Measuring Efflux and Permeability in Mycobacteria

  • Liliana Rodrigues
  • Miguel Viveiros
  • José A. AínsaEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1285)


The intrinsic resistance of mycobacteria to most antimicrobial agents is mainly attributed to the synergy between their relatively impermeable cell wall and efflux systems. The mycobacterial cell wall is rich in lipids and polysaccharides making a compact envelope that limits drug uptake. Changes in cell wall composition or structure lead to variations in susceptibility to drugs. Bacterial efflux pumps are membrane proteins that are capable of actively transporting a broad range of substrates, including drugs, from the cytoplasm to the extracellular environment. Increased expression of efflux pump genes confers a low level resistance phenotype, and under these conditions, bacteria may have greater chances of acquiring chromosomal mutation(s) conferring higher levels of drug resistance. In order to develop effective antimycobacterial therapeutic strategies, the contributions to drug resistance made by the limited permeability of the cell wall and the increased expression of efflux pumps must be understood. In this chapter, we describe a method that allows: (1) the quantification of general efflux activity of mycobacterial strains (clinical isolates, mutants impaired in efflux or permeability) by the study of the transport (influx and efflux) of fluorescent compounds, such as ethidium bromide; and (2) the screening of compounds in search of inhibitors of efflux pumps, which could restore the effectiveness of antimicrobials that are subject to efflux.

Key words

Mycobacterium Efflux pumps Efflux inhibitors Ethidium bromide Fluorometry Accumulation assay Efflux assay 



This work was supported by grants FP7-260872 (European Union) “More Medicines for Tuberculosis,” BIO-2009-09405 from the Spanish Ministry for Science and Education, and project PTDC/BIA-MIC/121859/2010 from Fundação para a Ciência e a Tecnologia (FCT) Portugal. L. Rodrigues was supported by Fundación Agencia Aragonesa para la Investigación y el Desarrollo (ARAID).


  1. 1.
    Greulich KO (2004) Single molecule techniques for biomedicine and pharmacology. Curr Pharm Biotechnol 5:243–259CrossRefPubMedGoogle Scholar
  2. 2.
    Jernaes MW, Steen HB (1994) Staining of Escherichia coli for flow cytometry: influx and efflux of ethidium bromide. Cytometry 17:302–309CrossRefPubMedGoogle Scholar
  3. 3.
    Lomovskaya O, Bostian KA (2006) Practical applications and feasibility of efflux pump inhibitors in the clinic – a vision for applied use. Biochem Pharmacol 71:910–918CrossRefPubMedGoogle Scholar
  4. 4.
    Viveiros M, Martins A, Paixão L et al (2008) Demonstration of intrinsic efflux activity of Escherichia coli K-12 AG100 by an automated ethidium bromide method. Int J Antimicrob Agents 31:458–462CrossRefPubMedGoogle Scholar
  5. 5.
    Ramón-García S, Martín C, Aínsa JA et al (2006) Characterization of tetracycline resistance mediated by the efflux pump Tap from Mycobacterium fortuitum. J Antimicrob Chemother 57:252–259CrossRefPubMedGoogle Scholar
  6. 6.
    Viveiros M, Martins M, Couto I et al (2008) New methods for the identification of efflux mediated MDR bacteria, genetic assessment of regulators and efflux pump constituents, characterization of efflux systems and screening for inhibitors of efflux pumps. Curr Drug Targets 9:760–778CrossRefPubMedGoogle Scholar
  7. 7.
    Paixão L, Rodrigues L, Couto I et al (2009) Fluorometric determination of ethidium bromide efflux kinetics in Escherichia coli. J Biol Eng 3:18CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Ramón-García S, Mick V, Dainese E et al (2012) Functional and genetic characterization of the tap efflux pump in Mycobacterium bovis BCG. Antimicrob Agents Chemother 56:2074–2083CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    De Rossi E, Aínsa JA, Riccardi G (2006) Role of mycobacterial efflux transporters in drug resistance: an unresolved question. FEMS Microbiol Rev 30:36–52CrossRefPubMedGoogle Scholar
  10. 10.
    Adams KN, Takaki K, Connolly LE et al (2011) Drug tolerance in replicating mycobacteria mediated by a macrophage-induced efflux mechanism. Cell 145:39–53CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Ramón-García S, Martín C, Thompson CJ et al (2009) Role of the Mycobacterium tuberculosis P55 efflux pump in intrinsic drug resistance, oxidative stress responses, and growth. Antimicrob Agents Chemother 53:3675–3682CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Lee RE, Hurdle JG, Liu J et al (2014) Spectinamides: a new class of semisynthetic antituberculosis agents that overcome native drug efflux. Nat Med 20:152–158CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Balganesh M, Dinesh N, Sharma S et al (2012) Efflux pumps of Mycobacterium tuberculosis play a significant role in antituberculosis activity of potential drug candidates. Antimicrob Agents Chemother 56:2643–2651CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Balganesh M, Kuruppath S, Marcel N et al (2010) Rv1218c, an ABC transporter of Mycobacterium tuberculosis with implications in drug discovery. Antimicrob Agents Chemother 54:5167–5172CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Viveiros M, Martins M, Rodrigues L et al (2012) Inhibitors of mycobacterial efflux pumps as potential boosters for anti-tubercular drugs. Expert Rev Anti Infect Ther 10:983–998CrossRefPubMedGoogle Scholar
  16. 16.
    Rodrigues L, Ramos J, Couto I et al (2011) Ethidium bromide transport across Mycobacterium smegmatis cell-wall: correlation with antibiotic resistance. BMC Microbiol 11:35CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Rodrigues L, Villellas C, Bailo R et al (2013) Role of the Mmr efflux pump in drug resistance in Mycobacterium tuberculosis. Antimicrob Agents Chemother 57:751–757CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Machado L, Spengler G, Evaristo M et al (2011) Biological activity of twenty-three hydantoin derivatives on intrinsic efflux pump system of Salmonella enterica serovar Enteritidis NCTC 13349. In Vivo 25:769–772PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Liliana Rodrigues
    • 1
    • 2
    • 3
  • Miguel Viveiros
    • 4
  • José A. Aínsa
    • 1
    • 2
    Email author
  1. 1.Grupo de Genética de Micobacterias, Departamento de Microbiología, Medicina Preventiva y Salud Publica, Facultad de MedicinaUniversidad de ZaragozaZaragozaSpain
  2. 2.Centro de Investigación Biomédica en Red de Enfermedades Respiratorias (CIBERES)ZaragozaSpain
  3. 3.Fundación Agencia Aragonesa para la Investigación y Desarrollo (ARAID)ZaragozaSpain
  4. 4.Grupo de Micobactérias, Unidade de Microbiologia Médica, Instituto de Higiene e Medicina TropicalUniversidade Nova de LisboaLisbonPortugal

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