rpoB公司
乙胺丁醇
英哈
结核分枝杆菌
生物
吡嗪酰胺
异烟肼
抗药性
微生物学
利福平
肺结核
病毒学
多重耐药
遗传学
抗生素
医学
病理
作者
Jun‐ichiro Sekiguchi,Tohru Miyoshi‐Akiyama,Ewa Augustynowicz–Kopeć,Zofia Zwolska,Fumiko Kirikae,Emiko Toyota,Ichiro Kobayashi,Koji Morita,Kohsuke Kudo,Seiya Kato,Tadatoshi Kuratsuji,Toru Mori,Teruo Kirikae
摘要
ABSTRACT We developed a DNA sequencing-based method to detect mutations in the genome of drug-resistant Mycobacterium tuberculosis . Drug resistance in M. tuberculosis is caused by mutations in restricted regions of the genome. Eight genome regions associated with drug resistance, including rpoB for rifampin (RIF), katG and the mabA ( fabG1 )- inhA promoter for isoniazid (INH), embB for ethambutol (EMB), pncA for pyrazinamide (PZA), rpsL and rrs for streptomycin (STR), and gyrA for levofloxacin, were amplified simultaneously by PCR, and the DNA sequences were determined. It took 6.5 h to complete all procedures. Among the 138 clinical isolates tested, 55 were resistant to at least one drug. Thirty-four of 38 INH-resistant isolates (89.5%), 28 of 28 RIF-resistant isolates (100%), 15 of 18 EMB-resistant isolates (83.3%), 18 of 30 STR-resistant isolates (60%), and 17 of 17 PZA-resistant isolates (100%) had mutations related to specific drug resistance. Eighteen of these mutations had not been reported previously. These novel mutations include one in rpoB , eight in katG , one in the mabA-inhA regulatory region, two in embB , five in pncA , and one in rrs. Escherichia coli isolates expressing individually five of the eight katG mutations showed loss of catalase and INH oxidation activities, and isolates carrying any of the five pncA mutations showed no pyrazinamidase activity, indicating that these mutations are associated with INH and PZA resistance, respectively. Our sequencing-based method was also useful for testing sputa from tuberculosis patients and for screening of mutations in Mycobacterium bovis . In conclusion, our new method is useful for rapid detection of multiple-drug-resistant M. tuberculosis and for identifying novel mutations in drug-resistant M. tuberculosis .
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