Journal List > Tuberc Respir Dis > v.63(2) > 1001122

Lin, Kim, Yun, Park, Kim, Park, and Kook: Mutations of katG and inhA in MDR M. tuberculosis

Abstract

Backgrounds

Mutations of katG and inhA (ORF and promoter) are known to be related to isoniazid (INH) resistance of Mycobacterium tuberculosis. Because reports on these mutations in Korean isolates are limited (i.e. only the frequency of katG codon 463 was evaluated.), we tried to know the kinds of mutations of two genes and their frequencies in INH resistant Korean M. tuberculosis strains.

Methods

PCR was performed to amplify katG (2,223 bp), inhA ORF (-77~897, 975 bp), and inhA promoter (-168~80, 248 bp) from 29 multidrug resistant M. tuberculosis (MDR-TB) DNAs prepared by bead beater-phenol method. Their sequences were determined and analyzed by ABI PRISM 3730 XL Analyzer and MegAlign package program, respectively.

Results

All of the isolates had more than one mutation in katG or inhA gene. Twenty seven (93%) of 29 tested strains had katG mutations, which suggests that katG is a critical gene determining INH resistance of M. tuberculosis. Amino acid substitutions, such as Arg463Leu and Ser315Thr, due to point mutations of the katG were the most frequent (62.1% and 55.2%) mutations. In addition, deletion of the katG gene was frequently observed (17.2%). Analyzed Korean MDR-TB isolates also had variable inhA mutations. Point mutation of inhA promoter region, such as -15 (C→T) was frequently found. Substitution of amino acid (Lsy8Asn) due to point mutation (AAA→AAC) of inhA ORF was found in 1 isolate. Interestingly, 14 point mutated types that were not previously reported were newly found. While four types resulted in amino acid change, the others were silent mutations.

Conclusions

Although it is not clear that the relationship of these newly found mutations with INH resistance, they show marked diversity in Korean MDR-TB strains. It also suggests their feasibility as a molecular target to supplement determining the INH resistance of clinical isolates because of the possible existence of low-level INH resistant strains.

Figures and Tables

Figure 1
Location of the primers on the katG gene
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Figure 2
Location of the primers on the inhA gene
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Figure 3
Diagram showing the distribution of mutant strains and their frequencies
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Table 1
Primers used for the amplification of katG and inhA DNA
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*The primer coordinates refer to the positions in the M. tuberculosis H37Rv genome (GenBank accession number, NC 000962).

Table 2
Results of PCR to amplify katG, inhA ORF, and inhA promoter in Korean multidrug resistant M. tuberculosis isolates.
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*Number of nucleotides in each gene.

Table 3
The mutations found in the katG of Korean multidrug resistant M. tuberculosis isolates.
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*Deletion.

Table 4
The mutations found in the inhA of Korean multidrug resistant M. tuberculosis isolates
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*Not applicable, Wild type.

Table 5
The mutations found in katG, inhA ORF and inhA promoter region in Korean multidrug resistant M. tuberculosis isolates
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*Deletion, Wild type, Not applicable.

Table 6
Mutations newly found in this study
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*Wild type.

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