Identification of Efflux-Mediated Multi-drug Resistance in Bacterial Clinical Isolates by Two Simple Methods

  • Marta Martins
  • Isabel Couto
  • Miguel Viveiros
  • Leonard AmaralEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 642)


Two simple, instrument-free, user-friendly methods that can readily be implemented by a routine microbiology laboratory are described for the detection of multi-drug-resistant (MDR) isolates that overexpress efflux pump (EP) systems responsible for the MDR phenotype. The first method employs the universal EP substrate ethidium bromide (EB) at varying concentrations in agar-containing plates upon which the contents of an overnight culture are swabbed as spokes of a wheel. In this method, named the EB–agar cartwheel method, it is assumed that the smallest concentration of EB that produces fluorescence of the bacterial mass represents the highest concentration of EB that the bacteria can exclude. Consequently, as the efflux system(s) of a given MDR clinical bacterial isolate is overexpressed relative to that of a reference strain, the minimal concentration of EB producing fluorescence is significantly greater. A simple formula is provided which affords the ranking of MDR clinical isolates with respect to the degree of their efflux capacity. The second method, which can be used after the first one, determines whether the MDR phenotype is based upon an overexpressed efflux system. This method employs a 24-well microplate with separate wells containing or lacking an efflux pump inhibitor (EPI) and Kirby–Bauer discs that correspond to the antibiotics to which the MDR strain is resistant. After the wells are inoculated with the MDR clinical isolate, the plate is incubated overnight and each well is evaluated by eye for evidence of growth. Comparison of growth to the relevant control enables the observer to determine the following outcomes: no growth produced by the EPI–antibiotic combination (i.e., reversal of antibiotic resistance); reduced growth produced by the EPI–antibiotic combination; no difference in growth, i.e., EPI does not affect the resistance to the given antibiotic. If the first method showed that there was a significant difference between the minimum concentrations of EB in agar that produced fluorescence for the clinical isolate and its reference strain, then one can conclude that if the EPI had no effect on reducing antibiotic resistance, the differences in the EB concentrations that produced fluorescence are probably due to differences in the permeability of the strain to EB, reflecting a downregulation of porins if the clinical isolate is a Gram-negative bacterium.

Key words

Multi-drug resistance Efflux pumps Efflux pump inhibitors Ethidium bromide Screening methods 



We thank Seamus Fanning and Jean-Marie Pagès for providing the Salmonella and Enterobacter strains, respectively. The help/participation of Gabriella Spengler, Liliana Rodrigues, Ana Martins, Ana Cardoso, Nadia Charepe, Celeste Falcão, Sofia Santos Costa, and Daniela Sampaio in the characterization of reference and clinical bacterial strains by the methods described is also acknowledged. This work was supported by grants EU-FSE/FEDER-POCI/SAU-MMO/59370/2004 and EU-FSE/FEDER-PTDC/BIA-MIC/71280/2006 provided by the Fundação para a Ciência e a Tecnologia (FCT) of Portugal. M. Martins was supported by grant SFRH/BD/14319/2003 (FCT, Portugal).


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Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Marta Martins
    • 1
  • Isabel Couto
    • 2
    • 3
  • Miguel Viveiros
    • 2
  • Leonard Amaral
    • 1
    Email author
  1. 1.Unit of Mycobacteriology and UPMM, Instituto de Higiene e Medicina TropicalUniversidade Nova de Lisboa (IHMT/UNL)LisboaPortugal
  2. 2.Unit of Mycobacteriology, Instituto de Higiene e Medicina TropicalUniversidade Nova de Lisboa (IHMT/UNL)LisboaPortugal
  3. 3.Centro de Recursos Microbiológicos (CREM), Faculdade de Ciências e TecnologiaUniversidade Nova de LisboaCaparicaPortugal

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