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

J Antimicrob Chemother. 2011 Apr;66(4):730-3. doi: 10.1093/jac/dkq519. Epub 2011 Jan 17.


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 β-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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Substitution / genetics
  • Antitubercular Agents / pharmacology*
  • Binding Sites
  • Cloning, Molecular
  • DNA-Directed RNA Polymerases / chemistry
  • DNA-Directed RNA Polymerases / genetics*
  • Drug Resistance, Bacterial*
  • Humans
  • Mutation, Missense*
  • Mycobacterium smegmatis / genetics
  • Mycobacterium tuberculosis / drug effects*
  • Mycobacterium tuberculosis / genetics*
  • Mycobacterium tuberculosis / isolation & purification
  • Rifampin / pharmacology*
  • Thermus thermophilus / genetics
  • Tuberculosis / microbiology


  • Antitubercular Agents
  • DNA-Directed RNA Polymerases
  • RNA polymerase beta subunit
  • Rifampin