Tuberculosis (TB) is an infectious respiratory disease caused by Mycobacterium tuberculosis and one of the top 10 causes of death worldwide. Treating TB is challenging; successful treatment requires a long course of multiple antibiotics. Rifampicin (RIF) is a first-line drug for treating TB, and the development of RIF-resistant M. tuberculosis makes treatment even more difficult. To determine the mechanism of RIF resistance in these strains, we searched for novel mutations by sequencing. Four isolates, CDC-1, CDC-2, CDC-3, and CDC-4, had high-level RIF resistance and unique mutations encoding RpoB G 158 R, RpoB V 168 A, RpoB S 188 P, and RpoB Q 432 insQ, respectively. To evaluate their correlation with RIF resistance, plasmids carrying rpoB genes encoding these mutant proteins were transfected into the H 37 Rv reference strain. The plasmid complementation of RpoB indicated that G 158 R, V 168 A, and S 188 P did not affect the MIC of RIF. However, the MIC of RIF was increased in H 37 Rv carrying RpoB Q 432 insQ. To confirm the correlation between RIF resistance and Q 432 insQ, we cloned an rpoB fragment carrying the insertion (encoding RpoB Q 432 insQ) into H 37 Rv by homologous recombination using a suicide vector. All replacement mutants expressing RpoB Q 432 insQ were resistant to RIF (MIC > 1 mg/L). These results indicate that RpoB Q 432 insQ causes RIF resistance in M. tuberculosis.
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