title: Increased expression of efflux pump genes in extensively drug-resistant isolates of Mycobacterium tuberculosis.
abstract: BACKGROUND
  Extensively drug-resistant tuberculosis (XDR-TB) is defined as tuberculosis (TB) caused by Mycobacterium tuberculosis (MTB) strains that are multidrug resistant (MDR) and also resistant to a fluoroquinolone and to one injectable aminoglycoside or capreomycin. Whilst resistance in MTB has been associated with single nucleotide polymorphisms (SNPs), efflux pumps are thought to play a role in conferring resistance to MTB but little is known about them.
  
  
  METHODS
  We studied XDR MTB (n=10) strains characterized by whole genome sequencing (WGS; http://www.ebi.ac.uk/ena/data/view/PRJEB7798). Phenotypic susceptibility testing was performed by the MGIT 960 (Becton, Dickinson and Co., NJ, USA) method. All XDR MTB strains were resistant to at least seven drugs whilst one XDR MTB strain, X54 was resistant to isoniazid, rifampicin, pyrazinamide, streptomycin, ethambutol, fluoroquinolones, capreomycin, kanamycin, amikacin, and ethionamide. The mRNA expression of efflux candidate genes Rv0194, Rv2688c, Rv1634, drrA, and drrB was determined in XDR MTB strains as compared with the ATCC reference strain, H37Rv, and drug-susceptible (DS) MTB (n=9) strains using the relative quantification method normalized to 16S rRNA.
  
  
  RESULTS
  The mRNA expression levels of efflux genes Rv2688c (p=0.0037), Rv1634 (p=0.0042), drrA (p=0.0078) and drrB (p=0.0003) were upregulated in XDR-TB strains as compared with DS MTB strains.
  
  
  CONCLUSIONS
  The differences between XDR-TB and drug-susceptible isolates suggest that the increased expression levels of MTB efflux pump genes may contribute to drug resistance in extensively drug-resistant tuberculosis. Future studies are needed to determine whether combining efflux pump inhibitors to antitubercular drugs would be effective to treat resistant tuberculosis.
authors.affiliation: Department of Pathology and Laboratory Medicine, Aga Khan University Hospital, Karachi, Pakistan. Electronic address: akbar.kanji@aku.edu.
authors.forename: Akbar
authors.initials: A
authors.lastname: Kanji
authors.affiliation: Department of Pathology and Laboratory Medicine, Aga Khan University Hospital, Karachi, Pakistan.
authors.forename: Rumina
authors.initials: R
authors.lastname: Hasan
authors.affiliation: Department of Molecular Microbiology and Immunology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD, USA.
authors.forename: Ying
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Department of Molecular Microbiology and Immunology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD, USA.
authors.forename: Wanliang
authors.initials: W
authors.lastname: Shi
authors.affiliation: Department of Pathology and Laboratory Medicine, Aga Khan University Hospital, Karachi, Pakistan.
authors.forename: Kehkashan
authors.initials: K
authors.lastname: Imtiaz
authors.affiliation: Department of Pathology and Laboratory Medicine, Aga Khan University Hospital, Karachi, Pakistan.
authors.forename: Kiran
authors.initials: K
authors.lastname: Iqbal
authors.affiliation: Department of Pathology and Laboratory Medicine, Aga Khan University Hospital, Karachi, Pakistan.
authors.forename: Samreen
authors.initials: S
authors.lastname: Shafiq
authors.affiliation: Department of Pathology and Laboratory Medicine, Aga Khan University Hospital, Karachi, Pakistan.
authors.forename: Zahra
authors.initials: Z
authors.lastname: Hasan