fluorescence) relative to the wild-type strain, indicating that efflux
pump activity is associated with the efflux of EtBr in M. bovis
BCG [25].
The protocols described in the previous edition of this publication have been used in several other reports, in which increased
accumulation of EtBr was found indicative of increased cell wall or
membrane permeability. In M. tuberculosis mutants defective in
enzymes responsible for ligating arabinogalactan to peptidoglycan,
an increase in EtBr accumulation rate was detected. This was
attributed to a general increase in cell wall permeability, rather
than to changes in efflux pump activity [26]. Similarly, an
M. tuberculosis mutant defective in a putative iron dicitrate-binding
protein accumulated significantly more EtBr than wild-type cells,
independently of the activity of efflux pumps [27]. In Mycobacterium marinum, a series of suppressor strains carrying defects in
diverse genes related with the function of Esx-1 secretion system
were shown to have higher levels of EtBr accumulation, hence
demonstrating that such alterations had resulted from increased
cell wall permeability [28]. Bacteriocin AS-48 kills M. tuberculosis
and other mycobacteria by interfering with cell membrane functions, and this was demonstrated since, in the presence of AS-48,
EtBr accumulation increased notably in comparison with
non-treated cells [29].
Also, these protocols were used to test whether diverse compounds could have any efflux inhibitory activity. It was hypothesized that pyronaridine, an antimalarial drug for which
antituberculosis activity was recently reported [30], could function
as an efflux inhibitor since it modulates the activity of P-glycoprotein. However, EtBr accumulation experiments could not demonstrate any significant activity of pyronaridine as an efflux inhibitor in
M. tuberculosis [30]. In another report, a series of inhibitors of the
sole signal peptidase in M. tuberculosis did not significantly alter
accumulation of EtBr, demonstrating their specificity against this
target [31]. Natural product carvacrol has both antimicrobial and
efflux inhibitory activities against several bacterial species. In
M. tuberculosis, EtBr accumulation was greatly increased in the
presence of carvacrol and derivatives, and this effect notably higher
than that produced by VP, hence demonstrating efflux inhibitory
activity of this series of compounds [32].
Finally, other reports have used these protocols with the aim of
assessing general efflux activity of mycobacterial strains. VP, TZ,
CPZ, flupentixol, and haloperidol increased accumulation and
reduced efflux of EtBr in a set of multidrug and extensively drugresistant strains of M. tuberculosis, hence demonstrating its activity
as efflux inhibitors in addition of being ion channel blockers
[33]. Similarly, VP, TZ, and CPZ were used to demonstrate innate
efflux activity in a series of clinical strains of M. tuberculosis having
different resistance-conferring mutations [24].
Efflux and Permeability
235
pump activity is associated with the efflux of EtBr in M. bovis
BCG [25].
The protocols described in the previous edition of this publication have been used in several other reports, in which increased
accumulation of EtBr was found indicative of increased cell wall or
membrane permeability. In M. tuberculosis mutants defective in
enzymes responsible for ligating arabinogalactan to peptidoglycan,
an increase in EtBr accumulation rate was detected. This was
attributed to a general increase in cell wall permeability, rather
than to changes in efflux pump activity [26]. Similarly, an
M. tuberculosis mutant defective in a putative iron dicitrate-binding
protein accumulated significantly more EtBr than wild-type cells,
independently of the activity of efflux pumps [27]. In Mycobacterium marinum, a series of suppressor strains carrying defects in
diverse genes related with the function of Esx-1 secretion system
were shown to have higher levels of EtBr accumulation, hence
demonstrating that such alterations had resulted from increased
cell wall permeability [28]. Bacteriocin AS-48 kills M. tuberculosis
and other mycobacteria by interfering with cell membrane functions, and this was demonstrated since, in the presence of AS-48,
EtBr accumulation increased notably in comparison with
non-treated cells [29].
Also, these protocols were used to test whether diverse compounds could have any efflux inhibitory activity. It was hypothesized that pyronaridine, an antimalarial drug for which
antituberculosis activity was recently reported [30], could function
as an efflux inhibitor since it modulates the activity of P-glycoprotein. However, EtBr accumulation experiments could not demonstrate any significant activity of pyronaridine as an efflux inhibitor in
M. tuberculosis [30]. In another report, a series of inhibitors of the
sole signal peptidase in M. tuberculosis did not significantly alter
accumulation of EtBr, demonstrating their specificity against this
target [31]. Natural product carvacrol has both antimicrobial and
efflux inhibitory activities against several bacterial species. In
M. tuberculosis, EtBr accumulation was greatly increased in the
presence of carvacrol and derivatives, and this effect notably higher
than that produced by VP, hence demonstrating efflux inhibitory
activity of this series of compounds [32].
Finally, other reports have used these protocols with the aim of
assessing general efflux activity of mycobacterial strains. VP, TZ,
CPZ, flupentixol, and haloperidol increased accumulation and
reduced efflux of EtBr in a set of multidrug and extensively drugresistant strains of M. tuberculosis, hence demonstrating its activity
as efflux inhibitors in addition of being ion channel blockers
[33]. Similarly, VP, TZ, and CPZ were used to demonstrate innate
efflux activity in a series of clinical strains of M. tuberculosis having
different resistance-conferring mutations [24].
Efflux and Permeability
235