DETECTION OF KANAMYCIN-RESISTANT MYCOBACTERIUM-TUBERCULOSIS BY IDENTIFYING MUTATIONS IN THE 16S RIBOSOMAL-RNA GENE

In Mycobacterium smegmatis and a limited number of Mycobacterium tuber culosis strains, the involvement of alterations of the 16S rRNA gene ( rrs) in resistance to kanamycin has been shown. To investigate the ext ent to which mutations in a specific region of the rrs gene and the ka namycin-resistant phenotype in clinically isolated M. tuberculosis str ains were correlated, 43 kanamycin-resistant strains (MICs, greater th an or equal to 200 mu g/ml), 71 kanamycin-susceptible strains, and 4 t ype strains were examined. The 300-bp DNA fragments carrying the rrs g ene and the intervening sequence between the rrs gene and 23S rRNA (rr l) gene fragments were amplified by PCR and were subjected to PCR-base d direct sequencing. By comparing the nucleotide sequences, substituti ons were found in 29 of 43 (67.4%) kanamycin-resistant clinical isolat es at positions 1400, 1401, and 1483 but in none of the 71 sensitive i solates or the 4 type strains. The most frequent substitution, from A to G, occurred at position 1400. A substitution from C to T at positio n 1401 as found once. Two clinical isolates carried the double mutatio n from C to A at position 1401 and from G to T at position 1483. In ad dition, we found that these mutants can be distinguished from wild-typ e strains by digestion with the restriction endonucleases TaiI and Tsp 45I. Furthermore, we found that the genotypes of kanamycin-resistant s trains can be discriminated from each other by digestion with a restri ction endonuclease, BstUI or DdeI.