title: Acquisition of rifabutin resistance by a rifampicin resistant mutant of Mycobacterium tuberculosis involves an unusual spectrum of mutations and elevated frequency.
abstract: BACKGROUND
  Mutations in a small region of the rpoB gene are responsible for most rifamycin resistance in Mycobacterium tuberculosis. In this study we have sequentially generated resistant strains to first rifampicin and then rifabutin. Portions of the rpoB gene were sequenced from 131 randomly selected mutants. Second round selection resulted in a changed frequency of specific mutations.
  
  
  METHODS
  Mycobacterium tuberculosis (strain Mtb72) rifamycin resistant mutants were selected in vitro with either rifampicin or rifabutin. One mutant R190 (rpoB S522L) selected with rifampicin had a rifampicin MIC of 32 microg/ml but remained sensitive to rifabutin (MIC<0.8 microg/ml). This mutant was subjected to a second round of selection with rifabutin.
  
  
  RESULTS
  All 105 first round resistant mutants derived from the parent strain (Mtb72) screened acquired mutations within the 81 bp rpoB hotspot. When the rifampicin resistant but rifabutin sensitive S522L mutant was subjected to a second round of selection, single additional rpoB mutations were identified in 24 (92%) of 26 second round mutants studied, but 14 (54%) of these strains contained mutations outside the 81 bp hotspot (codons 144, 146, 148, 505). Additionally, spontaneous rifabutin resistant mutants were produced at >10 times the frequency by the S522L mutant than the parent strain.
  
  
  CONCLUSIONS
  First round selection of mutation S522L with rifampicin increased the frequency and changed the spectrum of mutations identified after selection with rifabutin.
authors.affiliation: KIT (Royal Tropical Institute) Biomedical Research, Meibergdreef 39, 1105 AZ Amsterdam, The Netherlands. r.anthony@kit.nl
authors.forename: Richard M
authors.initials: RM
authors.lastname: Anthony
authors.forename: Anja R J
authors.initials: AR
authors.lastname: Schuitema
authors.forename: Indra L
authors.initials: IL
authors.lastname: Bergval
authors.forename: Tim J
authors.initials: TJ
authors.lastname: Brown
authors.forename: Linda
authors.initials: L
authors.lastname: Oskam
authors.forename: Paul R
authors.initials: PR
authors.lastname: Klatser
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics*; D003062:Codon; D012321:DNA-Directed RNA Polymerases; D024881:Drug Resistance, Bacterial; D008826:Microbial Sensitivity Tests; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000235:genetics*; D017828:Rifabutin / Q000494:pharmacology*; D012293:Rifampin / Q000494:pharmacology*
chemical_list: D001426:Bacterial Proteins; D003062:Codon; C505805:rpoB protein, Mycobacterium tuberculosis; D017828:Rifabutin; D012321:DNA-Directed RNA Polymerases; D012293:Rifampin