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Journal of Clinical Microbiology, January 2007, p. 179-192, Vol. 45, No. 1
0095-1137/07/$08.00+0     doi:10.1128/JCM.00750-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Detection of Multidrug Resistance in Mycobacterium tuberculosis{triangledown}

Jun-ichiro Sekiguchi,1 Tohru Miyoshi-Akiyama,1 Ewa Augustynowicz-Kopec,2 Zofia Zwolska,2 Fumiko Kirikae,1 Emiko Toyota,1 Intetsu Kobayashi,3 Koji Morita,4 Koichiro Kudo,1 Seiya Kato,5 Tadatoshi Kuratsuji,1,6 Toru Mori,5,7 and Teruo Kirikae1*

International Medical Center of Japan, 1-21-1 Toyama, Shinjuku, Tokyo 162-8655, Japan,1 National Research Institute of Tuberculosis and Lung Diseases, Plocka St. 26, Warsaw 01-138, Poland,2 Mitsubishi Kagaku Bio-Clinical Laboratories, Inc., 3-30-1 Shimura, Itabashi, Tokyo174-8555, Japan,3 Department of Microbiology, Kyorin University School of Health Sciences, 476 Miyashita, Hachioji, Tokyo 192-8508, Japan,4 Research Institute of Tuberculosis, Japan Anti-Tuberculosis Association, Matsuyama 3-1-24, Kiyose, Tokyo 204-8533, Japan,5 National Research Institute for Child Health and Development, Setagaya, Tokyo 157-8535, Japan,6 Leprosy Research Center, National Institute of Infectious Diseases, 4-2-1 Aoba-cho, Higashimurayama, Tokyo 189-0002, Japan7

Received 8 April 2006/ Returned for modification 3 July 2006/ Accepted 9 October 2006

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.

* Corresponding author. Mailing address: Department of Infectious Diseases, Research Institute, International Medical Center of Japan, 1-21-1 Toyama, Shinjuku, Tokyo 162-8655, Japan. Phone: (81) 3 3202 7181, ext. 2838. Fax: (81) 3 3202 7364. E-mail: tkirikae{at}ri.imcj.go.jp.

{triangledown} Published ahead of print on 15 November 2006.

Journal of Clinical Microbiology, January 2007, p. 179-192, Vol. 45, No. 1
0095-1137/07/$08.00+0     doi:10.1128/JCM.00750-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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