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Antimicrobial Agents and Chemotherapy, January 2001, p. 105-116, Vol. 45, No. 1
Microcide Pharmaceuticals, Inc., Mountain
View, California 94043,1 and Daiichi
Pharmaceutical Co., Ltd., Tokyo 134, Japan2
Received 24 July 2000/Returned for modification 9 September
2000/Accepted 9 October 2000
Whole-cell assays were implemented to search for efflux pump
inhibitors (EPIs) of the three multidrug resistance efflux pumps (MexAB-OprM, MexCD-OprJ, MexEF-OprN) that contribute to fluoroquinolone resistance in clinical isolates of Pseudomonas aeruginosa.
Secondary assays were developed to identify lead compounds with
exquisite activities as inhibitors. A broad-spectrum EPI which is
active against all three known Mex efflux pumps from P. aeruginosa and their close Escherichia coli efflux
pump homolog (AcrAB-TolC) was discovered. When this compound,
MC-207,110, was used, the intrinsic resistance of P. aeruginosa to fluoroquinolones was decreased significantly
(eightfold for levofloxacin). Acquired resistance due to the
overexpression of efflux pumps was also decreased (32- to 64-fold
reduction in the MIC of levofloxacin). Similarly, 32- to 64-fold
reductions in MICs in the presence of MC-207,110 were observed for
strains with overexpressed efflux pumps and various target mutations
that confer resistance to levofloxacin (e.g., gyrA and
parC). We also compared the frequencies of emergence of
levofloxacin-resistant variants in the wild-type strain at four times
the MIC of levofloxacin (1 µg/ml) when it was used either alone or in
combination with EPI. In the case of levofloxacin alone, the frequency
was ~10 Active efflux of toxic compounds out
of cells is a general mechanism that bacteria have developed to protect
themselves against the adverse effects of their environments.
Antibiotics that are used in clinical settings are among these toxic
compounds, and extrusion of antibiotics from bacterial cells
significantly decreases their clinical utility. Antibiotics are
expelled from the cells by membrane transporter proteins, the so-called
drug-efflux pumps. Of particular interest are efflux pumps capable of
extruding out of the cell a large variety of structurally unrelated
compounds with different antibacterial modes of action (13, 15,
30-32). Most of the genes encoding these multidrug resistance
(MDR) pumps are normal constituents of bacterial chromosomes. Some of
these genes have a relatively high level of constitutive expression and
confer so-called intrinsic resistance to antibiotics. Expression of
other genes that confer an efflux capability is not detected in
wild-type cells, but such genes can become expressed after the
acquisition of regulatory mutations.
In gram-negative bacteria, most of the efflux pumps that contribute to
both intrinsic and acquired resistance to clinically useful antibiotics
are three-component structures that traverse both inner membranes and
outer membranes. Each tripartite pump contains a transporter located in
the cytoplasmic membrane, an outer membrane channel, and a periplasmic
linker protein, which is thought to bring the other two components into
contact (54, 55). This structural organization allows
extrusion of substrates directly into the external medium, bypassing
the periplasm. Direct efflux as a mechanism of drug extrusion is
required since these rather slow tripartite MDR pumps (46)
rely heavily on the help of the outer membrane, which serves as a
permeability barrier for both hydrophobic and hydrophilic antibiotics
(33).
Several classes of MDR pumps have been identified on the basis of
sequence comparisons (42). Most of the inner membrane components of clinically relevant tripartite efflux pumps from gram-negative bacteria belong to a single class of transporters called
resistance-nodulation-division (RND) efflux pumps (5).
Pseudomonas aeruginosa is an important opportunistic
pathogen which is highly resistant to antibiotic therapy.
Fluoroquinolones, Besides efflux, resistance to fluoroquinolones is also conferred by
target mutations. These mutations mainly occur in quinolone resistance-determining regions (QRDRs) (11, 36, 52) in DNA gyrase (encoded by gyrA and gyrB) and
topoisomerase IV (encoded by parC and parE) in
many organisms including P. aeruginosa (26). Importantly, it has been demonstrated recently in P. aeruginosa (18) and Escherichia coli
(35) that when both target-based and efflux-mediated
resistance mechanisms are present in the same cell, they contribute
independently to fluoroquinolone resistance. As a result, deletion of
the MexAB-OprM efflux pump from a strain in which this pump is
overexpressed resulted in a 64-fold reduction in the MIC of
levofloxacin, regardless of the presence of additional resistance
mechanisms. It was also demonstrated that deletion of all described
pumps significantly reduces the frequency of emergence of
fluoroquinolone-resistant mutant strains (18).
On the basis of these genetic data, it appears that inhibition of
efflux pumps in P. aeruginosa may significantly improve the
clinical performance of fluoroquinolones. Inhibition of efflux pumps is
expected to (i) decrease the level of intrinsic resistance, (ii)
significantly reverse acquired resistance, and (iii) decrease the
frequency of emergence of P. aeruginosa mutants highly
resistant to fluoroquinolones.
The same considerations can also be applied to gram-positive bacteria.
Indeed, when reserpine (which was long ago identified as an inhibitor
of the mammalian MDR pump, P-glycoprotein, and which was later found to
inhibit MDRs from gram-positive bacteria) was added to the selection
media, it suppressed the emergence of Staphylococcus aureus
and Streptococcus pneumonia mutants resistant to
ciprofloxacin (19, 20). Recently, several new inhibitors of the NorA pump from S. aureus which potentiated the
activity of ciprofloxacin against both wild-type and
NorA-overexpressing strains have been reported (21). These
compounds decreased the MICs of ciprofloxacin up to fourfold (at
concentrations ranging from 0.2 to 1.5 µg/ml) for strains of S. aureus overexpressing the NorA pump. Some of these compounds also
inhibited efflux-mediated resistance in S. pneumoniae.
Importantly, all compounds dramatically suppressed the frequency of
emergence of ciprofloxacin-resistant strains when selection was
performed with 1 µg of ciprofloxacin per ml (21).
The potential benefits of broad-spectrum efflux pump inhibitors (EPIs)
prompted us to screen our synthetic compound and natural product
libraries to search for inhibitors of the Mex pumps from P. aeruginosa. Recently, we have described one such inhibitor, MC-207,110, and presented a description of a portion of our efforts to
optimize the biological and physiochemical properties of this lead
compound (41). Here we present an additional
characterization of MC-207,110: evidence of its mode of action as an
inhibitor of efflux pump activity, its potentiating effect of the
activity of levofloxacin against both laboratory strains with multiple target mutations and recent clinical isolates of P. aeruginosa, and its ability to decrease the emergence of P. aeruginosa strains with clinically relevant resistance to levofloxacin.
