An instrument-free method for the demonstration of efflux pump activity of bacteria.
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.
Martins
Marta
M
Unidade de Micobacterias, UPMM, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, Rua da Junqueira 96, 1349-008 Lisboa, Portugal.
Santos
Bruno
B
Martins
Ana
A
Viveiros
Miguel
M
Couto
Isabel
I
Cruz
Alexandra
A
Pagès
Jean-Marie
JM
Molnar
Joseph
J
Fanning
Seamus
S
Amaral
Leonard
L
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*
D000900:Anti-Bacterial Agents; D003470:Culture Media; D000362:Agar; D004996:Ethidium