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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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