A Simple Method for Assessment of MDR Bacteria for Over-Expressed Efflux Pumps

Marta Martins a , b , *, Matthew P McCusker a , b , Miguel Viveiros b , c , Isabel Couto c , d , Séamus Fanning a , b , Jean-Marie Pagès b , e , Leonard Amaral b , c
a School of Public Health, Physiotherapy and Population Science, Centre for Molecular Innovation and Drug Discovery, Centre for Food Safety, Science Centre South, Room S125, University College Dublin, Belfield, Dublin 4, Ireland
b Cost Action BM0701 (ATENS)
c Grupo de Micobactérias; Unidade de Microbiologia Médica, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa (IHMT/UNL), Rua da Junqueira, 100, 1349-008 Lisbon, Portugal
d Centro de Recursos Microbiológicos (CREM), Faculdade de Ciências e Tecnologia, UNL, 2829-516 Caparica, Portugal
e UMR_MD1, Aix-Marseille University, IRBA, Marseille, France

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© Martins et al.; Licensee Bentham Open.

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* Address correspondence to this author at the Centre for Molecular Innovation and Drug Discovery, School of Public Health, Physiotherapy and Population Science, Centre for Food Safety; Science Centre South, Room S125; University College Dublin, Belfield, Dublin 4, Ireland; Tel: +353 1 7162879/2871; Fax: +353 1 7161147; E-mail:


It is known that bacteria showing a multi-drug resistance phenotype use several mechanisms to overcome the action of antibiotics. As a result, this phenotype can be a result of several mechanisms or a combination of thereof. The main mechanisms of antibiotic resistance are: mutations in target genes (such as DNA gyrase and topoisomerase IV); over-expression of efflux pumps; changes in the cell envelope; down regulation of membrane porins, and modified lipopolysaccharide component of the outer cell membrane (in the case of Gram-negative bacteria). In addition, adaptation to the environment, such as quorum sensing and biofilm formation can also contribute to bacterial persistence. Due to the rapid emergence and spread of bacterial isolates showing resistance to several classes of antibiotics, methods that can rapidly and efficiently identify isolates whose resistance is due to active efflux have been developed. However, there is still a need for faster and more accurate methodologies. Conventional methods that evaluate bacterial efflux pump activity in liquid systems are available. However, these methods usually use common efflux pump substrates, such as ethidium bromide or radioactive antibiotics and therefore, require specialized instrumentation, which is not available in all laboratories.

In this review, we will report the results obtained with the Ethidium Bromide-agar Cartwheel method. This is an easy, instrument-free, agar based method that has been modified to afford the simultaneous evaluation of as many as twelve bacterial strains. Due to its simplicity it can be applied to large collections of bacteria to rapidly screen for multi-drug resistant isolates that show an over-expression of their efflux systems. The principle of the method is simple and relies on the ability of the bacteria to expel a fluorescent molecule that is substrate for most efflux pumps, ethidium bromide. In this approach, the higher the concentration of ethidium bromide required to produce fluorescence of the bacterial mass, the greater the efflux capacity of the bacterial cells. We have tested and applied this method to a large number of Gram-positive and Gram-negative bacteria to detect efflux activity among these multi-drug resistant isolates. The presumptive efflux activity detected by the Ethidium Bromide-agar Cartwheel method was subsequently confirmed by the determination of the minimum inhibitory concentration for several antibiotics in the presence and absence of known efflux pump inhibitors.

Keywords: Clinical isolates, Efflux activity, Efflux pumps, Ethidium bromide, Multi-drug resistance, Screening method.