kasA＋rpoB	121＋431	AGG-AAG＋AGC-AGG	精氨酸-赖氨酸＋丝氨酸-精氨酸	0	0.00	1	0.22	−	0.904^b
合计	−	−	−	35	−	6	−	−	−
注：a. 矫正χ²值，b. Fisher精确概率，−. 无数据

下载: 导出CSV

参考文献(26)

[1]	World Health Organization. Global tuberculosis report 2020[R]. Geneva: World Health Organization, 2020.
[2]	孔伟伟, 邢应如, 胡万发, 等. 耐药结核分枝杆菌与耐药基因突变的相关性分析[J]. 安徽医科大学学报,2019,54(2):329–332. DOI:10.19405/j.cnki.issn1000−1492.2019.02.035. Kong WW, Xing YR, Hu WF, et al. Correlation analysis between drug-resistant Mycobacterium tuberculosis and drug-resistant gene mutations[J]. Acta Univ Med Anhui, 2019, 54(2): 329–332. DOI: 10.19405/j.cnki.issn1000−1492.2019.02.035.
[3]	de Almeida IN, da Silva Carvalho W, Rossetti ML, et al. Evaluation of six different DNA extraction methods for detection of Mycobacterium tuberculosis by means of PCR-IS6110: preliminary study[J]. BMC Res Notes, 2013, 6: 561. DOI: 10.1186/1756−0500−6−561.
[4]	World Health Organization. WHO consolidated guidelines on drug-resistant tuberculosis treatment[M]. Geneva: World Health Organization, 2019.
[5]	Raja S, Prakash CR. Novel 1-(4-substituted benzylidene)-4-(1-(substituted methyl)-2, 3-dioxoindolin-5-yl)semicarbazide derivatives for use against Mycobacterium tuberculosis H37Rv (ATCC 27294) and MDR-TB strain[J]. Arch Pharm Res, 2013, 36(4): 411–422. DOI: 10.1007/s12272−013−0062−1.
[6]	Wright A, Zignol M, Van Deun A, et al. Epidemiology of antituberculosis drug resistance 2002-07: an updated analysis of the Global Project on Anti-Tuberculosis Drug Resistance Surveillance[J]. Lancet, 2009, 373(9678): 1861–1873. DOI: 10.1016/S0140−6736(09)60331−7.
[7]	Zignol M, Hosseini MS, Wright A, et al. Global incidence of multidrug-resistant tuberculosis[J]. J Infect Dis, 2006, 194(4): 479–485. DOI: 10.1086/505877.
[8]	李国利, 张灵霞, 王倩, 等. 结核分枝杆菌临床分离株利福平耐药表型及rpoB基因型分析[J]. 临床和实验医学杂志,2011,10(21):1649–1652. DOI:10.3969/j.issn.1671−4695.2011.21.001. Li GL, Zhang LX, Wang Q, et al. Analysis of rifampin-resistant phenotype and rpoB genetype in clinical Mycobacterium tuberculosis isolates[J]. J Clin Exp Med, 2011, 10(21): 1649–1652. DOI: 10.3969/j.issn.1671−4695.2011.21.001.
[9]	Seifert M, Catanzaro D, Catanzaro A, et al. Genetic mutations associated with isoniazid resistance in Mycobacterium tuberculosis: a systematic review[J]. PLoS One, 2015, 10(3): e0119628. DOI: 10.1371/journal.pone.0119628.
[10]	Martínez LMW, Castro GP, Guerrero MI. A molecular platform for the diagnosis of multidrug-resistant and pre-extensively drug-resistant tuberculosis based on single nucleotide polymorphism mutations present in Colombian isolates of Mycobacterium tuberculosis[J]. Mem Inst Oswaldo Cruz, 2016, 111(2): 93–100. DOI: 10.1590/0074−02760150306.
