Antimicrobial Agents and Chemotherapy, April 1998, p. 795-800, Vol. 42, No. 4
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Accumulation of Norfloxacin by Mycobacterium
aurum and Mycobacterium smegmatis
Kerstin J.
Williams,1
Gavin A. C.
Chung,2 and
Laura J. V.
Piddock1,*
Antimicrobial Agents Research Group,
Department of Infection, The Medical School, University of Birmingham,
Edgbaston, Birmingham B15 2TT,1 and
Glaxo Wellcome Medicines Research Center, Stevenage SG1
2NY, Hertfordshire,2 United Kingdom
Received 18 August 1997/Returned for modification 24 October
1997/Accepted 22 December 1997
 |
ABSTRACT |
The modified fluorescence method was used to determine the
accumulation of norfloxacin by Mycobacterium aurum A+ and
Mycobacterium smegmatis mc2155. By using an
exogenous norfloxacin concentration of 10 µg/ml, a steady-state
concentration (SSC) of 160 to 180 ng of norfloxacin/mg of cells was
obtained for M. aurum, and an SSC of 120 to 140 ng of
norfloxacin/mg of cells obtained for M. smegmatis. For both species of mycobacteria, the SSC was achieved within 5 min. The silicon
oil method was investigated and gave higher SSCs than the modified
fluorescence method. Further studies on the mechanism of norfloxacin
accumulation by M. aurum were performed. An increase in the
pH of the wash buffer from 7.0 to 9.0 did not significantly affect the
final SSC obtained. Accumulation was nonsaturated over a norfloxacin
concentration range of 0 to 100 µg/ml, and the proton motive force
inhibitor 2,4-dinitrophenol (1 and 2 mM), whether it was added before
or after norfloxacin was added, had no effect on the final SSC
obtained. 2,4-Dinitrophenol also had no effect on norfloxacin
accumulation by M. smegmatis. Furthermore, norfloxacin accumulation by M. aurum was unaffected by the presence of
either Tween 80 or subinhibitory concentrations of ethambutol in the growth medium. Therefore, it is proposed that norfloxacin accumulation by mycobacteria occurs by simple, energy-independent diffusion.
| |
INTRODUCTION |
Although several hundred
antimicrobial agents are available worldwide, very few are effective
against Mycobacterium tuberculosis and even fewer are
effective against atypical mycobacteria, such as Mycobacterium
avium complex. It has been widely proposed that the mycobacterial
cell wall has low permeability and that this reduced permeability plays
a major role in the intrinsic resistance of mycobacteria to most
antibiotics (4, 14, 15). However, few studies of the
accumulation or transport of antituberculous agents by mycobacteria or
the role that permeability plays in mycobacterial drug resistance have
been performed.
The fluoroquinolones are broad-spectrum, bactericidal antimicrobial
agents that were developed in the 1980s and that are active against
gram-positive bacteria, gram-negative bacteria, and some mycobacteria
(6, 29, 32). Ofloxacin, ciprofloxacin, and, in particular,
sparfloxacin show good in vitro activity against M. tuberculosis (3, 16, 18). The fluoroquinolones act by inhibiting the topoisomerase II enzyme DNA gyrase, which catalyzes the
unwinding or negative supercoiling of double-stranded DNA during
replication (12). Topoisomerase IV, which is encoded by
parC and parE (grlA and
grlB in Staphylococcus aureus), has been proposed
as a secondary target of fluoroquinolones in Escherichia coli when a susceptible DNA gyrase is lacking (13, 38).
However, for some gram-positive bacteria such as S. aureus
and Streptococcus pneumoniae, topoisomerase IV is thought to
be the primary target and DNA gyrase is thought to be the secondary
target of fluoroquinolones (25, 26). Both enzymes are
intracellular; therefore, in order to exert their antibacterial effect,
the fluoroquinolones must cross the bacterial cell envelope.
Fluoroquinolone resistance arises due to mutations in gyrA
and, less frequently, gyrB, which encode the A and B
subunits of DNA gyrase, respectively (30). Although other
mechanisms of resistance to fluoroquinolones have been documented for
other bacteria, e.g., parC mutations, reduced intracellular
accumulation, and enhanced drug efflux (30), only
gyrA mutations have been reported to give rise to
fluoroquinolone resistance in clinical M. tuberculosis
isolates (1, 39). Due to the moderate in vivo activity of
fluoroquinolones and increasing resistance to these drugs, the
fluoroquinolones are generally considered second-line agents against
tuberculosis (5). However, with the increasing incidence of
multiple-drug-resistant M. tuberculosis and the increased use of fluoroquinolones in combination with other agents for the treatment of tuberculosis, much attention has focused upon the therapeutic value of the fluoroquinolones for the treatment of tuberculosis and other mycobacterial diseases (42).