Bacterial strains and media.
The laboratory bacterial
strains used in this study are listed in Table
1. Bacterial cells were grown in L broth
(1% [wt/vol] tryptone, 0.5% [wt/vol] yeast extract, 0.5%
[wt/vol] NaCl) or L agar (L broth plus 1.5% agar) at 37°C.
Clinical isolates of P. aeruginosa were collected from the
United States, Canada, England, and France between 1995 and 1998. Levofloxacin was synthesized at Daiichi Pharmaceutical Co., Ltd.
(Tokyo, Japan). Nitrocefin was purchased from Calbiochem-Behring (La
Jolla, Calif.). All other antibiotics as well as
N-phenylnaphthylamine (NPN) and polymyxin B nonapeptide
(PMBN) were purchased from Sigma Chemical Co. (St. Louis, Mo).
MC-207,110 was from the synthetic compound library of Microcide
Pharmaceuticals, Inc. MC-002,595, which is a close analog of
MC-207,110, and MC-005,556 (alanine
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.1.105-116.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Identification and Characterization of Inhibitors
of Multidrug Resistance Efflux Pumps in Pseudomonas
aeruginosa: Novel Agents for Combination Therapy
ABSTRACT
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
7 CFU/ml. In contrast, with an EPI, the
frequency was below the level of detection (<1011). In
summary, we have demonstrated that inhibition of efflux pumps (i)
decreased the level of intrinsic resistance significantly, (ii)
reversed acquired resistance, and (iii) resulted in a decreased frequency of emergence of P. aeruginosa strains that are
highly resistant to fluoroquinolones.
INTRODUCTION
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
-lactams, and aminoglycosides are among the
primary agents available for treatment of infections caused by this
pathogen. Four multicomponent MDR RND efflux pumps have been identified
in P. aeruginosa, namely, MexAB-OprM (39),
MexCD-OprJ (38), MexEF-OprN (12), and
MexXY-OprM (1). These pumps have somewhat overlapping
spectra of antibiotic substrates. For example, all four pumps confer
various degrees of resistance to fluoroquinolones, and mutants that
overexpress three of these pumps have been isolated among
fluoroquinolone-resistant bacteria in clinical settings:
nalB mutants that overproduce the MexAB-OprM pump
(3), nfxB mutants that overproduce MexCD-OprJ (53), and nfxC mutants that overproduce the
MexEF-OprN efflux pump (6). So far, overexpression of
MexXY-OprM has not been reported as a cause of fluoroquinolone
resistance in P. aeruginosa; however, this pump has recently
been implicated in low-level resistance to aminoglycosides (1,
50). Of these four pumps, only MexAB-OprM is expressed at a
level sufficient to confer intrinsic MDR in wild-type cells. Deletion
of the mexA, mexB, or oprM gene
renders P. aeruginosa more susceptible to multiple
antibiotics, including fluoroquinolones (8, 39, 51).
Importantly, overexpression of the MexCD-OprJ or the MexEF-OprN efflux
pumps restores resistance to fluoroquinolones in strains lacking the
MexAB-OprM efflux pump (9, 14, 18).
MATERIALS AND METHODS
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
-naphthylamide [Ala-Nap]) were
synthesized at Microcide Pharmaceuticals, Inc.
TABLE 1.
Laboratory strains and plasmid used in this study
Construction of P. aeruginosa strains overexpressing a single Mex pump. The mutants overexpressing each of the three Mex pumps individually (MexAB-OprM, MexCD-OprJ, or MexEF-OprN) were selected on L-agar plates containing levofloxacin at 1 µg/ml from strains lacking the two other pumps. The frequency of resistance was determined as the ratio of the number of CFU per milliliter appearing after overnight incubation on antibiotic-containing L-broth or L-agar plates versus the number of CFU per milliliter appearing after overnight incubation on antibiotic-free L-broth or L-agar plates.
MICs. MIC determinations were carried out in 96-well microtiter plates by a twofold standard broth microdilution method (27) in Muller-Hinton broth (Difco) either with or without fixed concentrations of MC-207,110. The inocula in all experiments were 104 to 105 cells/ml.
Checkerboard titration assays. Interactions between levofloxacin and MC-207,110 were assessed by a checkerboard titration assay. Levofloxacin was tested at 11 concentrations (0.008 to 8 µg/ml), while MC-207,110 was tested at 7 concentrations (0.06 to 40 µg/ml). Levofloxacin was dispensed alone in the first row and was combined with MC-207,110 in the remaining rows. MC-207,110 was also dispensed alone in the first column. Drug interactions were classified as synergistic if the fractional inhibitory concentration (FIC) index was <0.5. The FIC index is the sum of the FIC of each of the drugs, which in turn is defined as the MIC of each drug when used in combination divided by the MIC of the drug when used alone.
MC-005,556 (Ala-Nap) uptake assay.
Ala-Nap, which is not
fluorescent in solution, is cleaved enzymatically inside the cells to
produce highly fluorescent -naphthylamine. The more Ala-Nap that
enters the cells, the more fluorescence that is produced. Importantly,
Ala-Nap is a substrate of the Mex pumps from P. aeruginosa
as well as the AcrAB-TolC pump from E. coli (see Results).
To assess the uptake of Ala-Nap, cultures of P. aeruginosa
were grown to an optical density at 600 nm (OD600) of ~1,
washed, and resuspended in buffer at pH 7.0 containing K2HPO4 (50 mM), MgSO4 (1 mM), and
glucose (0.4%) (buffer A). For treatment with carbonyl cyanide
m-chlorophenylhydrazone (CCCP), the cells were resuspended
in buffer A without glucose and mixed with 0.5 mM CCCP, and the mixture
was incubated for 15 min. After treatment, the cells were washed with
buffer A without glucose at least three times. Assays were performed in
96-well flat-bottom black plates (Applied Scientific or Costar) in a
final volume of 200 µl and were initiated by the addition of Ala-Nap
to suspensions of intact cells to a final concentration of 64 µg/ml.
Fluorescence was measured on an fMAX spectrofluorometer (Molecular
Devices) with excitation of 320 nm and emission of 460 nm. To measure
the effects of the EPI or PMBN on the rate of Ala-Nap uptake, the cells
were preincubated with different concentrations of these compounds
prior to Ala-Nap addition.
NPN efflux assays. NPN is an uncharged lipophilic molecule which fluoresces weakly in aqueous environments but which becomes strongly fluorescent in nonpolar environments such as the lipid bilayers of biological membranes. NPN is also a substrate of RND efflux pumps (34). Cultures of E. coli were grown in L broth to an OD600 of ~1, washed, and resuspended in buffer A without glucose. Cells were treated with CCCP (100 µM), washed, and then loaded with NPN at 9 µM for 10 min. NPN efflux was initiated by the addition of glucose and was recorded as the decay in fluorescence. Assays were performed in 96-well spectrofluorometer cuvettes in a final volume of 200 µl. When stated, the EPI was added to the cells together with glucose.
