利福霉素耐药结核分枝杆菌rpoB突变与利福布丁耐药水平的关系

Relationship between the mutations in rpoB gene and the level of rifabutin resistance of rifamycin-resistant Mycobacterium tuberculosis

目的研究利福霉素耐药结核分枝杆菌rpoB基因突变与利福布丁耐药水平的相关性。方法倍比稀释法测定64株利福霉素耐药及6株敏感菌株对利福布丁的最低抑菌浓度(MIC),并分析其对异烟肼的耐药情况。同时对rpoB全基因扩增后测序,分析rpoB突变位点和突变性质与利福布丁MICs高低及多重耐药的关系。结果 6株敏感株rpoB未突变,MICs为0.25～0.50 mg/L。64株耐药株rpoB突变率为100%。37株利福布丁高度耐药(MICs≥4 mg/L)株中,S531L突变27株,H526R突变和Y389C突变各2株,S531W、H526Y、Q513K、V176F、D516Y联合Q253R突变与D516G联合L511P突变各1株。17株中度耐药(MICs 2～4 mg/L)株中,S531L突变16株,D516G联合L511P和S509R突变1株。10株低度耐药(MICs 0.25～1 ng/L)株中,L533P、H526L、H526S、D516V、D516Y单点突变各2株。93.75%(60/64)的利福霉素耐药株对异烟肼耐药。结论检测rpoB突变即可初步筛选多重耐药结核分枝杆菌;中、高水平利福布丁耐药株以S531L突变占绝对优势,rpoB突变位点及突变类型与利福布丁耐药水平有一定相关性。

Objective To study the relationship between the mutations in rpoB gene of rifamycin-resistant Mycobacterium tuberculosis （M. tuberculosis） and the level of rifabutin resistance. Methods The minimal inhibition concentrations（MICs） of 64 rifamycin-resistant isolates and 6 sensitive isolates of M. tuberculosis to rifabutin were determined by the dilution method, their isonicotinyl hydrazide （INH） resistance were analyzed. The whole rpoB gene were amplified and sequenced, then the relationship between the mutations in rpoB gene and the level of rifabutin resistance and multidrug resistance were analyzed. Results The sensitive isolates had no mutations in rpoB gene and their MICs were 0. 25 mg/L - 0. 50 mg/L. All rifamycin-resistant isolates had mutations in rpoB gene. Among 37 high-level rifabutin-resistant strains （MICs≥4 rag/L）, mutations of S531L, H526R, and Y389C were found in 27, 2 and 2 isolates respectively, co-mutation of S531W, H526Y, QS13K, V176F, DS16Y and Q253R was in 1 strain, co-mutation of D516G and L511P was in 1 strain. Of 17 intermediated-level rifabutin-resistant strains （MICs 2 - 4 mg/L）, 16 isolates were with S531L mutation and 1 with D516G combined with L511P and S509R mutation, respec- tively. Of 10 low-level rifabutin-resistant strains （MICs 0. 25 - 1 mg/L）, single mutation of L533P, H526L, H526S, D516V, and D516Y were found in 2 isolates respectively. 93. 75% （60/64） of rifamycin-resistant isolates were also resistant to INH. Conclusion Detection of rpoB mutation can preliminarily screen multidrug-resistant M. tuberculosis; The predominant mutations of rpoB gene in high-and middle-level rifabutin-resistant isolates are S531L, the mutation positions and types of rpoB gene in M. tuberculosis correlates with the level of rifabutin resistance.