title: Contribution of efflux to the emergence of isoniazid and multidrug resistance in Mycobacterium tuberculosis.
abstract: Multidrug resistant (MDR) tuberculosis is caused by Mycobacterium tuberculosis resistant to isoniazid and rifampicin, the two most effective drugs used in tuberculosis therapy. Here, we investigated the mechanism by which resistance towards isoniazid develops and how overexpression of efflux pumps favors accumulation of mutations in isoniazid targets, thus establishing a MDR phenotype. The study was based on the in vitro induction of an isoniazid resistant phenotype by prolonged serial exposure of M. tuberculosis strains to the critical concentration of isoniazid employed for determination of drug susceptibility testing in clinical isolates. Results show that susceptible and rifampicin monoresistant strains exposed to this concentration become resistant to isoniazid after three weeks; and that resistance observed for the majority of these strains could be reduced by means of efflux pumps inhibitors. RT-qPCR assessment of efflux pump genes expression showed overexpression of all tested genes. Enhanced real-time efflux of ethidium bromide, a common efflux pump substrate, was also observed, showing a clear relation between overexpression of the genes and increased efflux pump function. Further exposure to isoniazid resulted in the selection and stabilization of spontaneous mutations and deletions in the katG gene along with sustained increased efflux activity. Together, results demonstrate the relevance of efflux pumps as one of the factors of isoniazid resistance in M. tuberculosis. These results support the hypothesis that activity of efflux pumps allows the maintenance of an isoniazid resistant population in a sub-optimally treated patient from which isoniazid genetically resistant mutants emerge. Therefore, the use of inhibitors of efflux should be considered in the development of new therapeutic strategies for preventing the emergence of MDR-TB during treatment.
authors.affiliation: Grupo de Micobactérias, Unidade de Microbiologia Médica, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa (IHMT/UNL), Lisboa, Portugal.
authors.forename: Diana
authors.initials: D
authors.lastname: Machado
authors.forename: Isabel
authors.initials: I
authors.lastname: Couto
authors.forename: João
authors.initials: J
authors.lastname: Perdigão
authors.forename: Liliana
authors.initials: L
authors.lastname: Rodrigues
authors.forename: Isabel
authors.initials: I
authors.lastname: Portugal
authors.forename: Pedro
authors.initials: P
authors.lastname: Baptista
authors.forename: Bruno
authors.initials: B
authors.lastname: Veigas
authors.forename: Leonard
authors.initials: L
authors.lastname: Amaral
authors.forename: Miguel
authors.initials: M
authors.lastname: Viveiros
mesh_terms: D000995:Antitubercular Agents / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000235:genetics; D001693:Biological Transport, Active; D002352:Carrier Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics; D002374:Catalase / Q000037:antagonists & inhibitors / Q000235:genetics; D002746:Chlorpromazine / Q000494:pharmacology; D012321:DNA-Directed RNA Polymerases; D024901:Drug Resistance, Multiple, Bacterial / Q000187:drug effects*; D015870:Gene Expression; D006801:Humans; D007538:Isoniazid / Q000494:pharmacology*; D008826:Microbial Sensitivity Tests; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D012293:Rifampin / Q000494:pharmacology; D013881:Thioridazine / Q000494:pharmacology; D018088:Tuberculosis, Multidrug-Resistant / Q000188:drug therapy / Q000382:microbiology; D014700:Verapamil / Q000494:pharmacology
chemical_list: D000995:Antitubercular Agents; D001426:Bacterial Proteins; D002352:Carrier Proteins; C505805:rpoB protein, Mycobacterium tuberculosis; D014700:Verapamil; D002374:Catalase; C480353:katG protein, Mycobacterium tuberculosis; D012321:DNA-Directed RNA Polymerases; D013881:Thioridazine; D002746:Chlorpromazine; D007538:Isoniazid; D012293:Rifampin