2005 Fiscal Year Final Research Report Summary
Genetic and Phenotypic Studies to Characterize Clonal Diversity of Mycobacterium tuberculosis and to Address the Prevalence of Multidrug-resistant Isolates
| Project/Area Number |
16590461
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Laboratory medicine
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| Research Institution | University of the Ryukyus |
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Principal Investigator |
YAMANE Nobuhisa University of the Ryukyus, Faculty of Medicine, Professor, 医学部, 教授 (80125682)
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| Co-Investigator(Kenkyū-buntansha) |
SHIOHIRA Chika M. University of the Ryukyus, Faculty of Medicine, Instructor, 医学部, 助手 (50325833)
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| Project Period (FY) |
2004 – 2005
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| Keywords | M.tuberculosis / drug-resistant mutation / mutidrug-resistant M.tuberculosis / in vitro synergy / synergy of the three-dimensional antimicrobial combination / M.tuberculosis clone / IS6110 restriction fragment length polymorphism / clonal diversity |
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| Research Abstract |
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.
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