Although it is thought that the hydrophobic mycobacterial cell wall
acts as an efficient barrier to hydrophilic molecules (4, 14,
15), hydrophilic nutrients must be taken up by the cell, and some
hydrophilic drugs, such as isoniazid and ethambutol, are extremely
effective against M. tuberculosis (23).
Mycobacterial porins have recently been characterized in
Mycobacterium chelonae (33, 34) and
Mycobacterium smegmatis (35), and it is thought that similar porins are widely distributed among mycobacteria (4). However, these porins are thought to be both less
abundant and less efficient than porins of other bacterial species,
such as OmpF in E. coli and OprD in Pseudomonas
aeruginosa (33, 34). For example, studies have shown
that the cell wall permeability of M. chelonae is about
10-fold lower than that of P. aeruginosa to cephalosporins
(14). However, there does appear to be significant differences in cell wall permeability between various mycobacterial species. For example, M. smegmatis and M. tuberculosis have been shown to be more permeable to 
-lactams
than M. chelonae (7, 35). Furthermore,
hydrophilic drugs have a wide range of MICs for mycobacteria and the
low levels of cell wall permeability to these drugs may play a major
role in this intrinsic drug resistance (15).
The accumulation of norfloxacin by E. coli and S. aureus is well documented (22, 24), and recently,
studies on the accumulation of norfloxacin by M. smegmatis
CNCM 7326 and M. tuberculosis H37Ra have been reported
(10, 17). Mycobacterium aurum and M. smegmatis are nonpathogenic, fast-growing, saprophytic
mycobacteria and give homogeneous cell suspensions without the use of
detergents, such as Tween 80. M. aurum A+ possesses a MIC
profile similar to that of M. tuberculosis for the
first-line antituberculous drugs and is used in a high-throughput
screenings for the detection of novel antituberculous agents
(8). M. smegmatis mc2155 is the
strain of M. smegmatis most commonly used in laboratory experiments and genetic manipulations. Therefore, in order to gain
further insight into the mechanisms of fluoroquinolone accumulation by
saprophytic mycobacteria and, by extrapolation, by M. tuberculosis, this study sought to determine and compare the
accumulation of norfloxacin by M. aurum and M. smegmatis and to characterize the mechanism of norfloxacin
accumulation by M. aurum.
| |
MATERIALS AND METHODS |
Bacterial strains and growth conditions.
M. aurum A+
(Pasteur Institute, Paris, France) and M. smegmatis
mc2155 (W. R. Jacobs, Albert Einstein College of
Medicine, New York, N.Y.) were maintained on Lowenstein-Jensen slopes
and were cultured on Middlebrook 7H11 agar (Difco, West Molesy, United
Kingdom) supplemented with 10% (vol/vol) OADC (oleic acid, albumin
fraction V, dextrose, and catalase). Cultures were grown aerobically in Middlebrook 7H9 broth (Difco) supplemented with 10% (vol/vol) ADC
(albumin fraction V, dextrose, and catalase) at 37°C.
Growth kinetics.
A total of 250 ml of 7H9 broth was
inoculated with a 3-day-old culture (25 ml) of M. aurum
followed by incubation at 37°C with a 1-min episode of shaking (180 rpm) every 4 h. For M. smegmatis, 250 ml of 7H9 broth
was inoculated with an overnight culture (25 ml), followed by
incubation at 37°C with shaking at 180 rpm. Growth was evaluated by
measuring the optical density (OD) of the growing culture at 550 nm.
The viable cell counts of the growing cultures were determined by
diluting culture samples in sterile distilled water and spreading 100 µl of each dilution over the surfaces of 7H11 agar plates. The plates
were incubated at 37°C, and the colonies were counted after 48 and
72 h. The numbers of CFU per milliliter were calculated by
counting the number of colonies on the agar plate and assuming that one
viable organism gives rise to one colony. Cell dry weights for M. aurum and M. smegmatis were determined by removing
20-ml aliquots of the growing culture at selected OD readings. The
cells were immediately centrifuged at 3,003 × g
(Mistral; MSE) for 15 min at 4°C. The cell pellets were washed with
10 ml of sterile distilled water and centrifuged as described above.
The cell pellets were dried overnight in a 60°C hot-air oven and
weighed. The tubes were kept under desiccative conditions and were
reweighed until a stable weight was obtained.
Chemicals.
Norfloxacin and ethambutol were obtained from
Sigma Chemical Co., Poole, United Kingdom, and were made up according
to the manufacturer's instructions. 2,4-Dinitrophenol (DNP; 50 mM;
Sigma) was dissolved in 3 parts dimethyl sulfoxide to 2 parts distilled water, and the solution was stored at 
20°C.
Antibiotic susceptibility testing.
MICs were determined in
7H9 broth by a standard microdilution technique with an inoculum of
105 CFU/ml. The plates were incubated at 37°C, and the
results were read after 48 and 72 h. The MIC was defined as the
lowest concentration of drug at which no visible growth was observed.
Measurement of norfloxacin accumulation.