Nitrocefin uptake assay.
Cells of P. aeruginosa
(PAM2005 or PAM2035) with constitutive expression of chromosomal
-lactamase were grown overnight in L broth, harvested, washed in
Mg2+-free buffer A, and resuspended in the same buffer at
an OD600 of ~0.5. To 100 µl of the cell suspension, 50 µl of either PMBN or MC-207,110 was added to give a final
concentration of 1 to 128 µg/ml. Next, 50 µl of nitrocefin was
added to give a final concentration of 32 µg/ml. Hydrolysis of
nitrocefin was monitored spectrophotometrically by measurement of the
increase in absorbance at 490 nm. Assays were performed in 96-well
plates in a SpectraMAX Plus spectrophotometer (Molecular Devices).
31P NMR assay. 31P nuclear magnetic resonance (NMR) measurements were conducted to probe the intracellular pH of E. coli cells under different conditions. Assignments of resonance were based upon previously published in vivo NMR studies of E. coli (2, 48). A total of 1,000 ml of E. coli ATCC 25922 cells was grown to the mid-logarithmic phase (OD600, ~1) in L broth. The cells were harvested (centrifugation at 3,000 × g for 10 min at 4°C) and washed twice with ice-cold phosphate-buffered saline buffer (pH 6.5). The cell pellet (7.5 ml) was resuspended in an equal volume of the following buffer at pH 6.5: KH2PO4 (2.5 mM), K2HPO4 (2.5 mM), morpholineethanesulfonic acid (100 mM), and NaCl (85 mM). The cells were kept on ice until needed (less than 2 h). Before use, the cells were allowed to reach room temperature. One milliliter of cell suspension was placed in a 5-mm NMR tube. The field was shimmed by using proton free induction decays. A total of 20 µl of 50% glucose was added to the cell suspension. When stated, 20 µl of either 20 mM MC-207,110 or CCCP was added to give a final concentration of 0.4 mM. 31P NMR spectra were obtained at 161.88 MHz. Free induction decays were accumulated at 5-min intervals for 30 min.
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RESULTS |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Screening for EPIs and criteria for efflux pump inhibitory mode of
action of screening hits.
To identify potential efflux pump
inhibitors, the growth inhibitory activity of synthetic compounds and
natural products extracts was assayed using strains of P. aeruginosa overexpressing each of the three efflux pumps:
MexAB-OprM (PAM1032), MexCD-OprJ (PAM1033), and MexEF-OprN (PAM1034)
that were grown with and without subinhibitory concentrations of
levofloxacin (47). Compounds or extracts that inhibited
the growth of all three strains only in the presence of levofloxacin
were chosen for follow-up assays to discriminate between compounds with
false-positive results and true broad-spectrum EPIs. To qualify as
EPIs, hit compounds had to satisfy several criteria: (i) must enhance
the activities of levofloxacin and other antibiotics that are effluxed
in strains containing functioning pumps, (ii) must not significantly
potentiate the activities of antibiotics in a strain that lacks efflux
pumps, (iii) must not potentiate the activities of antibiotics that are
not effluxed, (iv) must increase the level of accumulation and decrease
the level of extrusion of efflux pump substrates, and (iv) must not affect the proton gradient across the inner membrane. MC-207,110 (Fig.
1) was among the first efflux pump
inhibitors identified in our screening efforts.
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MC-207,110 series satisfies microbiological criteria for EPIs.
To assess the interaction between levofloxacin and MC-207,110, we
performed standard checkerboard assays in which the MICs of
levofloxacin were determined in the presence of different
concentrations of MC-207,110 (Table 2).
While MC-207,110 itself was devoid of significant antibacterial
activity (MICs, 64 or >512 µg/ml for the strains lacking or
overexpressing efflux pumps, respectively), it increased the
susceptibilities of the three pump-overexpressing mutants of P. aeruginosa to levofloxacin 64-fold. MC-207,110 also potentiated
the activity of levofloxacin against strain PAM2391 containing plasmid
pAGH97 (a gift from P. Plesiat) with the mexXY genes and
against wild-type strain PAM1020. The latter result was expected since
constitutive expression of the mexAB-oprM operon contributes
to the intrinsic resistance to levofloxacin in this strain.
Interactions between MC-207,110 and levofloxacin were clearly
synergistic for all the strains expressing efflux pumps, with FIC
indices ranging from 0.05 to 0.14. No synergy was observed against
strain PAM1626, which lacks all known Mex pumps.
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MC-207,110 series increases levels of accumulation of efflux pump substrates inside the cell. If MC-207,110 is an efflux pump inhibitor it should increase the level of accumulation of efflux pump substrates inside the cell. We (as well as others) have not been able to demonstrate directly, using several hydrophobic fluorescent dyes, efflux by Mex pumps from P. aeruginosa in membrane vesicles, probably due to the low levels of activity of pumps that rely on the outer membrane. Because of this, accumulation and efflux experiments had to be performed with intact cells. The interpretations of these assays could be complicated by the fact that both efflux pump inhibitors and permeabilizers of the outer membrane of P. aeruginosa may have similar effects on the accumulation of at least some test compounds (7, 16). To avoid such complications, we sought to identify a compound with the following features: (i) it is a substrate of efflux pumps, (ii) it is easily detected inside the cell, and (iii) its permeation is not restricted by the outer membrane in P. aeruginosa. If MC-207,110 increased the level of accumulation of such a compound, it could be interpreted to be a consequence only of inhibition of efflux.
MC-005,556 (Ala-Nap), synthesized at Microcide, satisfied all criteria for such a test compound. Ala-Nap, which is not fluorescent in solution, is cleaved enzymatically inside the cells to produce the highly fluorescent compound-naphthylamine. The fluorescence of
-naphthylamine itself is not quenched by the cells and is not
affected by the presence or absence of efflux pumps (data not shown).
The rate of production of -naphthylamine (recorded as an increase in
fluorescence) is limited by the rate of entry of Ala-Nap into the cell:
cell extracts prepared from PAM1723 (overexpressing the MexAB-OprM)
produced -naphthylamine at an approximately fivefold higher rate
than intact cells of the same strain (data not shown). The rate of
cleavage of Ala-Nap was much higher in PAM1626 than in strains
overexpressing an efflux pump, such as PAM1723. Importantly, the cells
of both strains (when treated with CCCP to inhibit active efflux)
cleaved Ala-Nap at the same rate (Fig.
