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

Identification of Gene Mutation Sites of Mycobacterium Tuberculosis and the Relationship Between Isoniazid and Rifampicin Resistance in Ningbo

关键词:  结核分枝杆菌  基因  突变位点  耐药机制

Key words:  Mycobacterium tuberculosis  gene  mutation site  drug resistance mechanism

摘要:

目的 阐明宁波地区初治肺结核患者结核分枝杆菌rpoB、katG、inhA基因突变特征，深入分析基因突变与异烟肼、利福平耐药关系，为临床抗结核治疗提供科学依据。方法 采用1%比例法对2 455例结核分枝杆菌临床分离株进行药敏检测，对其中的耐药菌株和部分全敏菌株提取DNA，PCR扩增，对耐药菌株katG、inhA、rpoB基因测序，分析3种基因特征及基因突变与单耐药、耐多药关联性。结果 2 455例结核分枝杆菌临床分离株中，发现108例耐药结核分枝杆菌，其中单耐药75例（单耐异烟肼51例，利福平24例），单耐药率为3.05%；33例耐多药，耐多药率为1.34%。33例耐多药菌株中，5例发生inhA基因错义突变，突变率为15.15%（5/33），rpoB基因450位点（ser450leu）突变率为51.52%（17/33），katG基因315位点（ser315thr）突变率为87.88%（29/33），463位点（arg463leu）突变率为78.79%（26/33）。24例单耐利福平菌株中有17例发生rpoB基因错义突变，突变率为70.83%（17/24），主要在446位点（lys446lysarg）发生，该位点突变率为45.83%（11/24）。51例单耐异烟肼菌株中有45例发生katG基因错义突变，突变率为88.23%（45/51），315位点（ser315thr）突变率为72.55%（37/51），463位点（arg463leu）突变率为64.71%（33/51）；22例发生inhA基因错义突变，突变率为43.14%（22/51），145位点（val145valgly）突变率为27.45%（14/51）；5例发生rpoB基因错义突变，突变率为9.80%（5/51），位点较分散。另外，108例耐药菌株中，有11例菌株发生rpoB、katG、inhA多基因联合突变，且在大多数位点发生错义突变，突变率为10.18%（11/108）。结论 rpoB、katG、inhA基因在450（ser450leu）、315（ser315thr）、446（lys446lysarg）、463（arg463leu）、145（val145valgly）等位点突变和宁波区结核分枝杆菌对异烟肼、利福平耐药密切相关，其中rpoB、inhA基因的主要突变位点和突变频率存在明显地区差异。

Abstract:

OBJECTIVE To elucidate the mutation characteristics of rpoB, katG and inhA genes of Mycobacterium tuberculosis in newly treated pulmonary tuberculosis patients in Ningbo, and to analyze the relationship between gene mutation and drug resistance to isoniazid and rifampicin, so as to provide scientific basis for clinical anti-tuberculosis treatment. METHODS Drug susceptibility of 2 455 clinical isolates of Mycobacterium tuberculosis was detected by 1% ratio method. DNA was extracted from drug-resistant strains and amplified by PCR. The katG, inhA and rpoB genes of drug-resistant strains were sequenced. The three gene characteristics and the association of gene mutations with single drug resistance and multi-drug resistance were analyzed. RESULTS Among 2 455 clinical isolates of Mycobacterium tuberculosis, 108 cases were found to be resistant to Mycobacterium tuberculosis, including 75 cases of single drug resistance (inh 51 cases, rfp 24 cases), the single drug resistance rate was 3.05%; 33 cases of multi-drug resistance, the multi-drug resistance rate was 1.34%. Among the 33 multi-drug resistance strains, 5 of them had mutations of inhA gene, the mutation rate was 15.15%(5/33). The mutation rate of rpoB gene at 450 locus(ser450leu) was 51.52%(17/33), the mutation rate of katG gene 315 locus(ser315thr) was 87.88%(29/33), the mutation rate of 463 locus(arg463leu) was 78.79%(26/33). Among the 24 rifampicin resistant strains, 17 of them had mutations in rpoB gene, the mutation rate was 70.83%(17/24), mainly at the 446 locus(lys446lysarg), the mutation rate was 45.83%(11/24). Among 51 isoniazid-resistant strains, 45 cases had missense mutation of katG gene, the mutation rate was 88.23%(45/51), the mutation rate of 315 locus(ser315thr) was 72.55%(37/51), the mutation rate of 463 locus(arg463leu) was 64.71%(33/51), 22 cases had inhA gene missense mutation, the mutation rate was 43.14%(22/51), the mutation rate of 145 locus(val145valgly) was 27.45%(14/51), 5 cases had rpoB gene missense, the mutation rate was 9.80%(5/51). In addition, 11 of 108 drug-resistant strains had combined mutations of rpoB, katG and inhA genes, and missense mutations occurred at most sites, the mutation rate was 10.18%(11/108). CONCLUSION The mutations of rpoB, katG and inhA genes at 450 locus(ser450leu), 315 locus(ser315thr), 446 locus(lys446lysarg), 463 locus(arg463leu), 145 locus(val145valgly) are closely related to the resistance of Mycobacterium tuberculosis to isoniazid and rifampicin in Ningbo. The main mutation sites and mutation frequencies of rpoB and inhA genes are significantly different.