体外诱导对氨基水杨酸高浓度耐药结核分枝杆菌及其突变位点研究

Study on high-concentration p-aminosalicylic acid resistant Mycobacterium tuberculosis induced in vitro and the mutation sites

目的:通过对实验室诱导产生的对氨基水杨酸(para-aminosalicylic acid,PAS)耐药结核分枝杆菌(Mycobacterium tuberculosis,MTB)和临床分离株进行全基因组测序,探索PAS的潜在作用机制和耐药性。方法:通过体外药物浓度梯度诱导法,将MTB标准菌株H37Rv诱导成标准耐PAS菌株及高水平耐药菌株,收集并保存每一代诱导菌株。用液体微孔板法检测上述诱导菌株的最低抑菌浓度(minimum inhibitory concentration,MIC)及交叉耐药情况,筛选耐药水平相似的临床分离株。通过对PAS耐药的实验室突变株和临床分离株进行全基因组测序,从基因水平上探索MTB对PAS的耐药机制。结果:本研究在体外建立了耐PAS的MTB耐药动态变化模型。全基因组测序结果显示,PAS耐药可能与3个位点突变直接相关,分别是plcC(Q462R)、folC(S150R)和1个thyA上游位点(3074495G→A)。MTB对高水平的PAS耐药的产生除了基因突变因素以外,尚存在其他的调控机制。对PAS敏感和耐药的MTB临床分离株测序发现某些folC、thyA和thyX突变并非由PAS引起。结论:本研究构建的PAS耐药MTB菌株模型,为PAS耐药结核病研究和临床实践提供了理想的生物模型。全基因组测序分析在DNA水平上阐明了MTB对PAS的耐药机制,尚存在其他的调控机制,仍需进一步研究。所得的与PAS耐药相关的基因突变也需要进一步的实验验证。

Objective:To explore the potential mechanisms of action and resistance to para-aminosalicylic acid(PAS)by sequencing the whole genome(WGS)of lab-induced PAS-resistant Mycobacterium tuberculosis(MTB)and clinical isolates.Methods:The MTB standard strain was induced into standard PAS resistant strain and high-level drug resistant strain by in vitro drug concentration gradient induction method,and the induced strains of each generation were collected and preserved.The minimum inhibitory concentration(MIC)and cross resistance of the above induced strains were detected by liquid microplate method,and clinical isolates with similar drug resistance levels were screened.By sequencing the whole genome of PAS resistant laboratory mutants and clinical isolates,the mechanism of resistance of MTB to PAS was explored at the gene level.Results:A dynamic PAS-resistant model of MTB was established in vitro.The WGS results showed that PAS resistance may be directly related to three site mutations,plcC(Q462R),folC(S150R)and a thyA upstream site(3074495G→A).Except for genetic mutations,there were other regulatory mechanisms for the resistance to high levels PAS of MTB.Moreover,WGS results of PAS-susceptible and resistant clinical isolates revealed that some folC,thyA and thyX mutations were not caused by PAS.Conclusion:The PAS-resistant model of MTB constructed in this study provides an ideal biological model for the research and clinical practice of PAS-resistant tuberculosis.WGS analysis has clarified the resistance mechanism of MTB to PAS at the DNA level,and other regulatory mechanisms still need further investigation.The gene mutations associated with PAS resistance obtained in this study also need further experimental validation.