2A). These data indicate that Ala-Nap is
a substrate of the MexAB-OprM efflux pump. (The observation of an increase in the level of accumulation of Ala-Nap in the presence of
CCCP in the strain from which the pump was deleted does not necessarily
imply the presence of an additional pump in this strain. Since Ala-Nap
is a weak base with a pKa of 8.2, the abolishment of the pH
gradient across the membrane with CCCP leads to the spontaneous influx
of Ala-Nap into the cell.) In analogous experiments it was demonstrated
that Ala-Nap is also a substrate of the MexCD-OprJ and the MexEF-OprN
pumps from P. aeruginosa (Fig. 2B) as well as the close
homolog from E. coli, the AcrAB-TolC pump (data not shown).
We then studied the uptake of Ala-Nap by PAM1723 cells in the presence
of various concentrations (1 to 128 µg/ml) of PMBN, which is a
polymyxin B derivative lacking the fatty acid moiety of the parent
compound. PMBN is a potent permeabilizer of the outer membrane in
P. aeruginosa (49). Assays were performed both
in the presence and in the absence of Mg2+, which inhibits
the activity of PMBN (37). In PAM1723 cells, PMBN did not
increase the rate of conversion (or uptake) of Ala-Nap, even in
Mg2+-free buffer (data not shown). The same result was
obtained with cells of PAM1626 lacking efflux pumps (data not shown).
Thus, Ala-Nap was an appropriate substrate for use in the assessment of
the broad-spectrum efflux pump inhibitory activities of our lead
compounds. It is also noteworthy that this particular compound apparently is a very good substrate for the efflux pumps since no help
from the outer membrane was needed to demonstrate an effect of the
efflux pump on its accumulation kinetics.
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acrAB::Km, data not shown) or when the
pump-overexpressing strains of P. aeruginosa or E. coli were treated with CCCP (data not shown).
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MC-207,110 series inhibits efflux of NPN.
To evaluate further
the modes of action of the MC-207,110 series of compounds, we studied
the efflux of NPN by E. coli in the presence of MC-002,595
(to demonstrate an effect with a pump substrate that is structurally
very dissimilar). Cells of E. coli either overexpressing
(ECM1642) or lacking (ECM1668) the AcrAB-TolC efflux pump were treated
with CCCP and loaded with NPN. When bound to the membranes, NPN becomes
intensely fluorescent. Its efflux can be followed in real time as a
decay of fluorescence after energization of the cells. Indeed, after
the addition of glucose, efflux of NPN was observed in the case of
ECM1642. Importantly, when MC-002,595 was added to the cells prior to
the addition of glucose, it inhibited efflux in a dose-dependent manner
(Fig. 4A). In fact, efflux was completely
abolished with the EPI at 128 µg/ml. Apparent noncomplete inhibition
is due to the quenching of NPN fluorescence by MC-002,595 (Fig. 4B).
PMBN, which strongly increases the level of accumulation of NPN even in
the presence of 1 mM Mg2+ (data not shown), did not have
any effect on its efflux (data not shown).
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MC-207,110 does not affect the proton gradient across the inner
membrane.
RND efflux pumps use the energy of the proton gradient
across the inner membrane for drug extrusion: drug efflux is
accompanied by the influx of protons (55). Compounds that
disrupt the proton gradient may appear as efflux pump inhibitors.
Therefore, it was important to demonstrate that the mode of action of
MC-207,110 was not based on such disruption. To assess the effects of
MC-207,110 on the proton gradient we used the method of determining the
change in pH (pH) based on measurement of the NMR spectrum of
inorganic phosphate (31P), which is a pH-sensitive NMR
probe. This particular method was chosen because many procedures of
measuring pH rely on the accumulation of pH-sensitive fluorescent
probes [for example, various acridines or 2',7'-bis-(2-carboxyethyl)-5
(and -6-)-carboxyfluorescein]. Many if not all of these compounds are
substrates of efflux pumps. Consequently, it would be difficult to
discriminate between the efflux pump inhibitory activity of the
compound and its effect on the proton gradient.
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MC-207,110 series has an additional mode of action.
We wanted
to know whether MC-207,110, besides efflux pump inhibition, might have
an additional mode of action, namely, the ability to permeabilize the
outer membrane. This possibility appeared to be feasible on the basis
of the structure of MC-207,110, which is a dipeptide amide
(41), with two positive charges at physiological pH
(pKa1, ~10.5; pKa2, ~8.0). The outer
membrane-permeabilizing effect of MC-207,110 at a wide range of
concentrations was assessed by examining the rates of hydrolysis of a
chromogenic -lactam, nitrocefin, by intact cells of P. aeruginosa. An increased rate of hydrolysis in intact cells is
indicative of increased permeation of nitrocefin to the periplasmic
-lactamase, since the rate of hydrolysis is limited by the rate of
diffusion across the outer membrane (10, 56). For these
experiments we used two strains of P. aeruginosa, PAM2005
and PAM2035. Both strains constitutively produce the -lactamase
AmpC, encoded by the corresponding gene normally present in the genome
of this bacterium. PAM2005 also overproduces the MexAB-OprM efflux
pump, while in PAM2035 this pump is nonfunctional due to insertional
inactivation of the gene mexA. PMBN was used as a positive
control, having a strong effect on the outer membrane permeability of
PAM2005 (50% inhibitory concentration, ~8 µg/ml), (Fig.
6A), as well as on that of PAM2035 (data
not shown). In contrast, MC-207,110 did not have a significant effect
on the outer membrane permeability of PAM2005 when it was tested over a
wide concentration range (1 to 128 µg/ml) (Fig. 6B). However, it
increased the permeability of the outer membrane of PAM2035, which
lacks the functional MexAB-OprM efflux pump (albeit with less potency
than PMBN [50% inhibitory concentration, ~70 µg/ml]), and of
that of PAM2005 when the strain was treated with CCCP (Fig. 6C and D).
The permeabilizing effects of both PMBN and MC-207,110 were completely
abolished by the addition of 1 mM Mg2+ (data not shown).
Results that were similar both qualitatively and quantitatively were
obtained for MC-002,595 (data not shown). In conclusion, it appears
that the MC-207,110 series is capable of altering the permeability of
the outer membrane in P. aeruginosa, but only in the absence
of a functional MexAB-OprM efflux pump.
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MC-207,110 enhances the activity of levofloxacin against laboratory
strains with multiple target mutations.
We demonstrated previously
that the deletion of efflux pump genes increased susceptibility to
fluoroquinolones even in strains containing mutations in genes encoding
the fluoroquinolone targets gyrAB and parCE
(18). Efflux pump inhibitors were expected to have a
similar effect. To test whether this was indeed the case, the MICs of
levofloxacin were determined for a series of isogenic laboratory
mutants in the presence or absence of MC-207,110 at 20 µg/ml. The
results are presented in Table 4.
