physiological processes such as redox potential and bacterial and
colony morphology [7, 8]. Therefore, efflux pumps must be considered in drug discovery programs [5, 9–11] as their inhibition
could contribute to new therapeutic strategies for tuberculosis and
drug-resistant tuberculosis [12, 13].
Several methods have been used to detect the transport of
radiolabeled, metal-labeled, or fluorescent substrates through the
bacterial cell wall, which can be correlated with altered permeability
and/or efflux activity of the bacteria [7, 14–21]. We have developed a fluorometric method for the assessment of permeability and
efflux activity in mycobacterial strains (reference strains, mutants
impaired in efflux or permeability, and clinical isolates) that use
ethidium bromide (EtBr), a common efflux substrate [18, 20–
25]. EtBr has been shown to be a particularly suitable probe for
these studies, since it emits weak fluorescence in aqueous solution
(extracellular medium) and becomes strongly fluorescent as it accumulates in non-polar and hydrophobic environments, such as the
periplasmic space of Gram-negative bacteria or cytoplasm of Grampositive bacteria [15]. This methodology allows easy and accurate
detection and quantification of the transport of EtBr through the
bacterial cell wall. Kinetics of EtBr accumulation and efflux can be
assayed separately, hence examining the contribution of both processes to EtBr transport [18, 20–25].
Evaluation of accumulation or efflux of EtBr is performed on a
real-time basis, with the possibility of testing many samples simultaneously and at different experimental conditions with the same
set of bacterial suspensions, hence maximizing reproducibility and
significance. Moreover, it also allows the screening of compounds
as potential efflux inhibitors or cell wall permeabilizers that could
be used in the future as adjuvants of antimycobacterial
therapy [12].
The EtBr accumulation assay assesses the ability of the strain to
handle increasing concentrations of EtBr. The concept behind this
assay is that when the ability of the efflux systems to extrude EtBr is
exceeded, EtBr will accumulate over a period of time, ultimately
reaching a quasi steady-state. Figure 1 shows an example of this
type of assay with M. bovis BCG. The accumulation of EtBr is
relatively stable (no significant increase in fluorescence over time)
at the concentration of 0.125 μg/mL, over a period of 60 min. In
the presence of an efflux inhibitor (Fig. 2), such as chlorpromazine
(CPZ), thioridazine (TZ), or verapamil (VP), accumulation of EtBr
increases due to the inhibition of efflux in comparison with
non-treated cells.
In an efflux assay (Fig. 3, adapted from Ref. 25), bacterial cells
are first de-energized with an efflux inhibitor and loaded with EtBr
during a period of time (usually 1 h) and then placed in an EtBr-free
solution. This assay allows the evaluation of efflux activity under
different conditions: (1) in the presence of a carbon source, such as
232
Liliana Rodrigues et al.
colony morphology [7, 8]. Therefore, efflux pumps must be considered in drug discovery programs [5, 9–11] as their inhibition
could contribute to new therapeutic strategies for tuberculosis and
drug-resistant tuberculosis [12, 13].
Several methods have been used to detect the transport of
radiolabeled, metal-labeled, or fluorescent substrates through the
bacterial cell wall, which can be correlated with altered permeability
and/or efflux activity of the bacteria [7, 14–21]. We have developed a fluorometric method for the assessment of permeability and
efflux activity in mycobacterial strains (reference strains, mutants
impaired in efflux or permeability, and clinical isolates) that use
ethidium bromide (EtBr), a common efflux substrate [18, 20–
25]. EtBr has been shown to be a particularly suitable probe for
these studies, since it emits weak fluorescence in aqueous solution
(extracellular medium) and becomes strongly fluorescent as it accumulates in non-polar and hydrophobic environments, such as the
periplasmic space of Gram-negative bacteria or cytoplasm of Grampositive bacteria [15]. This methodology allows easy and accurate
detection and quantification of the transport of EtBr through the
bacterial cell wall. Kinetics of EtBr accumulation and efflux can be
assayed separately, hence examining the contribution of both processes to EtBr transport [18, 20–25].
Evaluation of accumulation or efflux of EtBr is performed on a
real-time basis, with the possibility of testing many samples simultaneously and at different experimental conditions with the same
set of bacterial suspensions, hence maximizing reproducibility and
significance. Moreover, it also allows the screening of compounds
as potential efflux inhibitors or cell wall permeabilizers that could
be used in the future as adjuvants of antimycobacterial
therapy [12].
The EtBr accumulation assay assesses the ability of the strain to
handle increasing concentrations of EtBr. The concept behind this
assay is that when the ability of the efflux systems to extrude EtBr is
exceeded, EtBr will accumulate over a period of time, ultimately
reaching a quasi steady-state. Figure 1 shows an example of this
type of assay with M. bovis BCG. The accumulation of EtBr is
relatively stable (no significant increase in fluorescence over time)
at the concentration of 0.125 μg/mL, over a period of 60 min. In
the presence of an efflux inhibitor (Fig. 2), such as chlorpromazine
(CPZ), thioridazine (TZ), or verapamil (VP), accumulation of EtBr
increases due to the inhibition of efflux in comparison with
non-treated cells.
In an efflux assay (Fig. 3, adapted from Ref. 25), bacterial cells
are first de-energized with an efflux inhibitor and loaded with EtBr
during a period of time (usually 1 h) and then placed in an EtBr-free
solution. This assay allows the evaluation of efflux activity under
different conditions: (1) in the presence of a carbon source, such as
232
Liliana Rodrigues et al.