Chapter 9
Measuring Efflux and Permeability in Mycobacteria
Liliana Rodrigues, Jose ´ A. Aı ´nsa, and Miguel Viveiros
Abstract
Mycobacteria are intrinsically resistant to most antimicrobials, which is generally attributed to the impermeability of their cell wall that considerably limits drug uptake. Moreover, like in other pathogenic bacteria,
active efflux systems have been widely characterized from diverse mycobacterial species in laboratory
conditions, showing that they can promote resistance by extruding noxious compounds prior to their
reaching their intended targets. Therefore, the intracellular concentration of a given compound is determined by the balance between permeability, influx, and efflux.
Given the urgent need to discover and develop novel antimycobacterial compounds in order to design
effective therapeutic strategies, the contributions to drug resistance made by the controlled permeability of
the cell wall and the increased activity of efflux pumps must be determined. In this chapter, we will describe
a method that allows (1) the measuring of permeability and the quantification of general efflux activity of
mycobacteria, 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 agents that increase the permeability of the cell
wall and efflux inhibitors that could restore the effectiveness of antimicrobials that are subject to efflux.
Key words Mycobacterium, Permeability, Efflux pumps, Efflux inhibitors, Ethidium bromide, Fluorometry, Accumulation assay, Efflux assay
1 Introduction
Bacterial efflux pumps are membrane proteins that can transport a
broad range of substrates, including antimicrobials, from the cytoplasm to the exterior of the cell, fueled by the energy provided by
ATP hydrolysis or the transmembrane proton gradient [1]. Consequently, increased expression of efflux pump genes confers a
low-level resistance phenotype and it has been postulated that,
under these conditions, bacteria have greater chances of acquiring
chromosomal mutation(s) conferring stable and heritable higher
levels of drug resistance [2, 3]. Mycobacteria, similarly to other
bacterial genera, have a large number of active efflux pumps that
have a role in low-level resistance to drugs such as tetracycline,
aminoglycosides, fluoroquinolones [4, 5], macrophage-induced
drug tolerance to isoniazid and rifampicin [6], and other
Tanya Parish and Anuradha Kumar (eds.), Mycobacteria Protocols, Methods in Molecular Biology, vol. 2314,
https://doi.org/10.1007/978-1-0716-1460-0_9, © Springer Science+Business Media, LLC, part of Springer Nature 2021
231
Measuring Efflux and Permeability in Mycobacteria
Liliana Rodrigues, Jose ´ A. Aı ´nsa, and Miguel Viveiros
Abstract
Mycobacteria are intrinsically resistant to most antimicrobials, which is generally attributed to the impermeability of their cell wall that considerably limits drug uptake. Moreover, like in other pathogenic bacteria,
active efflux systems have been widely characterized from diverse mycobacterial species in laboratory
conditions, showing that they can promote resistance by extruding noxious compounds prior to their
reaching their intended targets. Therefore, the intracellular concentration of a given compound is determined by the balance between permeability, influx, and efflux.
Given the urgent need to discover and develop novel antimycobacterial compounds in order to design
effective therapeutic strategies, the contributions to drug resistance made by the controlled permeability of
the cell wall and the increased activity of efflux pumps must be determined. In this chapter, we will describe
a method that allows (1) the measuring of permeability and the quantification of general efflux activity of
mycobacteria, 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 agents that increase the permeability of the cell
wall and efflux inhibitors that could restore the effectiveness of antimicrobials that are subject to efflux.
Key words Mycobacterium, Permeability, Efflux pumps, Efflux inhibitors, Ethidium bromide, Fluorometry, Accumulation assay, Efflux assay
1 Introduction
Bacterial efflux pumps are membrane proteins that can transport a
broad range of substrates, including antimicrobials, from the cytoplasm to the exterior of the cell, fueled by the energy provided by
ATP hydrolysis or the transmembrane proton gradient [1]. Consequently, increased expression of efflux pump genes confers a
low-level resistance phenotype and it has been postulated that,
under these conditions, bacteria have greater chances of acquiring
chromosomal mutation(s) conferring stable and heritable higher
levels of drug resistance [2, 3]. Mycobacteria, similarly to other
bacterial genera, have a large number of active efflux pumps that
have a role in low-level resistance to drugs such as tetracycline,
aminoglycosides, fluoroquinolones [4, 5], macrophage-induced
drug tolerance to isoniazid and rifampicin [6], and other
Tanya Parish and Anuradha Kumar (eds.), Mycobacteria Protocols, Methods in Molecular Biology, vol. 2314,
https://doi.org/10.1007/978-1-0716-1460-0_9, © Springer Science+Business Media, LLC, part of Springer Nature 2021
231