MC-207,110 enhanced the activity of levofloxacin against all strains
containing a functional efflux pump (in this case, MexAB-OprM),
regardless of the presence of one or even multiple target mutations.
The magnitude of the potentiating effect was determined exclusively by
the level of expression of the MexAB-OprM pump: an 8-fold decrease in
the levofloxacin MIC for all strains expressing the
mexAB-oprM operon at the wild-type level and a 32-fold
decrease in the levofloxacin MIC for those strains overexpressing this
operon.
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MC-207,110 enhances the activity of levofloxacin against clinical
isolates of P. aeruginosa.
Fifty recent strains of P. aeruginosa for which the levofloxacin MIC range was wide (0.008 to
>128 µg/ml) and that were collected from the United States, Canada,
England, and France were used to determine whether MC-207,110 enhances
the activity of levofloxacin. MC-207,110 was tested at a fixed
concentration of 10 µg/ml. MC-207,110 decreased both the MIC at which
50% of strains are inhibited (MIC50) and the
MIC90 of levofloxacin by ca. 16-fold (Fig.
7). The nearly parallel character of the
curves constituting the cumulative susceptibility to levofloxacin with
or without MC-207,110 indicates that this compound enhanced the
activity of the antibiotic irrespective of the level of resistance of
individual isolates. These results are in good accordance with the data
reported above for laboratory mutants with multiple mechanisms of
levofloxacin resistance.
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MC-207,110 decreases the emergence of P. aeruginosa
strains with clinically relevant resistance to levofloxacin.
We
have previously demonstrated (18) that deletion of known
efflux pumps in P. aeruginosa significantly decreased the
frequency of emergence of resistant variants when selection with the
deletion mutant was performed with 1 µg of levofloxacin per ml
(strains of P. aeruginosa for which the levofloxacin MIC is
2 µg/ml or higher are considered to be clinically resistant). On the
basis of these data we have predicted that an efflux pump inhibitor should also affect the frequency of emergence of resistant bacteria. To
test this prediction we performed selection experiments by plating
cells of wild-type strain PAM1020 (MIC, 0.125 µg/ml) onto L-agar
plates containing either levofloxacin at 1 µg/ml (eight times the
MIC) or levofloxacin at 1 µg/ml in combination with MC-207,110 at 20 µg/ml. When the selection was performed with levofloxacin alone, the
frequency of resistant variants was as high as 107
CFU/ml. However, when MC-207,110 was added to the selective medium, the
frequency of emergence of strains with a clinically relevant level of
resistance was less than 1011 CFU/ml.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Inhibition of efflux pumps appears to be an attractive approach to improvement of the clinical efficacies of antibiotics that are substrates of such pumps. However, it is important to properly identify the antibiotics and target bacteria for which this approach would be the most productive. One should consider the prevalence of efflux-mediated resistance, other mechanisms besides efflux that contribute to resistance to a particular antibiotic, and interactions between different mechanisms of resistance. For example, inhibition of the transposon-encoded macrolide-specific efflux pumps MefA and MefE (4, 45) from gram-positive bacteria will enhance the efficacies of macrolides, but only against a population of resistant organisms containing the mef genes. Inhibitors of the predominant transporters TetA and TetB in gram-negative bacteria would reverse one type of tetracycline resistance. Such inhibitors have been identified among certain semisynthetic tetracycline analogs (28, 29). On the other hand, inhibition of a single RND MDR pump in Haemophilus influenzae that is encoded by a constitutively expressed housekeeping gene (43) should make the entire population of bacteria more susceptible to macrolides such as azithromycin or clarithromycin.
In P. aeruginosa, inhibitors of the MexAB-OprM efflux pump
will enhance the activities of a variety of -lactams (17, 24, 51), but only in strains that do not express high levels of -lactamase (22, 25). In this important and clinically
refractory organism, the efflux pump inhibitor approach appears to be
particularly compelling when it is applied to fluoroquinolones.
Constitutively expressed efflux pumps contribute to intrinsic resistance to fluoroquinolones. The high-level acquired resistance is achieved due to independent contributions by both increased efflux and target modification. Therefore, efflux pump inhibitors are expected to enhance the activities of fluoroquinolones even against strains with target-based mutations (18). All of the Mex pumps from P. aeruginosa that confer resistance to fluoroquinolones belong to the RND family and are similar at the protein level. Fluoroquinolone pumps from other gram-negative bacteria also belong to the same family of transporters. This increases the possibility that a single inhibitor might be active against multiple efflux pumps.
In the study described in this report we expanded the characterization of the first inhibitor of multiple RND transporters from P. aeruginosa, MC-207,110, which was discovered as such by empiric screening of a small molecule library (41). The mode of action of efflux pump inhibition of MC-207,110 was confirmed in a series of microbiological assays. The activity of MC-207,110 was shown to be synergistic with that of levofloxacin when they were tested either against the wild-type strain or against strains overexpressing each of the known efflux pumps. MC-207,110 also potentiated the activities of other antibiotics that are substrates of the MexAB-OprM pump but did not potentiate the activities of antibiotics that are not. MC-207,110 did not have significant activity against strains lacking known efflux pumps. Interestingly, the magnitude of the effect of MC-207,110 was strongly dependent on the nature of a particular substrate, perhaps indicating that different antibiotics may have different binding sites on the pump and that inhibition by MC-207,110 is binding site specific. The possibility of multiple substrate binding sites was recently demonstrated for several MDR pumps (23, 40). According to this interpretation, when the pump is exposed to MC-207,110, it can still extrude some, but not all, substrates. Furthermore, MC-207,110 itself may be efficiently extruded by efflux pumps. Indeed, the Mex pumps conferred resistance to the weak antibacterial effect of MC-207,110: 64 or >512 µg of MC-207,110 per ml was needed to inhibit the growth of triple-pump-deletion strain PAM1626 or Mex pump-overexpressing strains, respectively (Table 2). It is noteworthy that many compounds that enhance the activities of anticancer agents against mammalian cells overexpressing the MDR pump P-glycoprotein have been shown to be efficiently extruded by this pump (44).
Additional confirmation that the mode of action of the MC-207,110 series is as an EPI was obtained in accumulation and efflux assays. The EPI increased the level of accumulation of the efflux pump substrate Ala-Nap inside cells of P. aeruginosa and decreased the level of efflux of another substrate, NPN, out of NPN-loaded cells of E. coli. In both assays, the EPI had activity only against cells expressing functional efflux pumps.
