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Genetic and Phenotypic Studies to Characterize Clonal Diversity of Mycobacterium tuberculosis and to Address the Prevalence of Multidrug-resistant Isolates

表征结核分枝杆菌克隆多样性并解决多重耐药菌株流行问题的遗传和表型研究

基本信息

批准号：
16590461
负责人：
YAMANE Nobuhisa
金额：
$ 2.3万
依托单位：
University of the Ryukyus
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2004
资助国家：
日本
起止时间：
2004 至 2005
项目状态：
已结题

项目摘要

Genetic characterization of drug-resistant isolates of Mycobacterium tuberculosis was performed. Pyrazinamide (PZA)-resistant isolates of M.tuberculosis were genetically analyzed, the results indicating significant correlation between PZA-susceptibility, pyrazinamidase (PZase) activity and mutations on pncA gene. However, there found PZase-positive but PZA-resistant isolates and a PZase-negative isolate with no mutation on pncA. Also, an interpretive breakpoint of minimum inhibitory concentration (MIC) for rifampicin (RFP) was established. All the isolates with less than 0.06μg/ml of MICs against RFP were found to belong to wild strain with no mutation on rpoB gene.Synergy when three antimicrobial agents were combined against multidrug-resistant M.tuberculosts (MDR-TB) was determined by using three-dimensional microdilution chequerboard assay. Of 28 antimicrobial agents tested combined with RFP and isoniazide (INH), nine agents, including non-antituberculosis agents such as fluoroquinolones and clarithromycin, were significantly synergistic. The synergy of the combinations against MDR-TB was confined by the standard time-kill assay.Clonal diversity of M.tuberculosis in a single clinical specimen was analyzed. After plating clinical sputum specimens onto Middlebrook 7H11 agar plates, well-separated individual colonies were determined for antimycobacterial susceptibilities and DNA fingerprinting using IS6110 insertions. The results indicated that, in some patients of tuberculosis including the newly diagnosed, the isolates of M.tuberculosis revealed clonal diversity in phenotypic and genotypic characterizations, and that polyclonal infection with M.tuberculosis in patients not previously treated is often the case.

对耐药结核分枝杆菌分离株进行遗传鉴定。对耐药结核分枝杆菌PZA （Pyrazinamide， PZA）分离株进行遗传分析，结果表明PZA敏感性、pzinamidase （pyrazinamidase, PZase）活性与pncA基因突变有显著相关性。然而，有pzase阳性但耐药的分离株和pncA无突变的pzase阴性分离株。此外，还建立了利福平（RFP）最小抑制浓度（MIC）的解释断点。对RFP mic < 0.06μg/ml的分离株均为野生菌株，rpoB基因未发生突变。采用三维微量稀释棋盘法测定了3种抗菌药物联合抗多药结核分枝杆菌（MDR-TB）的协同作用。在与RFP和异烟肼（INH）联合使用的28种抗菌药物中，包括氟喹诺酮类药物和克拉霉素等非抗结核药物在内的9种药物具有显著的协同作用。联合治疗耐多药结核病的协同作用受到标准时间杀伤试验的限制。分析了单个临床标本中结核分枝杆菌的克隆多样性。将临床痰标本置于Middlebrook 7H11琼脂板上，利用IS6110插入检测分离良好的单个菌落的抗真菌敏感性和DNA指纹图谱。结果表明，在包括新诊断结核病患者在内的部分结核病患者中，结核分枝杆菌在表型和基因型特征上表现出克隆多样性，未接受治疗的结核分枝杆菌多克隆感染较为常见。