The modified
fluorometric method of Mortimer and Piddock (24) was altered
slightly to accommodate the growth characteristics of the mycobacteria.
Cells were grown to the mid-exponential phase in 7H9 broth
(A550 of 0.1 to 0.12 for M. aurum and
A550 of 0.4 to 0.5 for M. smegmatis)
and were harvested by centrifugation in a Mistral centrifuge (MSE) at
3,003 × g for 20 min at 15°C. The cells were washed
in 10 ml of 50 mM sodium phosphate buffer (pH 7) and were concentrated
with the same buffer to give the bacterial suspension of M. aurum an A550 of 5 and that of M. smegmatis an A550 of 20. The bacterial
suspension was placed in a stirring 37°C water bath and was left for
10 min to equilibrate. Norfloxacin was added to the required final
concentration (10 µg/ml), and 1-ml samples were removed at timed
intervals. The cells were immediately centrifuged at 12,000 × g (Jouan MR1812) for 3 min at 4°C. The cell pellets were
washed once with ice-cold sodium phosphate buffer (50 mM; pH 7) and
were resuspended in 1 ml of 0.1 M glycine hydrochloride (pH 3). The
samples were left overnight at room temperature with agitation to lyse
the cells. The efficacy of this lysis procedure has been assessed
previously by transmission electron microscopy (10). On the
following day, the samples were centrifuged at 12,000 × g (Jouan MR1812), and the fluorescence of the supernatants was determined at an excitation wavelength of 281 nm and an emission wavelength of 440 nm. Background fluorescence (i.e., that of
norfloxacin-free controls) possibly due to the porphyrins present in
mycobacterial cells (10) was subtracted from the
fluorescence for all samples taken. A standard curve of norfloxacin
fluorescence in the presence of 0.1 M glycine hydrochloride (pH 3) was
constructed, and the results are expressed as nanograms of norfloxacin
per milligram (dry weight) of cells.
To test for the possible saturation of transport, the accumulation of
norfloxacin over a concentration range of 0 to 100 µg/ml was studied
with M. aurum. The cells were incubated at 37°C for 5 min
in the presence of each norfloxacin concentration.
To study the effect of the pH of the wash buffer on norfloxacin
adsorption to the cell wall, accumulation experiments were performed,
as described above, except that the cell pellets were washed with
either pH 7 or pH 9 sodium phosphate buffer. The pH 9 buffer was
prepared by adding a few drops of 0.1 M sodium hydroxide to the pH 7 buffer.
To study the effect of DNP on norfloxacin accumulation by mycobacteria,
cells were treated with 1 or 2 mM DNP either 10 min before or 5 min
after the addition of norfloxacin to the cell culture.
The silicon oil method was performed essentially as described by Li et
al. (19). The cells were prepared as described above. Samples (0.5 ml) were removed at timed intervals and were placed on
0.5-ml aliquots of silicon oil (a 6:5 [vol/vol] mixture; Dow Corning
silicon oils) in Eppendorf tubes. The tubes were immediately centrifuged at 13,249 × g (Microcentaur; MSE), and the
top aqueous layer was removed. The tubes were then snap frozen in
liquid nitrogen for 1 s and the oil was removed by careful
pipetting. The cell pellet was resuspended in 1 ml of 0.1 M glycine
hydrochloride (pH 3) and left overnight with agitation to lyse the
cells. The accumulated norfloxacin was quantified as described above.
Statistical analysis.
The differences in the accumulation
data obtained by the modified fluorescence method and the silicon oil
method were analyzed by Student's t test. A P
value of <0.05 was considered significant.
| |
RESULTS |
Mycobacterial growth curves.
An example of a growth curve
obtained for M. aurum is shown in Fig.
1. For M. aurum, no lag phase
was observed and the logarithmic phase lasted about 3 days (72 h); the
generation time was 21 h. For M. smegmatis, a lag phase
of less than 2 h was observed and the logarithmic phase lasted
12 h; the generation time was 2 h.
Accumulation of norfloxacin.
The MIC of norfloxacin for both
M. aurum and M. smegmatis was 2 µg/ml. The
accumulation of norfloxacin (10 µg/ml) by M. aurum resulted in a steady-state concentration (SSC) of 160 to 180 ng of
norfloxacin/mg of cells (Fig. 2).
Accumulation was rapid; approximately 80% of the final concentration
of norfloxacin was accumulated within the first minute of drug
exposure. A further 30 ng of norfloxacin/mg of cells was accumulated at
between 1 and 5 min. The accumulation of norfloxacin by M. smegmatis resulted in an SSC of 120 to 140 ng of norfloxacin/mg of
cells (Fig. 2). Accumulation kinetics very similar to those for
M. aurum were observed for M. smegmatis. By the
modified fluorescence method (24), the cell cultures are
concentrated to an OD of 20. However, due to the low density of
M. aurum cells, the cells were concentrated to an OD of 5, because an OD of 20 could not be achieved. This ensured both a sufficient volume of culture for performance of an experiment and
sufficient numbers of cells for intracellular norfloxacin detection.