[11]	Ocheretina O, Escuyer VE, Mabou MM, et al. Correlation between genotypic and phenotypic testing for resistance to rifampin in Mycobacterium tuberculosis clinical isolates in Haiti: investigation of cases with discrepant susceptibility results[J]. PLoS One, 2014, 9(3): e90569. DOI: 10.1371/journal.pone.0090569.
[12]	Thirumurugan R, Kathirvel M, Vallayyachari K, et al. Molecular analysis of rpoB gene mutations in rifampicin resistant Mycobacterium tuberculosis isolates by multiple allele specific polymerase chain reaction in Puducherry, South India[J]. J Infect Public Health, 2015, 8(6): 619–625. DOI: 10.1016/j.jiph.2015.05.003.
[13]	Mekonnen D, Admassu A, Mulu W, et al. Multidrug-resistant and heteroresistant Mycobacterium tuberculosis and associated gene mutations in Ethiopia[J]. Int J Infect Dis, 2015, 39: 34–38. DOI: 10.1016/j.ijid.2015.06.013.
[14]	杨建东, 陈阳贵, 马丽, 等. 乌鲁木齐市耐多药结核分枝杆菌基因突变特征分析[J]. 中华疾病控制杂志,2017,21(1):22–25. DOI: 10.16462/j.cnki.zhjbkz.2017.01.005. Yang JD, Chen YG, Ma L, et al. Analysis on gene mutation characteristics of multi-drug resistant Mycobacterium tuberculosis in Urumqi city[J]. Chin J Dis Control Prev, 2017, 21(1): 22–25. DOI: 10.16462/j.cnki.zhjbkz.2017.01.005.
[15]	Zhang Y, Heym B, Allen B, et al. The catalase-peroxidase gene and isoniazid resistance of Mycobacterium tuberculosis[J]. Nature, 1992, 358(6387): 591–593. DOI: 10.1038/358591a0.
[16]	王希江, 谭云洪, 贺文从, 等. 结核分枝杆菌中利福平和异烟肼耐药相关基因突变与耐药水平的相关性研究[J]. 中国防痨杂志,2021,43(3):248–254. DOI:10.3969/j.issn.1000−6621.2021.03.010. Wang XJ, Tan YH, He WC, et al. The correlation between rifampicin and isoniazid resistance-related gene mutations and resistance level in Mycobacterium tuberculosis[J]. Chin J Antituberc, 2021, 43(3): 248–254. DOI: 10.3969/j.issn.1000−6621.2021.03.010.
[17]	张满娥, 黄文滨, 卢志华, 等. 福建省龙岩市411例肺结核患者对利福平和异烟肼耐药状况的研究[J]. 中国防痨杂志,2020,42(1):54–59. DOI:10.3969/j.issn.1000−6621.2020.01.013. Zhang ME, Huang WB, Lu ZH, et al. Study on resistance to rifampicin and isoniazid in 411cases of pulmonary tuberculosis in Longyan city, Fujian province[J]. Chin J Antituberc, 2020, 42(1): 54–59. DOI: 10.3969/j.issn.1000−6621.2020.01.013.
[18]	戚应杰, 刘婷, 查晓丹, 等. 合肥地区耐多药结核分枝杆菌异烟肼、链霉素及乙胺丁醇耐药相关基因位点表达研究[J]. 国际检验医学杂志,2019,40(14):1713–1716. DOI:10.3969/j.issn.1673−4130.2019.14.012. Qi YJ, Liu T, Cha XD, et al. Expression studies of drug-resistence related genes loci of isoniazid, streptomycin and ethambutol in multi-drug resistant Mycobacterium tuberculosis in Hefei area[J]. Int J Lab Med, 2019, 40(14): 1713–1716. DOI: 10.3969/j.issn.1673−4130.2019.14.012.
[19]	张炜煜, 杨修军, 王慧, 等. 吉林省耐多药结核分枝杆菌rpoB及katG基因突变特征分析[J]. 中国实验诊断学,2017,21(10):1731–1735. DOI:10.3969/j.issn.1007−4287.2017.10.016. Zhang WY, Yang XJ, Wang H, et al. Mutations of rpoB and katG drug genes in multidrug-resistant Mycobacterium tuberculosis isolates from Jilin province[J]. Chin J Lab Diagn, 2017, 21(10): 1731–1735. DOI: 10.3969/j.issn.1007−4287.2017.10.016.
