title: Ethidium bromide transport across Mycobacterium smegmatis cell-wall: correlation with antibiotic resistance.
abstract: BACKGROUND
  Active efflux systems and reduced cell-wall permeability are considered to be the main causes of mycobacterial intrinsic resistance to many antimicrobials. In this study, we have compared the Mycobacterium smegmatis wild-type strain mc2155 with knockout mutants for porins MspA (the main porin of M. smegmatis) and MspC, the efflux pump LfrA (the main efflux pump system of M. smegmatis) and its repressor LfrR for their ability to transport ethidium bromide (EtBr) on a real-time basis. This information was then correlated with minimum inhibitory concentrations (MICs) of several antibiotics in the presence or absence of the efflux inhibitors chlorpromazine, thioridazine and verapamil.
  
  
  RESULTS
  In the absence of porins MspA and MspC, accumulation of ethidium bromide decreased and the cells became more resistant to several antibiotics, whereas the knockout mutant for the LfrA pump showed increased accumulation of EtBr and increased susceptibility to EtBr, rifampicin, ethambutol and ciprofloxacin. Moreover, the efflux inhibitors caused a reduction of the MICs of streptomycin, rifampicin, amikacin, ciprofloxacin, clarithromycin and erythromycin in most of the strains tested.
  
  
  CONCLUSIONS
  The methodology used in this study demonstrated that porin MspA plays an important role in the influx of quaternary ammonium compounds and antibiotics and that efflux via the LfrA pump is involved in low-level resistance to several antimicrobial drugs in M. smegmatis. The results obtained with this non-pathogenic mycobacterium will be used in future studies as a model for the evaluation of the activity of the same efflux inhibitors on the susceptibility of multidrug resistant strains of Mycobacterium tuberculosis to isoniazid and rifampicin.
authors.affiliation: Unit of Mycobacteriology, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, Rua da Junqueira 100, 1349-008 Lisboa, Portugal.
authors.forename: Liliana
authors.initials: L
authors.lastname: Rodrigues
authors.forename: Jorge
authors.initials: J
authors.lastname: Ramos
authors.forename: Isabel
authors.initials: I
authors.lastname: Couto
authors.forename: Leonard
authors.initials: L
authors.lastname: Amaral
authors.forename: Miguel
authors.initials: M
authors.lastname: Viveiros
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology; D017920:Antiporters / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001692:Biological Transport; D002473:Cell Wall / Q000378:metabolism*; D024901:Drug Resistance, Multiple, Bacterial; D004996:Ethidium / Q000378:metabolism*; D008826:Microbial Sensitivity Tests; D020102:Mycobacterium smegmatis / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D018272:Porins / Q000235:genetics / Q000378:metabolism*; D017384:Sequence Deletion
chemical_list: D000900:Anti-Bacterial Agents; D017920:Antiporters; D001426:Bacterial Proteins; C097688:LfrA protein, Mycobacterium smegmatis; C434272:MspC protein, Mycobacterium smegmatis; D018272:Porins; C121838:mspA protein, Mycobacterium smegmatis; D004996:Ethidium