The accumulation of norfloxacin by M. smegmatis by the
silicon oil method described by Li et al. (19) was studied
at 0 and 37°C (Table 1). Higher
readings were obtained by the silicon oil method, but these were
subsequently determined to be due to high levels of norfloxacin
adsorption onto the cell. Intracellular accumulation values, i.e.,
accumulation at 37°C minus binding at 0°C, were similar by both
methods (Table 1) (19) and were not found to be
statistically significantly different (P > 0.05 for
all comparisons).
Effect of exogenous drug concentration on norfloxacin accumulation
by M. aurum.
To determine if norfloxacin transport
into M. aurum was saturable, the effect of the exogenous
norfloxacin concentration on accumulation was studied over a
concentration range of 0 to 100 µg/ml; Fig.
3 shows that norfloxacin accumulation by
M. aurum was not saturated. The concentration of norfloxacin
accumulated increased at a rate proportional to the increase in the
exogenous norfloxacin concentration. An increase in the exogenous
norfloxacin concentration of 20 µg/ml resulted in an increase of the
accumulated norfloxacin concentration of 190 ng of norfloxacin/mg of
cells.
Effect of the pH of the wash buffer on norfloxacin accumulation by
M. aurum.
According to Corti et al. (10), an
increase in the pH of the cell washing buffer from 7 to 9 reduces the
amount of norfloxacin adsorption to the cell wall of M. smegmatis during an uptake experiment. Therefore, the effect of an
increase in the wash buffer pH from 7 to 9 on the accumulation of
norfloxacin by M. aurum was studied. Experiments were
performed at 0°C with either pH 7 or pH 9 wash buffer to determine
the level of adsorption of norfloxacin to M. aurum cells.
The concentration of norfloxacin absorbed to the M. aurum
cell wall at 0°C was greater when a pH 7 wash buffer was used than
when a pH 9 wash buffer was used (data not shown). However, at 37°C
the pH of the wash buffer did not significantly affect the final
concentration of norfloxacin accumulated by M. aurum (Fig.
4).
Effect of metabolic inhibitors on norfloxacin accumulation by
M. aurum.
To determine whether the accumulation of
norfloxacin by M. aurum is energy independent or whether
norfloxacin is actively effluxed from M. aurum, cells were
treated with 1 or 2 mM DNP either 10 min before, 1 h before, or 5 min after the addition of norfloxacin (10 µg/ml) to the cell culture.
DNP at 2 mM has previously been used to study the effect of metabolic
inhibitors on quinolone accumulation by bacteria (9, 20).
Although 10 min should be sufficient to dissipate the proton motive
force in M. aurum, because of the longer generation time of
M. aurum (21 h), DNP was also added 1 h prior to the
addition of norfloxacin. Norfloxacin accumulation by M. aurum was unaffected by the presence of 1 and 2 mM DNP. In all
experiments (n = 3), whether the metabolic inhibitor
was added before or after the addition of norfloxacin, an SSC of 160 to
180 ng of norfloxacin/mg of cells was obtained (data not shown).
To determine if norfloxacin is actively effluxed from M. smegmatis, 2 mM DNP was added 5 min after the addition of
norfloxacin and the effect on accumulation was monitored. As with
M. aurum, norfloxacin accumulation in M. smegmatis was unaffected by the addition of DNP; an SSC of 120 to
140 ng of norfloxacin/mg of cells was obtained (data not shown).
Effect of ethambutol on norfloxacin accumulation by M. aurum.
To study the effect of ethambutol on norfloxacin
accumulation, ethambutol (0.5 or 1 µg/ml) was added to the growing
M. aurum culture 12, 24, or 48 h prior to cell
harvesting and was added 10 min prior to the addition of norfloxacin to
ensure the presence of ethambutol throughout the experiment. These
times should produce an M. aurum culture that had undergone
one, two, or three replication cycles in the presence of
ethambutol. In all experiments, an SSC of approximately 180 ng of
norfloxacin/mg of cells (data not shown) was obtained. The activity of
norfloxacin, with or without ethambutol (0.5 or 1 µg/ml), was studied
to determine if ethambutol affected the MIC of norfloxacin for M. aurum; the MIC was unaltered.
Effect of Tween 80 on norfloxacin accumulation by M. aurum.
To determine the effect of Tween 80 in the growth medium on
norfloxacin accumulation, cells were grown either in 7H9 alone or in
7H9 supplemented with 0.05% Tween 80. In all experiments, an SSC of
approximately 180 ng of norfloxacin/mg of cells was obtained (data not
shown) whether Tween 80 was present or not. Furthermore, the MIC of
norfloxacin for M. aurum was unaltered in the presence of
Tween 80.
| |
DISCUSSION |
A modified fluorescence method was used to determine the level of
accumulation of norfloxacin by M. aurum A+ and M. smegmatis mc2155 and to study the mechanism of
norfloxacin accumulation by M. aurum. The fluorescence
method exploits the natural fluorescence of the quinolone nucleus and
has been used extensively for measuring the level of quinolone
accumulation by bacteria (11, 22, 24).