[20]	龙茜. 广西边境与非边境地区结核分枝杆菌耐药及基因型分析[D]. 南宁: 广西医科大学, 2019. Long X. Drug resistance and genotype analysis of Mycobacterium tuberculosis in borderand non-border areas of Guangxi[D]. Nanjing: Guangxi Medical University, 2019.
[21]	赛依旦·吐尔逊, 刘金宝, 阿尔泰, 等. 2013年新疆436例不同民族住院结核病患者的耐药情况分析[J]. 中华疾病控制杂志,2015,19(7):675–678. DOI: 10.16462/j.cnki.zhjbkz.2015.07.008. Talson S, Liu JB, A ET, et al. Analysis of drug-resistance of hospitalized tuberculosis patients in 436 different nationalities of Xinjiang in 2013[J]. Chin J Dis Control Prev, 2015, 19(7): 675–678. DOI: 10.16462/j.cnki.zhjbkz.2015.07.008.
[22]	Sun HH, Zhang CC, Xiang L, et al. Characterization of mutations in streptomycin-resistant Mycobacterium tuberculosis isolates in Sichuan, China and the association between Beijing-lineage and dual-mutation in gidB[J]. Tuberculosis (Edinb) , 2016, 96: 102–106. DOI: 10.1016/j.tube.2015.09.004.
[23]	曹翌明, 李丽, 孟繁荣, 等. 中国结核杆菌利福平耐药株rpoB基因531、526、516位点碱基突变特点[J]. 现代医院,2015,15(7):7–9,12. DOI:10.3969/j.issn.1671−332X.2015.07.003. Cao YM, Li L, Meng FR, et al. Base mutation characteristics in the points of 531, 526 and 516 of rpoB gene of rifampicin-resistant tuberculosis bacili strains[J]. Mod Hosp, 2015, 15(7): 7–9,12. DOI: 10.3969/j.issn.1671−332X.2015.07.003.
[24]	胡族琼, 刘燕文, 周文, 等. 结核分枝杆菌rpoB基因突变特征与利福平耐药水平关系的研究[J]. 中国人兽共患病学报,2016,32(1):39–44,50. DOI:10.3969/j.issn.1002−2694.2016.01.009. Hu ZQ, Liu YW, Zhou W, et al. Relationship between rpoB mutations and the levels of rifampicin resistance in M. tuberculosis[J]. Chin J Zoon, 2016, 32(1): 39–44,50. DOI: 10.3969/j.issn.1002−2694.2016.01.009.
[25]	郑丹薇, 石洁, 苏茹月, 等. 结核分枝杆菌rpoB基因突变和利福平耐药水平相关研究[J]. 河南预防医学杂志,2020,31(11):805–806,818. DOI:10.13515/j.cnki.hnjpm.1006−8414.2020.11.002. Zheng DW, Shi J, Su RY, et al. Relationship between rpoB mutations and the levels of rifampicin resistance in M. tuberculosis[J]. Henan J Prev Med, 2020, 31(11): 805–806,818. DOI: 10.13515/j.cnki.hnjpm.1006−8414.2020.11.002.
[26]	徐峰, 饶跃峰, 张幸国, 等. 宁波地区结核分枝杆菌基因突变位点确证及与异烟肼、利福平耐药关系研究[J]. 中国现代应用药学,2020,37(20):2511–2515. DOI:10.13748/j.cnki.issn1007−7693.2020.20.014. Xu F, Rao YF, Zhang XG, et al. Identification of gene mutation sites of Mycobacterium tuberculosis and the relationship between isoniazid and rifampicin resistance in Ningbo[J]. Chin J Mod Appl Pharm, 2020, 37(20): 2511–2515. DOI: 10.13748/j.cnki.issn1007−7693.2020.20.014.