Characterization of combined mutation of isoniazid and rifampicin resistance genes in Mycobacterium tuberculosis in Guangxi
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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。 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. -
Key words:
- Mycobacteria /
- Tuberculosis /
- Multidrug resistance /
- Gene /
- Combined mutation
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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)耐多药 全敏感 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 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 突变(株) 突变率(%) 突变(株) 突变率(%) 单基因突变 7 14.29 22 4.79 5.753 0.016a katG 4 8.16 6 1.31 7.524 0.006a kasA 0 0.00 3 0.65 0.000 1.000a fabG1 0 0.00 8 1.74 0.108 0.743a rpoB 3 6.12 5 1.09 4.353 0.037a 基因联合突变 35 71.43 6 1.31 284.034 <0.001a katG+rpoB 33 67.35 4 0.87 279.956 <0.001a inhA+rpoB 1 2.04 0 0.00 1.872 0.171a ahpC+rpoB 1 2.04 1 0.00 0.543 0.461a kasA+rpoB 0 0.00 1 0.22 − 1.000b 注:a. 矫正χ2值,b. Fisher精确概率,−. 无数据 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 菌株数(株) 突变率
(%)菌株数(株) 突变率(%) 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 0.000a 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 0.000a 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 − 0.171b 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 − 0.171b 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 − 0.171b 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 − 0.171b 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 − 0.171b 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 − 0.171b 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 − 0.171b 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 − 0.171b 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 − 0.171b 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 − 0.171b 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 − 0.171b 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 − 0.171b 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 − 0.171b 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 − 0.171b ahpC+rpoB -81+450 C-T+TCG-TTG −+丝氨酸-亮氨酸 1 2.04 1 0.22 0.543 0.461a 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 − 0.904b 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 − 0.904b 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 − 0.904b 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 − 0.904b 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 − 0.904b 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 − 0.904b 合计 − − − 35 − 6 − − − 注:a. 矫正χ2值,b. Fisher精确概率,−. 无数据 -
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