We have already reported that for 14C-rifampin accumulation
by S. aureus and E. coli, the silicon oil method
gave rise to SSCs higher than those obtained by a chilled
centrifugation method similar to that used for fluoroquinolones
(40). Recently, Lopez-Hernandez et al. (21) also
reported higher accumulation values when using the silicon oil method
compared with those obtained by the fluorescence method to measure the
level of accumulation of fluoroquinolones in Acinetobacter
baumannii. Therefore, the silicon oil method described by Li et
al. (19) was used to study the level of accumulation of
norfloxacin by M. smegmatis. The silicon oil method gave
much greater values at 37 and 0°C than the fluorescence method.
Therefore, although the two methods give similar results for
intracellular accumulation, cell adsorption (values at 0°C) must be
calculated when the silicon oil method is used. Although the silicon
oil method has the advantage of quick separation of cells from drug, the high SSCs obtained as a result of not including a cell washing stage can be misleading.
Norfloxacin accumulation by mycobacteria showed kinetics similar to
those reported for other bacterial species, such as E. coli
and S. aureus (28). Furthermore, the
concentration of norfloxacin accumulated by both species of
mycobacteria falls within the range of concentrations previously
reported for gram-positive and gram-negative bacteria (2, 22,
24).
The permeability of the mycobacterial cell wall, at least to
hydrophilic agents, appears to vary greatly. For example, the permeability of M. chelonae to -lactams is about 3 orders
of magnitude lower than that of the E. coli outer membrane
and 10 times lower than the permeability of P. aeruginosa
(4). However, this study shows that the level of
accumulation of norfloxacin by M. aurum and M. smegmatis is similar to or even slightly higher than that by
E. coli and S. aureus. The SSCs of norfloxacin in M. smegmatis mc2155 reported by Liu et al.
(20) were similar to those obtained in the current study.
However, Corti et al. (10) reported an SSC of 35 ng of
norfloxacin/mg of cells for a different strain of M. smegmatis (strain CNCM 7326; MIC, 4 or 5 µg/ml). Kocagoz et al.
(17) reported an SSC of 110 pmol of norfloxacin/mg of cells
(approximately 35 ng of norfloxacin/mg of cells) for M. tuberculosis H37Ra, suggesting that M. tuberculosis is
less permeable to norfloxacin than M. aurum and M. smegmatis. Clearly, the permeability of mycobacteria to
norfloxacin, and presumably other agents, varies not only between
mycobacterial species but also between different strains of the same
species.
The uptake of quinolones by gram-negative bacteria is proposed to occur
by passive diffusion through porin channels or the lipid bilayer,
depending on the hydrophobicity of the quinolone (28), and
the uptake of quinolones by gram-positive bacteria is thought to
involve simple diffusion across the cytoplasmic membrane
(11). Norfloxacin is a very hydrophilic quinolone, with a
partition coefficient in n-octanol-phosphate buffer (pH 7.2) of 0.022 (2). Norfloxacin accumulation by M. aurum was nonsaturable over the concentration range studied (0 to
100 µg/ml), and transport was apparently unaffected by the presence
of DNP. Therefore, it is proposed that norfloxacin enters M. aurum by an energy-independent, nonsaturable porin pathway.
Mycobacterial porins have been characterized in M. chelonae
and M. smegmatis and are thought to exist in all
mycobacterial species, but they are proposed to be both less abundant
and less efficient than other bacterial porins (33, 34).
However, at least for norfloxacin, the mycobacterial cell wall does not
act as a significant barrier to drug accumulation.
Although the major form of quinolone resistance found in bacteria is
altered DNA gyrase and/or topoisomerase IV (30),
energy-dependent quinolone efflux has been shown to be a major
contributory factor in quinolone resistance in S. aureus
(22). In S. aureus the gene encoding this system,
norA, has been cloned (36). The NorA protein is a
multidrug efflux pump that confers an 8- to 64-fold increase in the
MICs of hydrophilic quinolones and a 2-fold increase in the MICs of
hydrophobic quinolones. Recently, active efflux of norfloxacin has been
reported in a quinolone-resistant strain of M. smegmatis
mc2155, and the gene encoding this system, lfrA,
has been cloned (20). The LfrA efflux pump is homologous to
QacA from S. aureus but not to NorA. QacA confers resistance
to ethidium and other organic cations, such as chlorhexidine, via
proton motive force-dependent efflux (27). However, as for
NorA, hydrophilic quinolones are preferentially pumped out by LfrA. In
the present study, DNP had no effect on norfloxacin accumulation by
M. aurum or M. smegmatis, and therefore, a
quinolone efflux system is not suspected of having significant activity
in wild-type mycobacteria.
