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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Acknowledgement
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).
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Rodrigues, L., Viveiros, M., Aínsa, J.A. (2015). Measuring Efflux and Permeability in Mycobacteria. In: Parish, T., Roberts, D. (eds) Mycobacteria Protocols. Methods in Molecular Biology, vol 1285. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2450-9_13
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DOI: https://doi.org/10.1007/978-1-4939-2450-9_13
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