title: Evidence for the critical role of a secondary site rpoB mutation in the compensatory evolution and successful transmission of an MDR tuberculosis outbreak strain.
abstract: BACKGROUND
  MDR Mycobacterium tuberculosis clinical strains that cause large outbreaks, particularly among HIV-negative patients, are likely to have undergone the most successful compensatory evolution. Hence, mutations secondary to the acquisition of drug resistance are worthy of consideration in these highly transmissible strains. Here, we assessed the role of a mutation within rpoB, rpoB V615M, secondary to the rifampicin resistance-conferring mutation rpoB S531L, which is associated with a major MDR tuberculosis outbreak strain that evolved in an HIV-negative context in northern Tunisia.
  
  
  METHODS
  Using BCG as a model organism, we engineered strains harbouring either the rpoB S531L mutation alone or the double mutation rpoB S531L, V615M. Individual and competitive in vitro growth assays were performed in order to assess the relative fitness of each BCG mutant.
  
  
  RESULTS
  The rpoB V615M mutation was found to be invariably associated with rpoB S531L. Structural analysis mapped rpoB V615M to the same bridge helix region as rpoB compensatory mutations previously described in Salmonella. Compared with the rpoB single-mutant BCG, the double mutant displayed improved growth characteristics and fitness rates equivalent to WT BCG. Strikingly, the rpoB double mutation conferred high-level resistance to rifampicin.
  
  
  CONCLUSIONS
  Here, we demonstrated the fitness compensatory role of a mutation within rpoB, secondary to the rifampicin resistance mutation rpoB S531L, which is characteristic of an MDR M. tuberculosis major outbreak strain. The finding that this secondary mutation concomitantly increased the resistance level to rifampicin argues for its significant contribution to the successful transmission of the MDR-TB strain.
authors.affiliation: Unit of Typing & Genetics of Mycobacteria, Laboratory of Molecular Microbiology, Vaccinology, and Biotechnology Development, Institut Pasteur de Tunis, Université de Tunis El Manar, Tunis, Tunisia.
authors.forename: Nedra
authors.initials: N
authors.lastname: Meftahi
authors.affiliation: Unit of Typing & Genetics of Mycobacteria, Laboratory of Molecular Microbiology, Vaccinology, and Biotechnology Development, Institut Pasteur de Tunis, Université de Tunis El Manar, Tunis, Tunisia.
authors.forename: Amine
authors.initials: A
authors.lastname: Namouchi
authors.affiliation: Unit of Typing & Genetics of Mycobacteria, Laboratory of Molecular Microbiology, Vaccinology, and Biotechnology Development, Institut Pasteur de Tunis, Université de Tunis El Manar, Tunis, Tunisia.
authors.forename: Besma
authors.initials: B
authors.lastname: Mhenni
authors.affiliation: Department of Medical Biochemistry and Microbiology, Box 582 Biomedical Center, Uppsala University, Uppsala, Sweden.
authors.forename: Gerrit
authors.initials: G
authors.lastname: Brandis
authors.affiliation: Department of Medical Biochemistry and Microbiology, Box 582 Biomedical Center, Uppsala University, Uppsala, Sweden.
authors.forename: Diarmaid
authors.initials: D
authors.lastname: Hughes
authors.affiliation: Unit of Typing & Genetics of Mycobacteria, Laboratory of Molecular Microbiology, Vaccinology, and Biotechnology Development, Institut Pasteur de Tunis, Université de Tunis El Manar, Tunis, Tunisia helmi.merdassi@pasteur.rns.tn.
authors.forename: Helmi
authors.initials: H
authors.lastname: Mardassi
mesh_terms: D000328:Adult; D012321:DNA-Directed RNA Polymerases / Q000235:genetics*; D004196:Disease Outbreaks; D019143:Evolution, Molecular; D005260:Female; D006801:Humans; D008297:Male; D020125:Mutation, Missense; D009163:Mycobacterium bovis / Q000201:enzymology / Q000235:genetics / Q000254:growth & development; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification; D059386:Reverse Genetics; D018088:Tuberculosis, Multidrug-Resistant / Q000453:epidemiology* / Q000382:microbiology* / Q000635:transmission; D014416:Tunisia / Q000453:epidemiology
chemical_list: D012321:DNA-Directed RNA Polymerases; C105078:RNA polymerase beta subunit