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Journal of Clinical Microbiology, May 1998, p. 1220-1225, Vol. 36, No. 5
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Detection of Kanamycin-Resistant Mycobacterium tuberculosis by Identifying Mutations in the 16S rRNA Gene

Yasuhiko Suzuki,1,* Chihiro Katsukawa,2 Aki Tamaru,2 Chiyoji Abe,3 Masanao Makino,4 Yasuo Mizuguchi,5 and Hatsumi Taniguchi6

Department of Pathology1 and Department of Microbiology,2 Osaka Prefectural Institute of Public Health, Nakamichi 1-3-69, Higashinari-ku, Osaka 537, The Research Institute of Tuberculosis, Japan Anti-Tuberculosis Association, 3-1-24 Matsuyama, Kiyose, Tokyo 204,3 National Leprosarium, Oku-Komyo-en, 6253 Mushiake, Okayama 701-45,4 Public Health Laboratory of Chiba Prefecture, 666-2, Chuo-ku, Chiba 260,5 and Department of Microbiology, School of Medicine, University of Occupational and Environmental Health, Iseigaoka, Yahatanishi-ku, Kitakyusyu 807,6 Japan

Received 2 October 1997/Returned for modification 15 December 1997/Accepted 17 February 1998

In Mycobacterium smegmatis and a limited number of Mycobacterium tuberculosis strains, the involvement of alterations of the 16S rRNA gene (rrs) in resistance to kanamycin has been shown. To investigate the extent to which mutations in a specific region of the rrs gene and the kanamycin-resistant phenotype in clinically isolated M. tuberculosis strains were correlated, 43 kanamycin-resistant strains (MICs, >= 200 µg/ml), 71 kanamycin-susceptible strains, and 4 type strains were examined. The 300-bp DNA fragments carrying the rrs gene and the intervening sequence between the rrs gene and 23S rRNA (rrl) gene fragments were amplified by PCR and were subjected to PCR-based direct sequencing. By comparing the nucleotide sequences, substitutions were found in 29 of 43 (67.4%) kanamycin-resistant clinical isolates at positions 1400, 1401, and 1483 but in none of the 71 sensitive isolates or the 4 type strains. The most frequent substitution, from A to G, occurred at position 1400. A substitution from C to T at position 1401 was found once. Two clinical isolates carried the double mutation from C to A at position 1401 and from G to T at position 1483. In addition, we found that these mutants can be distinguished from wild-type strains by digestion with the restriction endonucleases TaiI and Tsp45I. Furthermore, we found that the genotypes of kanamycin-resistant strains can be discriminated from each other by digestion with a restriction endonuclease, BstUI or DdeI.

* Corresponding author. Mailing address: Department of Pathology, Osaka Prefectural Institute of Public Health, Nakamichi 1-3-69, Higashinari-ku, Osaka 537, Japan. Phone: 81-6-972-1321, ext. 268. Fax: 81-6-972-0772. E-mail: suzuki{at}iph.pref.osaka.jp.

Journal of Clinical Microbiology, May 1998, p. 1220-1225, Vol. 36, No. 5
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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