Measuring efflux and permeability in Mycobacteria

Research output: Chapter in Book/Report/Conference proceedingChapterpeer-review

20 Citations (Scopus)

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.
Original languageEnglish
Title of host publication Methods in molecular biology
Subtitle of host publicationmycobacteria protocols
EditorsTanya Parish, David M Roberts
Place of PublicationNew York
PublisherSpringer Science Business Media
Pages227-239
Number of pages13
Volume1285
Edition2015
ISBN (Electronic)978-1-49392450-9
DOIs
Publication statusPublished - 2015

Keywords

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

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