title: Rifampicin reduces susceptibility to ofloxacin in rifampicin-resistant Mycobacterium tuberculosis through efflux.
abstract: BACKGROUND
  Central dogma suggests that rifampicin resistance in Mycobacterium tuberculosis develops solely through rpoB gene mutations.
  
  
  OBJECTIVE
  To determine whether rifampicin induces efflux pumps activation in rifampicin resistant M. tuberculosis strains thereby defining rifampicin resistance levels and reducing ofloxacin susceptibility.
  
  
  METHODS
  Rifampicin and/or ofloxacin minimum inhibitory concentrations (MICs) were determined in rifampicin resistant strains by culture in BACTEC 12B medium. Verapamil and reserpine were included to determine their effect on rifampicin and ofloxacin susceptibility. RT-qPCR was applied to assess expression of efflux pump/transporter genes after rifampicin exposure. To determine whether verapamil could restore susceptibility to first-line drugs, BALB/c mice were infected with a MDR-TB strain and treated with first-line drugs with/without verapamil.
  
  
  RESULTS
  Rifampicin MICs varied independently of rpoB mutation and genetic background. Addition reserpine and verapamil significantly restored rifampicin susceptibility (p = 0.0000). RT-qPCR demonstrated that rifampicin induced differential expression of efflux/transporter genes in MDR-TB isolates. Incubation of rifampicin mono-resistant strains in rifampicin (2 μg/ml) for 7 days induced ofloxacin resistance (MIC > 2 μg/ml) in strains with an rpoB531 mutation. Ofloxacin susceptibility was restored by exposure to efflux pump inhibitors. Studies in BALB/c mice showed that verapamil in combination with first-line drugs significantly reduced pulmonary CFUs after 1 and 2 months treatment (p < 0.05).
  
  
  CONCLUSIONS
  Exposure of rifampicin resistant M. tuberculosis strains to rifampicin can potentially compromise the efficacy of the second-line treatment regimens containing ofloxacin, thereby emphasising the need for rapid diagnostics to guide treatment. Efflux pump inhibitors have the potential to improve the efficacy of anti-tuberculosis drug treatment.
authors.affiliation: Department of Biomedical Sciences, Faculty of Health Sciences, Stellenbosch University, Cape Town, South Africa.
authors.forename: Gail E
authors.initials: GE
authors.lastname: Louw
authors.forename: Robin M
authors.initials: RM
authors.lastname: Warren
authors.forename: Nicolaas C
authors.initials: NC
authors.lastname: Gey van Pittius
authors.forename: Rosalba
authors.initials: R
authors.lastname: Leon
authors.forename: Adelina
authors.initials: A
authors.lastname: Jimenez
authors.forename: Rogelio
authors.initials: R
authors.lastname: Hernandez-Pando
authors.forename: Christopher R E
authors.initials: CR
authors.lastname: McEvoy
authors.forename: Melanie
authors.initials: M
authors.lastname: Grobbelaar
authors.forename: Megan
authors.initials: M
authors.lastname: Murray
authors.forename: Paul D
authors.initials: PD
authors.lastname: van Helden
authors.forename: Thomas C
authors.initials: TC
authors.lastname: Victor
mesh_terms: D018759:Adrenergic Uptake Inhibitors / Q000494:pharmacology; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D000904:Antibiotics, Antitubercular / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000187:drug effects / Q000235:genetics; D002121:Calcium Channel Blockers / Q000494:pharmacology; D018929:Cell Culture Techniques; D012321:DNA-Directed RNA Polymerases; D004195:Disease Models, Animal; D051379:Mice; D008807:Mice, Inbred BALB C; D008826:Microbial Sensitivity Tests; D009169:Mycobacterium tuberculosis / Q000187:drug effects / Q000235:genetics; D015242:Ofloxacin / Q000494:pharmacology*; D012110:Reserpine / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012293:Rifampin / Q000494:pharmacology*; D018088:Tuberculosis, Multidrug-Resistant / Q000188:drug therapy* / Q000235:genetics; D014700:Verapamil / Q000494:pharmacology
chemical_list: D018759:Adrenergic Uptake Inhibitors; D000900:Anti-Bacterial Agents; D000904:Antibiotics, Antitubercular; D001426:Bacterial Proteins; D002121:Calcium Channel Blockers; C505805:rpoB protein, Mycobacterium tuberculosis; D012110:Reserpine; D015242:Ofloxacin; D014700:Verapamil; D012321:DNA-Directed RNA Polymerases; D012293:Rifampin