Quinolones are zwitterionic and may exist in several forms. Corti et
al. (10) suggest that the cationic form of norfloxacin may
bind to negatively charged compounds in the mycobacterial cell wall,
such as the phospholipids, and this could contribute to imprecise
measurements of intracellular norfloxacin. Those investigators showed
that by increasing the pH of the wash buffer from 7 to 9, the amount of
norfloxacin adsorbed by the M. smegmatis cell wall during an
uptake experiment was reduced. In this study, the amount of norfloxacin
adsorbed by the M. aurum cell wall at 0°C was reduced when
a pH 9 wash buffer was used. However, at 37°C the concentration of
accumulated norfloxacin was slightly higher with a pH 9 wash buffer
compared to that with a pH 7 wash buffer (Fig. 4). If the pH 9 buffer
was reducing the amount of norfloxacin adsorption to the mycobacterial
cell wall, the opposite effect would be expected. Therefore, pH 7 wash
buffer was used throughout.
It is thought that ethambutol may increase the activities of some
antimycobacterial drugs by increasing the permeability of the
mycobacterial cell wall to these agents (31). Indeed, the MICs of rifampin for M. aurum and M. smegmatis
were reduced in the presence of sub-MICs of ethambutol (41).
Therefore, the effect of ethambutol on norfloxacin accumulation was
studied. However, no difference in the final concentration of
norfloxacin accumulated by M. aurum was observed. Ethambutol
concentrations of 0.5 and 1 µg/ml were chosen as sub-MICs (MIC, 2 µg/ml) such that the cell wall structure may be altered, but
mycobacterial viability was not affected. Tween 80 is also thought to
increase drug permeation in mycobacteria by disrupting the cell wall
structure (37). The MICs of rifampin for M. aurum
and M. smegmatis grown in 7H9 with 0.05% Tween 80 were
reduced compared to the MICs for control strains grown without Tween 80 (41). Tween 80 (0.05%) had no effect on the final
concentration of norfloxacin accumulated by M. aurum. The
recommended concentration for mycobacterial growth is 0.05% Tween 80. Because it is proposed that norfloxacin enters via a porin pathway,
these results are not surprising. Disruption of the lipid bilayer with
Tween 80 or disruption of the mycolic acid structure with ethambutol
may not be expected to affect norfloxacin transport. Furthermore, the
MIC of norfloxacin for M. aurum was unaltered in the
presence of ethambutol or Tween 80.
With the increased use of the quinolones as second-line agents in for
the treatment of tuberculosis caused by multidrug-resistant strains,
knowledge of the transport of these agents will assist in the
development of novel agents and will provide a useful weapon in the
fight against the resurgence of tuberculosis. Further studies of the
levels of accumulation of other agents by mycobacteria are warranted to
support and extend the data for fluoroquinolones.
| |
ACKNOWLEDGMENT |
K.J.W. was funded by a studentship from the Glaxo Wellcome Action
TB initiative.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Antimicrobial
Agents Research Group, Department of Infection, The Medical School,
University of Birmingham, Vincent Dr., Edgbaston, Birmingham B15 2TT,
United Kingdom. Phone: 44 121 414 6969. Fax: 44 121 414 6966. E-mail: l.j.v.piddock{at}bham.ac.uk.
| |
REFERENCES |
| 1.
|
Alangaden, G. J.,
E. K. Manavathu,
S. B. Vakulenko,
N. M. Zvonok, and S. A. Lerner.
1995.
Characterization of fluoroquinolone-resistant mutant strains of Mycobacterium tuberculosis selected in the laboratory and isolated from patients.
Antimicrob. Agents Chemother.
39:1700-1703[Abstract].
|
| 2.
|
Asuquo, A. E., and L. J. V. Piddock.
1993.
Accumulation and killing of fifteen quinolones for Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa.
J. Antimicrob. Chemother.
31:865-880[Abstract/Free Full Text].
|
| 3.
|
Berlin, O. G. W.,
L. S. Young, and D. A. Bruckner.
1987.
In vitro activity of six fluorinated quinolones against Mycobacterium tuberculosis.
J. Antimicrob. Chemother.
19:611-615[Abstract/Free Full Text].
|
| 4.
|
Brennan, P. J., and H. Nikaido.
1995.
The envelope of mycobacteria.
Annu. Rev. Biochem.
64:26-63.
|
| 5.
|
Cambau, E.,
W. Sougakoff,
M. Besson,
C. Truffot-Pernot,
J. Grosset, and V. Jarlier.
1994.
Selection of a gyrA mutant of Mycobacterium tuberculosis resistant to fluoroquinolones during treatment with ofloxacin.
J. Infect. Dis.
170:479-483[Medline].
|
| 6.
|
Canton, E.,
J. Peman,
M. T. Jimenez,
M. S. Ramon, and M. Gobernado.
1992.
In vitro activity of sparfloxacin compared with those of five other quinolones.
Antimicrob. Agents Chemother.
