title: Detection of multidrug resistance in Mycobacterium tuberculosis.
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.
authors.affiliation: International Medical Center of Japan, 1-21-1 Toyama, Shinjuku, Tokyo 162-8655, Japan.
authors.forename: Jun-ichiro
authors.initials: J
authors.lastname: Sekiguchi
authors.forename: Tohru
authors.initials: T
authors.lastname: Miyoshi-Akiyama
authors.forename: Ewa
authors.initials: E
authors.lastname: Augustynowicz-Kopeć
authors.forename: Zofia
authors.initials: Z
authors.lastname: Zwolska
authors.forename: Fumiko
authors.initials: F
authors.lastname: Kirikae
authors.forename: Emiko
authors.initials: E
authors.lastname: Toyota
authors.forename: Intetsu
authors.initials: I
authors.lastname: Kobayashi
authors.forename: Koji
authors.initials: K
authors.lastname: Morita
authors.forename: Koichiro
authors.initials: K
authors.lastname: Kudo
authors.forename: Seiya
authors.initials: S
authors.lastname: Kato
authors.forename: Tadatoshi
authors.initials: T
authors.lastname: Kuratsuji
authors.forename: Toru
authors.initials: T
authors.lastname: Mori
authors.forename: Teruo
authors.initials: T
authors.lastname: Kirikae
mesh_terms: D000995:Antitubercular Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000235:genetics; D004269:DNA, Bacterial / Q000032:analysis; D024901:Drug Resistance, Multiple, Bacterial / Q000235:genetics*; D029968:Escherichia coli Proteins; D006801:Humans; D008826:Microbial Sensitivity Tests; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D000091243:Ribosomal Protein S9; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D013696:Temperature; D018088:Tuberculosis, Multidrug-Resistant / Q000382:microbiology*; D014397:Tuberculosis, Pulmonary / Q000382:microbiology*
chemical_list: D000995:Antitubercular Agents; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D000091243:Ribosomal Protein S9; C000717190:RpsI protein, E coli