We have also demonstrated that the MC-207,110 series of EPIs may have an additional mode of action, namely, permeabilization of the outer membrane of P. aeruginosa. However, this permeabilizing effect of MC-207,110 was seen only for the strain of P. aeruginosa that lacked efflux pumps. This result is consistent with our hypothesis that MC-207,110 is an efflux pump substrate, whereupon the compound would be extruded by multicomponent Mex pumps directly into the external medium. Therefore, the active Mex pumps should decrease the concentration of MC-207,110 in all intracellular compartments, including the cytoplasm, the periplasm, and the outer membrane. By decreasing the MC-207,110 concentration in the outer membrane, the pump is expected to protect the cells from the outer membrane-permeabilizing effect of MC-207,110. We are exploring techniques that can be used to measure the efflux of MC-207,110 more directly.
In this report we have also demonstrated the numerous potential benefits of using MC-207,110 in combination with levofloxacin against P. aeruginosa. MC-207,110 decreased the intrinsic level of resistance to levofloxacin ca. 8-fold in the wild-type strain of P. aeruginosa, while in strains overexpressing efflux pumps, susceptibility was increased up to 64-fold. As expected, MC-207,110 potentiated the activity of levofloxacin irrespective of the presence of target-based mutations.
Recent clinical isolates of P. aeruginosa with a wide range of resistance phenotypes also showed increased susceptibilities to levofloxacin in the presence of MC-207,110. Remarkably, both the MIC50 and the MIC90 were decreased to the same extent (16-fold) by MC-207,110, providing additional evidence that the effect of MC-207,110 is not dependent on the absolute level of resistance but, rather, that the magnitude of this effect is determined only by the level of the efflux pump expression.
A major beneficial consequence of inhibition of multiple efflux pumps
demonstrated in this report was a dramatic decrease in the frequency of
emergence of P. aeruginosa strains with clinically relevant
levels of resistance to fluoroquinolones. Resistant mutants for which
the levofloxacin MIC was 1 µg/ml emerged at a frequency less than
1011 (versus a resistance frequency of 107
in the absence of efflux pump inhibitors). This result was expected, since MC-207,110 decreases the MIC of levofloxacin for wild-type strain
PAM1020 from 0.125 to 0.015 µg/ml, i.e., to the same level as that
for the strain lacking efflux pumps. As we have demonstrated previously
(18), for bacteria that lack efflux pumps (or in which
such pumps are completely inhibited) to emerge under the selection
conditions (levofloxacin at 1 µg/ml), they must acquire multiple
target-based mutations: a strain that acquired a single target-based
mutation that conferred only a four- to eightfold increase in
resistance could not emerge under the selection conditions. Therefore,
the frequency of emergence of mutants is decreased since the
simultaneous acquisition of multiple mutations is an extremely rare
event. Similar effects have been demonstrated with reserpine, which is
an inhibitor of the efflux pumps NorA and PmrA from S. aureus and S. pneumoniae, respectively, that are implicated in intrinsic and acquired resistance to ciprofloxacin (19, 20). Recently, several novel inhibitors of NorA have been described and their in vitro activities have been characterized (21).
From our experience with clinical isolates of P. aeruginosa, it is evident that besides the utility of EPIs in combination with antibacterial agents for antibacterial therapy, these compounds may prove to be very useful for study of the contribution and prevalence of efflux in acquired and intrinsic resistance to multiple antibiotics in gram-negative bacteria.
While initial reports have demonstrated the multifactorial benefits of the EPI approach in combating drug resistance, substantial efforts are needed before inhibitors can be used clinically. Our efforts are focused on the optimization of both the activities and the pharmacological properties of the MC-207,110 series of compounds.
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ACKNOWLEDGMENTS |
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We are grateful to G. Miller, Pat Martin, Paul Nakane, and Mike Dudley for critical reading of the manuscript. We are indebted to Hiroshi Nikaido for scientific advice and insightful comments.
This work was supported by Daiichi Pharmaceutical Co., Ltd., and Microcide Pharmaceuticals, Inc.
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FOOTNOTES |
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* Corresponding author. Mailing address: Microcide Pharmaceuticals, Inc., 850 Maude Ave., Mountain View, CA 94043. Phone: (650) 428-3548. Fax: (650) 428-3550. E-mail: olga{at}microcide.com.
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Antimicrobial Agents and Chemotherapy, January 2001, p. 105-116, Vol. 45, No. 1
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.1.105-116.2001
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Quinn, T., Bolla, J.-M., Pages, J.-M., Fanning, S.
(2007). Antibiotic-resistant Campylobacter: could efflux pump inhibitors control infection?. J Antimicrob Chemother
59: 1230-1236
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(2007). Bacterial efflux pump inhibitors from natural sources. J Antimicrob Chemother
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Mahamoud, A., Chevalier, J., Alibert-Franco, S., Kern, W. V., Pages, J.-M.
(2007). Antibiotic efflux pumps in Gram-negative bacteria: the inhibitor response strategy. J Antimicrob Chemother
59: 1223-1229
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Kehrenberg, C., de Jong, A., Friederichs, S., Cloeckaert, A., Schwarz, S.
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59: 886-892
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Hocquet, D., Roussel-Delvallez, M., Cavallo, J.-D., Plesiat, P.
(2007). MexAB-OprM- and MexXY-Overproducing Mutants Are Very Prevalent among Clinical Strains of Pseudomonas aeruginosa with Reduced Susceptibility to Ticarcillin. Antimicrob. Agents Chemother.
51: 1582-1583
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Reinhardt, A., Kohler, T., Wood, P., Rohner, P., Dumas, J.-L., Ricou, B., van Delden, C.
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51: 1341-1350
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Perez-Vazquez, M., Roman, F., Garcia-Cobos, S., Campos, J.
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51: 1566-1569
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Chan, Y. Y., Ong, Y. M., Chua, K. L.
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Lin, J., Martinez, A.
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(2006). Genes Involved in Intrinsic Antibiotic Resistance of Acinetobacter baylyi. Antimicrob. Agents Chemother.
50: 3562-3567
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Kumar, A., Chua, K.-L., Schweizer, H. P.
(2006). Method for Regulated Expression of Single-Copy Efflux Pump Genes in a Surrogate Pseudomonas aeruginosa Strain: Identification of the BpeEF-OprC Chloramphenicol and Trimethoprim Efflux Pump of Burkholderia pseudomallei 1026b. Antimicrob. Agents Chemother.
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Jiang, X., Zhang, Z., Li, M., Zhou, D., Ruan, F., Lu, Y.
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50: 2990-2995
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Gibreel, A., Taylor, D. E.
