title: High-level resistance to isoniazid and ethionamide in multidrug-resistant Mycobacterium tuberculosis of the Lisboa family is associated with inhA double mutations.
abstract: OBJECTIVE
  The purpose of this study was to determine the levels of isoniazid and ethionamide resistance and to identify associated mutations in endemic multidrug-resistant (MDR) strains of Mycobacterium tuberculosis from the Lisbon metropolitan area, Portugal.
  
  
  METHODS
  Seventeen clinical MDR tuberculosis (TB) strains were characterized by standard and semi-quantitative drug susceptibility testing to assess the level of isoniazid and ethionamide resistance. The genes katG, inhA, ethA and ndh were screened for mutations. All strains were genotyped by 24 loci mycobacterial interspersed repetitive unit-variable number of tandem repeats (MIRU-VNTR) analysis.
  
  
  RESULTS
  All strains showed high-level resistance to both isoniazid (>1 mg/L) and ethionamide (>25 mg/L). MIRU-VNTR typing revealed the presence of two main clusters, Lisboa3 and Q1, in 16/17 strains, all of which showed the C-15T mutation in the promoter region of the inhA gene. The 16 strains belong to the Latino-American-Mediterranean (LAM) genotype and the other strain belongs to the Beijing genotype. Sequencing of the inhA open reading frame revealed that the 16 strains also had mutations in the structural region of the gene, leading to the S94A substitution in 9 strains and the I194T substitution in 7 strains.
  
  
  CONCLUSIONS
  The results reveal that the presence of a mutation in the inhA regulatory region together with a mutation in the inhA coding region can lead to the development of high-level isoniazid resistance and cross-resistance to ethionamide among the MDR-TB strains circulating in Lisbon. This mutational pattern also hints to a possible involvement of strain-specific factors that could be a feature of the Portuguese MDR-TB strains where the LAM family is the major circulating genotype.
authors.affiliation: Grupo de Micobactérias, Unidade de Microbiologia Médica, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa (IHMT/UNL), Rua da Junqueira 100, 1349-008 Lisboa, Portugal.
authors.forename: Diana
authors.initials: D
authors.lastname: Machado
authors.forename: João
authors.initials: J
authors.lastname: Perdigão
authors.forename: Jorge
authors.initials: J
authors.lastname: Ramos
authors.forename: Isabel
authors.initials: I
authors.lastname: Couto
authors.forename: Isabel
authors.initials: I
authors.lastname: Portugal
authors.forename: Claudia
authors.initials: C
authors.lastname: Ritter
authors.forename: Erik C
authors.initials: EC
authors.lastname: Boettger
authors.forename: Miguel
authors.initials: M
authors.lastname: Viveiros
mesh_terms: D000995:Antitubercular Agents / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000235:genetics*; D024901:Drug Resistance, Multiple, Bacterial; D005000:Ethionamide / Q000494:pharmacology*; D005838:Genotype; D006801:Humans; D007538:Isoniazid / Q000494:pharmacology*; D008826:Microbial Sensitivity Tests; D058889:Molecular Typing; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000145:classification / Q000187:drug effects* / Q000235:genetics / Q000302:isolation & purification; D010088:Oxidoreductases / Q000235:genetics*; D011174:Portugal; D018088:Tuberculosis, Multidrug-Resistant / Q000382:microbiology*
chemical_list: D000995:Antitubercular Agents; D001426:Bacterial Proteins; D010088:Oxidoreductases; C085257:InhA protein, Mycobacterium; D005000:Ethionamide; D007538:Isoniazid
keywords: LAM family; MDR-TB; resistance levels; tuberculosis