title: An instrument-free method for the demonstration of efflux pump activity of bacteria.
abstract: The aim of the study was to develop a simple, inexpensive, reproducible ethidium bromide (EB)-agar based method that is independent of any specialized instrumentation, for the demonstration of efflux pump activity, which is responsible for antibiotic resistance of bacteria.
  
  
  METHODS
  A series of agar plates containing varying concentrations of EB were swabbed with strains of Escherichia coli or Staphylococcus aureus, which differed with respect to efflux pump activity. The plates were incubated at different temperatures and time periods and the measurements of fluorescence were used to evaluate the efflux activity of each culture.
  
  
  RESULTS
  This simple assay allowed us to identify the efflux of EB in different bacteria following an overnight incubation. The minimal concentration of EB that produced fluorescence was significantly greater at 37 degrees C than at 4 degrees C, suggesting the presence of an energy-dependent pump. The method was shown to simultaneously identify strains of a mixed culture that differed from each other with respect to the activity of their efflux pumps.
  
  
  CONCLUSIONS
  The method, in conjunction with the use of antibiotic-containing disks, provides an additional advantage for the easy identification and selection of colonies that differ with respect to antibiotic susceptibility and degree of efflux pump activity. Because the method is very reproducible it may form the basis for interlaboratory standardization of efflux pump activity of multi-drug resistant (MDR) clinical isolates.
authors.affiliation: Unidade de Micobacterias, UPMM, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, Rua da Junqueira 96, 1349-008 Lisboa, Portugal.
authors.forename: Marta
authors.initials: M
authors.lastname: Martins
authors.forename: Bruno
authors.initials: B
authors.lastname: Santos
authors.forename: Ana
authors.initials: A
authors.lastname: Martins
authors.forename: Miguel
authors.initials: M
authors.lastname: Viveiros
authors.forename: Isabel
authors.initials: I
authors.lastname: Couto
authors.forename: Alexandra
authors.initials: A
authors.lastname: Cruz
authors.forename: Jean-Marie
authors.initials: JM
authors.lastname: Pagès
authors.forename: Joseph
authors.initials: J
authors.lastname: Molnar
authors.forename: Seamus
authors.initials: S
authors.lastname: Fanning
authors.forename: Leonard
authors.initials: L
authors.lastname: Amaral
mesh_terms: D000362:Agar / Q000737:chemistry*; D000900:Anti-Bacterial Agents; D003470:Culture Media; D024881:Drug Resistance, Bacterial; D024901:Drug Resistance, Multiple, Bacterial; D004926:Escherichia coli / Q000254:growth & development / Q000502:physiology; D004996:Ethidium / Q000737:chemistry*; D005453:Fluorescence; D008828:Microbiological Techniques / Q000379:methods*; D015203:Reproducibility of Results; D013211:Staphylococcus aureus / Q000187:drug effects / Q000254:growth & development / Q000502:physiology*
chemical_list: D000900:Anti-Bacterial Agents; D003470:Culture Media; D000362:Agar; D004996:Ethidium