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

Abstract

In Mycobacterium smegmatis and a limited number ofMycobacterium 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 therrs gene and the intervening sequence between therrs 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 andTsp45I. Furthermore, we found that the genotypes of kanamycin-resistant strains can be discriminated from each other by digestion with a restriction endonuclease, BstUI orDdeI.

Directed mutagenesis of Mycobacterium smegmatis 16S rRNA to reconstruct the in vivo evolution of aminoglycoside resistance in Mycobacterium tuberculosis

“…Tobramycin differs from kanamycin A in two substituents (kanamycin A 2′OH, 3′OH; tobramycin 2′NH, 3′H), which, however, do not affect the overall structure of the drug target complex (François et al, 2005). Suzuki et al (1998). b.…”

“…g. Jugheli et al (2009). Clinical drug-resistant strains from Suzuki et al (1998) were isolated from patients receiving kanamycin; clinical drug-resistant strains from Sander et al (1996), Alangaden et al (1998), Krüüner et al (2003) and Maus et al (2005) were isolated from patients receiving kanamycin or amikacin. Information on the patients' drug regimen prior to isolation of the mutant clinical strains is not provided by Feuerriegel et al (2009) Drug resistance and fitness in M. tuberculosis 833 absence of these sequences from prokaryotic or eukaryotic small subunit rRNA To understand the development of aminoglycoside resistance in clinical M. tuberculosis strains, we made use of the above information where the impact on drug susceptibility and fitness cost was determined in M. smegmatis.…”

Usefulness of resistant gene markers for predicting treatment outcome on second-line anti-tuberculosis drugs

“…Although rrs nt1401 mutations had been reported to be mainly associated with in vitro KM resistance (Taniguchi et al 1997;Alangaden et al 1998;Suzuki et al 1998), early evidence (Alangaden et al 1998) showed that 6 of the 12 rrs nt1401 mutants showed CPM resistance. Further study (Maus et al 2005b) demonstrated that all rrs nt1401 mutants (14 strains) tested were resistant to CPM despite having remarkable MIC variation.…”

“…Previous works showed that different gene loci may offer some rapid alternatives for conventional DST (Bodmer et al 1995;Zhang et al 2007). Considerable progress had been achieved on aminoglycosides (Ramaswamy and Musser 1998;Suzuki et al 1998) and fluoroquinolones (Mokrousov et al 2008) where hot-spot mutations on 16S rDNA (rrs) and gyrA gene had been identified and evaluated, respectively. Feuerriegel et al (2009) demonstrated the usefulness of four genes (gyrA, gyrB, rrs and tlyA) for the rapid detection of XDR-TB among strains isolated from patients undergoing MDR-TB treatment.…”

Drug resistance mechanisms and drug susceptibility testing for tuberculosis

“…Meanwhile, uncommon mutations at codon 1484 were also reported to confer resistance in KAN and AMK. 83,84 Mutations at positons −10 and −35 of the promoter region of eis have been also been shown to confer resistance to KAN. 19,85 Mutation at nucleotide position 1401 of rrs is the major molecular mechanism of CAP resistance. 36 rpoA T187P N/A~1.20 36 rpoB S531L (S450L) Competition >1.00 37 rpoB S531W (S450W) Competition 0.67-0.88 37,38 Independent 0.71 38 In macrophage 0.28 38 rpoB H526Y (H445Y) Competition 0.81-0.89 37,38 Independent 0.86 38 In macrophage 0.63 38 rpoB S522L (S441L) Competition 0.54-0.88 38 Independent 0.95 38 In macrophage 0.50 38 rpoB S531L (S450L) Competition 0.91, 0.96, 37,38 rpoB H526D (H445D) Competition 0.78-0.81 37,38 rpoB H526R (H445R) Competition 0.82 37,38 rpoB Q513L (Q432L) Competition 0.83 37,38 rpoB H526P (H445P) Competition 0.84 37 42 for rpoB MTB numbering system reported in parenthesis.…”