(2006). Macrolide resistance in Campylobacter jejuni and Campylobacter coli. J Antimicrob Chemother
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50: 2506-2515
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Griffith, D. C., Corcoran, E., Lofland, D., Lee, A., Cho, D., Lomovskaya, O., Dudley, M. N.
(2006). Pharmacodynamics of Levofloxacin against Pseudomonas aeruginosa with Reduced Susceptibility Due to Different Efflux Pumps: Do Elevated MICs Always Predict Reduced In Vivo Efficacy?. Antimicrob. Agents Chemother.
50: 1628-1632
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Pannek, S., Higgins, P. G., Steinke, P., Jonas, D., Akova, M., Bohnert, J. A., Seifert, H., Kern, W. V.
(2006). Multidrug efflux inhibition in Acinetobacter baumannii: comparison between 1-(1-naphthylmethyl)-piperazine and phenyl-arginine-{beta}-naphthylamide. J Antimicrob Chemother
57: 970-974
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Piddock, L. J. V.
(2006). Clinically Relevant Chromosomally Encoded Multidrug Resistance Efflux Pumps in Bacteria. Clin. Microbiol. Rev.
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Teran, W., Krell, T., Ramos, J. L., Gallegos, M.-T.
(2006). Effector-Repressor Interactions, Binding of a Single Effector Molecule to the Operator-bound TtgR Homodimer Mediates Derepression. J. Biol. Chem.
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Kern, W. V., Steinke, P., Schumacher, A., Schuster, S., Baum, H. v., Bohnert, J. A.
(2006). Effect of 1-(1-naphthylmethyl)-piperazine, a novel putative efflux pump inhibitor, on antimicrobial drug susceptibility in clinical isolates of Escherichia coli. J Antimicrob Chemother
57: 339-343
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Schumacher, A., Steinke, P., Bohnert, J. A., Akova, M., Jonas, D., Kern, W. V.
(2006). Effect of 1-(1-naphthylmethyl)-piperazine, a novel putative efflux pump inhibitor, on antimicrobial drug susceptibility in clinical isolates of Enterobacteriaceae other than Escherichia coli. J Antimicrob Chemother
57: 344-348
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Ricci, V., Tzakas, P., Buckley, A., Coldham, N. C., Piddock, L. J. V.
(2006). Ciprofloxacin-Resistant Salmonella enterica Serovar Typhimurium Strains Are Difficult To Select in the Absence of AcrB and TolC. Antimicrob. Agents Chemother.
50: 38-42
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Tam, V. H., Schilling, A. N., Neshat, S., Poole, K., Melnick, D. A., Coyle, E. A.
(2005). Optimization of Meropenem Minimum Concentration/MIC Ratio To Suppress In Vitro Resistance of Pseudomonas aeruginosa. Antimicrob. Agents Chemother.
49: 4920-4927
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Gillis, R. J., White, K. G., Choi, K.-H., Wagner, V. E., Schweizer, H. P., Iglewski, B. H.
(2005). Molecular Basis of Azithromycin-Resistant Pseudomonas aeruginosa Biofilms. Antimicrob. Agents Chemother.
49: 3858-3867
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Mamelli, L., Prouzet-Mauleon, V., Pages, J.-M., Megraud, F., Bolla, J.-M.
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Gibreel, A., Kos, V. N., Keelan, M., Trieber, C. A., Levesque, S., Michaud, S., Taylor, D. E.
(2005). Macrolide Resistance in Campylobacter jejuni and Campylobacter coli: Molecular Mechanism and Stability of the Resistance Phenotype. Antimicrob. Agents Chemother.
49: 2753-2759
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Ueda, O., Wexler, H. M., Hirai, K., Shibata, Y., Yoshimura, F., Fujimura, S.
(2005). Sixteen Homologs of the Mex-Type Multidrug Resistance Efflux Pump in Bacteroides fragilis. Antimicrob. Agents Chemother.
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Valdezate, S., Vindel, A., Saez-Nieto, J. A., Baquero, F., Canton, R.
(2005). Preservation of topoisomerase genetic sequences during in vivo and in vitro development of high-level resistance to ciprofloxacin in isogenic Stenotrophomonas maltophilia strains. J Antimicrob Chemother
56: 220-223
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Baucheron, S., Mouline, C., Praud, K., Chaslus-Dancla, E., Cloeckaert, A.
(2005). TolC but not AcrB is essential for multidrug-resistant Salmonella enterica serotype Typhimurium colonization of chicks. J Antimicrob Chemother
55: 707-712
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Thiolas, A., Bollet, C., La Scola, B., Raoult, D., Pages, J.-M.
(2005). Successive Emergence of Enterobacter aerogenes Strains Resistant to Imipenem and Colistin in a Patient. Antimicrob. Agents Chemother.
49: 1354-1358
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Kriengkauykiat, J., Porter, E., Lomovskaya, O., Wong-Beringer, A.
(2005). Use of an Efflux Pump Inhibitor To Determine the Prevalence of Efflux Pump-Mediated Fluoroquinolone Resistance and Multidrug Resistance in Pseudomonas aeruginosa. Antimicrob. Agents Chemother.
49: 565-570
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Bohnert, J. A., Kern, W. V.
(2005). Selected Arylpiperazines Are Capable of Reversing Multidrug Resistance in Escherichia coli Overexpressing RND Efflux Pumps. Antimicrob. Agents Chemother.
49: 849-852
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Laatu, M., Rautelin, H., Hanninen, M.-L.
(2005). Susceptibility of Campylobacter hyointestinalis subsp. hyointestinalis to antimicrobial agents and characterization of quinolone-resistant strains. J Antimicrob Chemother
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Kaatz, G. W., Thyagarajan, R. V., Seo, S. M.
(2005). Effect of Promoter Region Mutations and mgrA Overexpression on Transcription of norA, Which Encodes a Staphylococcus aureus Multidrug Efflux Transporter. Antimicrob. Agents Chemother.
49: 161-169
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Olliver, A., Valle, M., Chaslus-Dancla, E., Cloeckaert, A.
(2005). Overexpression of the Multidrug Efflux Operon acrEF by Insertional Activation with IS1 or IS10 Elements in Salmonella enterica Serovar Typhimurium DT204 acrB Mutants Selected with Fluoroquinolones. Antimicrob. Agents Chemother.
49: 289-301
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Gruger, T., Nitiss, J. L., Maxwell, A., Zechiedrich, E. L., Heisig, P., Seeber, S., Pommier, Y., Strumberg, D.
(2004). A Mutation in Escherichia coli DNA Gyrase Conferring Quinolone Resistance Results in Sensitivity to Drugs Targeting Eukaryotic Topoisomerase II. Antimicrob. Agents Chemother.
