结核分枝杆菌乙胺丁醇高水平耐药的分子机制研究

To understand the resistance mechanism of Mycobacterium tuberculosis (MTB) is critical for the success of drug-resistant tuberculosis control and the development of new drugs. Until now, most resistance mechanisms of first-line antituberculosis drugs are fully understood. However, embB306, which is related to ethambutol (EMB) resistance, was found to occur in EMB susceptible clinical strains. And embB306 mutants are also associated with multiple antituberculosis drug resistance. Subsequent studies found that embB306 mutants alone can only lead to low-level resistance to EMB, but not sufficient for high-level resistance. Therefore, in this study we will firstly analyze the drug resistance associated mutations in clinical strains with different resistance level to EMB, and find the possible reasons which can cause high-level resistance to EMB. Then the recombinant strains (based on standard strain H37Rv) containing various mutations related to different drug resistance will be constructed and the minimum inhibitory concentration (MIC) to EMB will be detected. Secondly, we will analyze the difference of genetic background in clinical strains with different resistance level to EMB. Then the recombinant strains (based on Beijing genotype strain and non-Beijing genotype strain, separately) containing various mutations related to different drug resistance will be constructed and the MIC to EMB will be detected. Our study will make clear how the combined mutations lead to high-level EMB resistance, and whether the genetic background of strain has the effects during the process of EMB high-level resistance.

明确结核分枝杆菌（MTB）耐药产生机制对控制耐药结核病的流行和新药研发极为重要。目前多数一线抗结核药物的耐药机制已非常明确，但乙胺丁醇（EMB）耐药相关embB306突变却被发现也存在于EMB敏感株中，且与多种抗结核药物耐药相关。随后发现，该位点单独突变仅可引起EMB耐药水平小幅升高，不足以导致高水平耐药。鉴于此，本项目拟通过对MTB分离株EMB耐药水平高低和耐药相关基因突变的关联分析，寻找可能导致EMB高水平耐药的联合作用分子基础；然后以H37Rv为模板构建包含耐药相关基因突变的重组菌株，观察重组菌株对EMB耐药水平的变化，明确耐药相关基因联合突变在EMB高水平耐药产生中的作用；通过对MTB分离株EMB耐药水平高低与菌株遗传背景的关联分析，再分别以非北京基因型菌株和北京基因型菌株为模板构建重组菌株，观察菌株重组前后对EMB耐药水平的变化，明确不同遗传背景在EMB高水平耐药产生中的作用。

背景.乙胺丁醇（ethambutol, EMB）是抗结核治疗的一线药物，近年来EMB耐药率呈现上升趋势，因此明确EMB的耐药分子机制具有非常重要的意义。结核分枝杆菌（Mycobacterium tuberculosis, MTB）对EMB耐药性的产生与阿拉伯糖基转移酶编码基因embCAB操纵子突变有关，其中以embB306突变最常见。但目前认为embB306突变仅能导致EMB低水平耐药，而EMB高水平耐药株的分子机制目前尚不明确。.目的.1、明确我国MTB分离株的EMB耐药水平；.2、明确EMB不同耐药水平菌株的分子特点及高水平耐药的分子机制。.方法.选取304株MTB临床分离株，采用比例法和最小抑菌浓度（minimum inhibitory concentration, MIC）法进行药物敏感性检测，同时进行spoligotyping基因分型和耐药相关基因突变（包括利福平与rpoB，异烟肼与katG，EMB与embB，链霉素与rpsL）检测，进而比较不同EMB耐药水平菌株的特点。.选取EMB不同耐药水平的同源菌株，进行细菌全基因组测序，并采用液相与质谱联用技术（LC-MS）进行代谢产物检测，比较不同耐药水平菌株的基因组和代谢组差异，以明确EMB高水平耐药的分子机制。.结果与结论.1、81.6%（248/304）的入组菌株为EMB低水平耐药菌株（MIC＜8g/mL），提示我国MTB分离株以EMB低水平耐药为主；.2、在EMB耐药菌株中，MDR菌株中发生高水平耐药的风险显著增高（P＜0.001）；且四联耐药菌株的发生高水平耐药的风险高于三联耐药菌株（相对风险值为24和62.4）；.3、embB306突变与EMB耐药性的产生相关（P＜0.001），而与耐药水平无关（P=0.234）；而rpoB、rpsL和embB三个基因联合突变更容易发生EMB高水平耐药（P＜0.05）；.4、北京和非北京基因型菌株中EMB高水平耐药比例无显著差异（P=1.000），提示基因背景对EMB耐药水平影响较小；.5、与EMB低水平耐药菌株相比，高水平耐药菌株中发生了5个基因改变，分别是Rv0532、Rv0747、Rv0833、Rv1883c和Rv3619c，且EMB不同水平耐药菌株的代谢产物存在显著差异。