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

结核分枝杆菌耐药菌株的遗传特征进行。对结核分枝杆菌吡嗪酰胺（PZA）耐药株进行了遗传学分析，结果表明PZA敏感性、吡嗪酰胺酶（PZase）活性与pncA基因突变显著相关。然而，也发现了PZase阳性但对PZA耐药的分离株和一株PZase阴性但pncA无突变的分离株。建立了利福平最低抑菌浓度（MIC）的判读折点。对RFP MIC <0.06μg/ml的菌株均为rpoB基因未发生突变的野生型菌株。在28种与RFP和异烟肼（INH）联合使用的抗微生物药物中，有9种药物（包括氟喹诺酮类和克拉霉素等非抗结核药物）具有显著的协同作用。通过标准的时间-杀灭试验确定联合用药对耐多药结核病的协同作用。分析单个临床标本中结核分枝杆菌的克隆多样性。将临床痰液标本接种到Middlebrook 7 H11琼脂平板上后，使用IS 6110插入测定分离良好的单个菌落的抗分枝杆菌亲和性和DNA指纹图谱。结果表明，在部分结核病患者（包括新诊断的结核病患者）中，结核分枝杆菌分离株在表型和基因型特征上均表现出克隆性多样性，而在既往未接受过治疗的患者中，结核分枝杆菌多克隆感染的情况较为常见。

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.