Mycobacteria Protocols pp 227-239

Part of the Methods in Molecular Biology book series (MIMB, volume 1285) | Cite as

Measuring Efflux and Permeability in Mycobacteria

  • Liliana Rodrigues
  • Miguel Viveiros
  • José A. Aínsa
Protocol

Abstract

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 

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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
  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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