title: Detection of mutations associated with isoniazid resistance in multidrug-resistant Mycobacterium tuberculosis clinical isolates.
abstract: OBJECTIVE
  To determine the prevalence of isoniazid resistance-conferring mutations among multidrug-resistant (MDR) isolates of Mycobacterium tuberculosis from Poland.
  
  
  METHODS
  Nine genetic loci, including structural genes (katG, inhA, ahpC, kasA, ndh, nat and mshA) and regulatory regions (i.e. the mabA-inhA promoter and oxyR-ahpC intergenic region) of 50 MDR M. tuberculosis isolates collected throughout Poland were PCR-amplified in their entirety and screened for mutations by direct sequencing methodology.
  
  
  RESULTS
  Forty-six (92%) MDR M. tuberculosis isolates had mutations in the katG gene, and the katG Ser315Thr substitution predominated (72%). Eight (16%) isolates (six with a mutated katG allele) had mutations in the inhA promoter region and two such isolates also had single inhA structural gene mutations. Mutations in the oxyR-ahpC locus were found in five (10%) isolates, of which all but one had at least one additional mutation in katG. Mutations in the remaining genetic loci (kasA, ndh, nat and mshA) were detected in 12 (24%), 4 (8%), 5 (10%) and 17 (34%) MDR isolates, respectively. All non-synonymous mutants for these genes harboured mutations in katG. One isolate had no mutations in any of the analysed loci.
  
  
  CONCLUSIONS
  This study accentuates the usefulness of katG and inhA promoter mutations as predictive markers of isoniazid resistance. Testing only for katG 315 and inhA -15 mutations would detect isoniazid resistance in 84% of the MDR M. tuberculosis sample. This percentage would increase to 96% if the sequence analysis was extended to the entire katG gene. Analysis of the remaining genetic loci did not contribute greatly to the identification of isoniazid resistance.
authors.affiliation: Department of Applied Microbiology, Institute of Microbiology, Faculty of Biology, University of Warsaw, I. Miecznikowa 1, 02-096 Warsaw, Poland t.jagielski@biol.uw.edu.pl.
authors.forename: Tomasz
authors.initials: T
authors.lastname: Jagielski
authors.affiliation: Department of Applied Microbiology, Institute of Microbiology, Faculty of Biology, University of Warsaw, I. Miecznikowa 1, 02-096 Warsaw, Poland.
authors.forename: Zofia
authors.initials: Z
authors.lastname: Bakuła
authors.affiliation: Department of Applied Microbiology, Institute of Microbiology, Faculty of Biology, University of Warsaw, I. Miecznikowa 1, 02-096 Warsaw, Poland.
authors.forename: Katarzyna
authors.initials: K
authors.lastname: Roeske
authors.affiliation: Department of Applied Microbiology, Institute of Microbiology, Faculty of Biology, University of Warsaw, I. Miecznikowa 1, 02-096 Warsaw, Poland.
authors.forename: Michał
authors.initials: M
authors.lastname: Kamiński
authors.affiliation: Department of Microbiology, National Tuberculosis and Lung Diseases Research Institute, Płocka 26, 01-138 Warsaw, Poland.
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Napiórkowska
authors.affiliation: Department of Microbiology, National Tuberculosis and Lung Diseases Research Institute, Płocka 26, 01-138 Warsaw, Poland.
authors.forename: Ewa
authors.initials: E
authors.lastname: Augustynowicz-Kopeć
authors.affiliation: Department of Microbiology, National Tuberculosis and Lung Diseases Research Institute, Płocka 26, 01-138 Warsaw, Poland.
authors.forename: Zofia
authors.initials: Z
authors.lastname: Zwolska
authors.affiliation: Department of Applied Microbiology, Institute of Microbiology, Faculty of Biology, University of Warsaw, I. Miecznikowa 1, 02-096 Warsaw, Poland.
authors.forename: Jacek
authors.initials: J
authors.lastname: Bielecki
mesh_terms: D000328:Adult; D000368:Aged; D000995:Antitubercular Agents / Q000494:pharmacology*; D004269:DNA, Bacterial; D024901:Drug Resistance, Multiple, Bacterial; D005260:Female; D005798:Genes, Bacterial; D005838:Genotype; D060005:Genotyping Techniques / Q000379:methods; D006801:Humans; D007538:Isoniazid / Q000494:pharmacology*; D008297:Male; D008826:Microbial Sensitivity Tests / Q000379:methods; D008875:Middle Aged; D008969:Molecular Sequence Data; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000302:isolation & purification; D011044:Poland; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D018088:Tuberculosis, Multidrug-Resistant / Q000382:microbiology*
chemical_list: D000995:Antitubercular Agents; D004269:DNA, Bacterial; D007538:Isoniazid
keywords: M. tuberculosis; TB; first-line anti-TB drugs; multidrug resistance; sequence analysis; spoligotyping; tuberculosis