Mutations outside the rifampicin resistance-determining region associated with rifampicin resistance in Mycobacterium tuberculosis

Objectives: Ninety-six percent of rifampicin resistance in Mycobacterium tuberculosis was shown to be associated with mutations inside the 81 bp rifampicin resistance-determining region (RRDR) located in the centre of the rpoB gene. The detection of rifampicin resistance by targeting the RRDR failed to match with a resistant phenotype in 4% of all cases. Our study aims to identify the mutations outside the RRDR that are associated with rifampicin resistance in M. tuberculosis. Methods and results: Among 50 rifampicin-resistant and 20 rifampicin-susceptible clinical isolates of M. tuberculosis, 2 of the rifampicin-resistant isolates did not harbour any known mutations in the RRDR. Sequencing analysis of the whole rpoB gene identified two rare mutations, V146F and I572F. A molecular structure model based on Thermus thermophilus RpoB revealed that both these substituted amino acids are located in close proximity to the rifampicin-binding pocket of the beta-subunit. Substitutions of simple amino acids for bulky ones are likely to affect the protein-drug interaction. Cloning and transformation of the mutated rpoB gene into wild-type Mycobacterium smegmatis and M. tuberculosis successfully elevated the MIC of rifampicin and conferred the rifampicin resistance phenotype. Conclusions: Our study showed that amino acid positions 146 and 572 are associated with rifampicin resistance in M. tuberculosis in addition to the RRDR. Molecular assays for identifying rifampicin-resistant M. tuberculosis might be improved in terms of accuracy by including these two positions.