36:558-565[Abstract/Free Full Text].
|
| 7.
|
Chambers, H. F.,
D. Moreau,
D. Yajko,
C. Miick,
C. Wagner,
C. Hackbarth,
S. Kocagoz,
E. Rosenberg,
W. K. Hadley, and H. Nikaido.
1995.
Can penicillins and other -lactams be used to treat tuberculosis?
Antimicrob. Agents Chemother.
39:2620-2624[Abstract].
|
| 8.
|
Chung, G. A. C.,
Z. Aktar,
S. Jackson, and K. Duncan.
1995.
High-throughput screening for detecting antimycobacterial agents.
Antimicrob. Agents Chemother.
39:2235-2238[Abstract].
|
| 9.
|
Cohen, S. P.,
D. C. Hooper,
J. S. Wolfson,
K. S. Souza,
L. M. McMurray, and S. B. Levy.
1988.
Endogenous active efflux of norfloxacin in susceptible Escherichia coli.
Antimicrob. Agents Chemother.
32:1187-1191[Abstract/Free Full Text].
|
| 10.
|
Corti, S.,
J. Chevalier, and A. Cremieux.
1995.
Intracellular accumulation of norfloxacin in Mycobacterium smegmatis.
Antimicrob. Agents Chemother.
39:2466-2471[Abstract].
|
| 11.
|
Furet, Y. X.,
J. Deshusses, and J. C. Pechere.
1992.
Transport of perfloxacin across the bacterial cytoplasmic membrane in quinolone-susceptible Staphylococcus aureus.
Antimicrob. Agents Chemother.
36:2506-2511[Abstract/Free Full Text].
|
| 12.
|
Hooper, D. C., and J. S. Wolfson.
1991.
Mode of action of the new quinolones: new data.
Eur. J. Clin. Microbiol. Infect. Dis.
10:223-231[Medline].
|
| 13.
|
Hoshino, K.,
A. Kitamura,
I. Morrissey,
K. Sato,
J. Kato, and H. Ikeda.
1994.
Comparison of inhibition of Escherichia coli topoisomerase IV by quinolones with DNA gyrase inhibition.
Antimicrob. Agents Chemother.
38:2623-2627[Abstract/Free Full Text].
|
| 14.
|
Jarlier, V., and H. Nikaido.
1990.
Permeability barrier to hydrophilic solutes in Mycobacterium chelonae.
J. Bacteriol.
172:1418-1423[Abstract/Free Full Text].
|
| 15.
|
Jarlier, V., and H. Nikaido.
1994.
Mycobacterial cell wall: structure and role in natural resistance to antibiotics.
FEMS Microbiol. Lett.
123:11-18[Medline].
|
| 16.
|
Ji, B.,
C. Truffot-Pernot, and J. Grosset.
1991.
In vitro and in vivo activities of sparfloxacin (AT-4140) against Mycobacterium tuberculosis.
Tubercle
72:181-186[Medline].
|
| 17.
|
Kocagoz, T.,
C. J. Hackbarth,
I. Unsal,
E. Y. Rosenberg,
H. Nikaido, and H. F. Chambers.
1996.
Gyrase mutations in laboratory-selected, fluoroquinolone-resistant mutants of Mycobacterium tuberculosis H37Ra.
Antimicrob. Agents Chemother.
40:1768-1774[Abstract].
|
| 18.
|
Lalande, V.,
C. Truffot-Pernot,
A. Paccaly-Moulin,
J. H. Grosset, and B. Ji.
1993.
Powerful bactericidal activity of sparfloxacin (AT-4140) against Mycobacterium tuberculosis in mice.
Antimicrob. Agents Chemother.
37:407-413[Abstract/Free Full Text].
|
| 19.
|
Li, X. Z.,
D. Ma,
D. M. Livermore, and H. Nikaido.
1994.
Role of efflux pump(s) in intrinsic resistance of Pseudomonas aeruginosa: active efflux as a contributing factor to -lactam resistance.
Antimicrob. Agents Chemother.
38:1732-1742[Abstract/Free Full Text].
|
| 20.
|
Liu, J.,
H. E. Takiff, and H. Nikaido.
1996.
Active efflux of fluoroquinolones in Mycobacterium smegmatis mediated by LfrA, a multidrug efflux pump.
J. Bacteriol.
178:3791-3795[Abstract/Free Full Text].
|
| 21.
|
Lopez-Hernandez, I.,
L. Martinez-Martinez,
A. Pascual,
I. I. Garcia, and E. J. Perea.
1997.
Accumulation of fluoroquinolones in Acinetobacter baumannii.
J. Clin. Microbiol. Infect.
3:176.
|
| 22.
|
McCaffrey, C.,
A. Bertasso,
J. Pace, and N. H. Georgopapadakou.
1992.
Quinolone accumulation in Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus.
Antimicrob. Agents Chemother.
36:1601-1605[Abstract/Free Full Text].
|
| 23.
|
Mitchison, D. A.
1992.
The Garrod Lecture. Understanding the chemotherapy of tuberculosis.
J. Antimicrob. Chemother.
29:477-493[Free Full Text].
|
| 24.
|
Mortimer, P. G. S., and L. J. V. Piddock.
1991.
A comparison of methods used for measuring the accumulation of quinolones by Enterobacteriaceae, Pseudomonas aeruginosa and Staphylococcus aureus.
J. Antimicrob. Chemother.
28:639-653[Abstract/Free Full Text].
|
| 25.
|
Munoz, R., and A. G. Delacampa.
1996.
ParC subunit of DNA topoisomerase IV of Streptococcus pneumoniae is a primary target of fluoroquinolones and cooperates with DNA gyrase-A subunit in forming resistance phenotype.
Antimicrob. Agents Chemother.
40:2252-2257[Abstract].
|
| 26.
|
Pan, X. S., and L. M. Fisher.
1997.
Targeting of DNA gyrase in Streptococcus pneumoniae by sparfloxacin: selective targeting of gyrase or topoisomerase IV by quinolones.
Antimicrob. Agents Chemother.
41:471-474[Abstract].
|
| 27.
|
Paulsen, I. T.,
M. H. Brown, and R. A. Skurray.
1996.
Proton-dependent multidrug efflux systems.
Microbiol. Rev.
60:575-608[Abstract/Free Full Text].
|
| 28.
|
Piddock, L. J. V.
1991.
Mechanism of quinolone uptake into bacterial cells.
J. Antimicrob. Chemother.
27:399-403[Free Full Text].
|
| 29.
|
Piddock, L. J. V.
1994.
New quinolones and gram-positive bacteria.
Antimicrob. Agents Chemother.
38:163-169[Free Full Text].
|
| 30.
|
Piddock, L. J. V.
1995.
Mechanisms of resistance to fluoroquinolones: state-of-the-art 1992-1994.
Drugs
49:29-35.
|
| 31.
|
Rastogi, N.,
V. Labrousse, and A. Bryskier.
1995.
Intracellular activities of roxithromycin used alone and in association with other drugs against Mycobacterium avium complex in human macrophages.
Antimicrob. Agents Chemother.
39:976-978[Abstract].
|
| 32.
|
Tomioka, H.,
H. Saito, and K. Sato.
1993.
Comparative antimycobacterial activities of the newly synthesized quinolone AM-1155, sparfloxacin, and ofloxacin.
Antimicrob. Agents Chemother.
37:1259-1263[Abstract/Free Full Text].
|
| 33.
|
Trias, J.,
V. Jarlier, and R. Benz.
1992.
Porins in the cell wall of mycobacteria.
Science
258:1479-1481[Abstract/Free Full Text].
|
| 34.
|
Trias, J., and R. Benz.
1993.
Characterization of the channel formed by the mycobacterial porin in lipid bilayer membranes.
J. Biol. Chem.
268:6234-6240[Abstract/Free Full Text].
|
| 35.
|
Trias, J., and R. Benz.
1994.
Permeability of the cell wall of Mycobacterium smegmatis.
Mol. Microbiol.
14:283-290[Medline].
|
| 36.
|
Ubukata, K.,
N. Itoh-Yamashita, and M. Konno.
1989.
Cloning and expression of the norA gene for fluoroquinolone resistance in Staphylococcus aureus.
Antimicrob. Agents Chemother.
33:1535-1539[Abstract/Free Full Text].
|
| 37.
|
Van Boxtel, R. M.,
R. S. Lambrecht, and M. T. Collins.
1990.
Effects of colonial morphology and Tween 80 on antimicrobial susceptibility of Mycobacterium paratuberculosis.
Antimicrob. Agents Chemother.
34:2300-2303[Abstract/Free Full Text].
|
| 38.
|
Vila, J.,
J. Ruiz,
P. Goni, and M. T. J. Deanta.
1996.
Detection of mutations in parC in quinolone-resistant clinical isolates of Escherichia coli.
Antimicrob. Agents Chemother.
40:491-493[Abstract].
|
| 39.
|
Williams, K. J., and L. J. V. Piddock.
1996.
gyrA of ofloxacin-resistant clinical isolates of Mycobacterium tuberculosis from Hong Kong.
J. Antimicrob. Chemother.
37:1032-1034[Free Full Text].
|
| 40.
| Williams, K. J., and L. J. V. Piddock. Submitted for publication.
|
| 41.
| Williams, K. J., and L. J. V. Piddock. Unpublished data.
|
| 42.
|
Young, L. S.
1993.
The Garrod Lecture. Mycobacterial diseases in the 1990s.
J. Antimicrob. Chemother.
32:179-194[Free Full Text].
|
Antimicrobial Agents and Chemotherapy, April 1998, p. 795-800, Vol. 42, No. 4
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
Top
Abstract
Introduction
) and M. smegmatis (
).
, pH 7; 
, pH 9.