48: 4495-4504
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Mullin, S., Mani, N., Grossman, T. H.
(2004). Inhibition of Antibiotic Efflux in Bacteria by the Novel Multidrug Resistance Inhibitors Biricodar (VX-710) and Timcodar (VX-853). Antimicrob. Agents Chemother.
48: 4171-4176
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Baucheron, S., Tyler, S., Boyd, D., Mulvey, M. R., Chaslus-Dancla, E., Cloeckaert, A.
(2004). AcrAB-TolC Directs Efflux-Mediated Multidrug Resistance in Salmonella enterica Serovar Typhimurium DT104. Antimicrob. Agents Chemother.
48: 3729-3735
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Chollet, R., Chevalier, J., Bryskier, A., Pages, J.-M.
(2004). The AcrAB-TolC Pump Is Involved in Macrolide Resistance but Not in Telithromycin Efflux in Enterobacter aerogenes and Escherichia coli. Antimicrob. Agents Chemother.
48: 3621-3624
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Giraud, E., Blanc, G., Bouju-Albert, A., Weill, F.-X., Donnay-Moreno, C.
(2004). Mechanisms of quinolone resistance and clonal relationship among Aeromonas salmonicida strains isolated from reared fish with furunculosis. J Med Microbiol
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Chollet, R., Chevalier, J., Bollet, C., Pages, J.-M., Davin-Regli, A.
(2004). RamA Is an Alternate Activator of the Multidrug Resistance Cascade in Enterobacter aerogenes. Antimicrob. Agents Chemother.
48: 2518-2523
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Hasdemir, U. O., Chevalier, J., Nordmann, P., Pages, J.-M.
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Chan, Y. Y., Tan, T. M. C., Ong, Y. M., Chua, K. L.
(2004). BpeAB-OprB, a Multidrug Efflux Pump in Burkholderia pseudomallei. Antimicrob. Agents Chemother.
48: 1128-1135
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Chevalier, J., Bredin, J., Mahamoud, A., Mallea, M., Barbe, J., Pages, J.-M.
(2004). Inhibitors of Antibiotic Efflux in Resistant Enterobacter aerogenes and Klebsiella pneumoniae Strains. Antimicrob. Agents Chemother.
48: 1043-1046
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Chang, L.-L., Chen, H.-F., Chang, C.-Y., Lee, T.-M., Wu, W.-J.
(2004). Contribution of integrons, and SmeABC and SmeDEF efflux pumps to multidrug resistance in clinical isolates of Stenotrophomonas maltophilia. J Antimicrob Chemother
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Saenz, Y., Ruiz, J., Zarazaga, M., Teixido, M., Torres, C., Vila, J.
(2004). Effect of the efflux pump inhibitor Phe-Arg-{beta}-naphthylamide on the MIC values of the quinolones, tetracycline and chloramphenicol, in Escherichia coli isolates of different origin. J Antimicrob Chemother
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Ricci, V., Piddock, L.
(2003). Accumulation of garenoxacin by Bacteroides fragilis compared with that of five fluoroquinolones. J Antimicrob Chemother
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Lin, J., Sahin, O., Michel, L. O., Zhang, Q.
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Gayet, S., Chollet, R., Molle, G., Pages, J.-M., Chevalier, J.
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47: 1555-1559
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Van Bambeke, F., Glupczynski, Y., Plesiat, P., Pechere, J. C., Tulkens, P. M.
(2003). Antibiotic efflux pumps in prokaryotic cells: occurrence, impact on resistance and strategies for the future of antimicrobial therapy. J Antimicrob Chemother
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Sanchez, P., Le, U., Martinez, J. L.
(2003). The efflux pump inhibitor Phe-Arg-{beta}-naphthylamide does not abolish the activity of the Stenotrophomonas maltophilia SmeDEF multidrug efflux pump. J Antimicrob Chemother
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Dean, C. R., Visalli, M. A., Projan, S. J., Sum, P.-E., Bradford, P. A.
(2003). Efflux-Mediated Resistance to Tigecycline (GAR-936) in Pseudomonas aeruginosa PAO1. Antimicrob. Agents Chemother.
47: 972-978
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Kaatz, G. W., Moudgal, V. V., Seo, S. M., Kristiansen, J. E.
(2003). Phenothiazines and Thioxanthenes Inhibit Multidrug Efflux Pump Activity in Staphylococcus aureus. Antimicrob. Agents Chemother.
47: 719-726
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Webber, M. A., Piddock, L. J. V.
(2003). The importance of efflux pumps in bacterial antibiotic resistance. J Antimicrob Chemother
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(2002). Interactions among Strategies Associated with Bacterial Infection: Pathogenicity, Epidemicity, and Antibiotic Resistance. Clin. Microbiol. Rev.
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Payne, D. J., Miller, W. H., Berry, V., Brosky, J., Burgess, W. J., Chen, E., DeWolf, W. E. Jr., Fosberry, A. P., Greenwood, R., Head, M. S., Heerding, D. A., Janson, C. A., Jaworski, D. D., Keller, P. M., Manley, P. J., Moore, T. D., Newlander, K. A., Pearson, S., Polizzi, B. J., Qiu, X., Rittenhouse, S. F., Slater-Radosti, C., Salyers, K. L., Seefeld, M. A., Smyth, M. G., Takata, D. T., Uzinskas, I. N., Vaidya, K., Wallis, N. G., Winram, S. B., Yuan, C. C. K., Huffman, W. F.
(2002). Discovery of a Novel and Potent Class of FabI-Directed Antibacterial Agents. Antimicrob. Agents Chemother.
46: 3118-3124
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Shapiro, E., Baneyx, F.
(2002). Stress-Based Identification and Classification of Antibacterial Agents: Second-Generation Escherichia coli Reporter Strains and Optimization of Detection. Antimicrob. Agents Chemother.
46: 2490-2497
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Hirakata, Y., Srikumar, R., Poole, K., Gotoh, N., Suematsu, T., Kohno, S., Kamihira, S., Hancock, R. E. W., Speert, D. P.
(2002). Multidrug Efflux Systems Play an Important Role in the Invasiveness of Pseudomonas aeruginosa. JEM
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Miyamae, S., Ueda, O., Yoshimura, F., Hwang, J., Tanaka, Y., Nikaido, H.
(2001). A MATE Family Multidrug Efflux Transporter Pumps out Fluoroquinolones in Bacteroides thetaiotaomicron. Antimicrob. Agents Chemother.
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Zhang, L., Li, X.-Z., Poole, K.
(2001). Fluoroquinolone susceptibilities of efflux-mediated multidrug-resistant Pseudomonas aeruginosa, Stenotrophomonas maltophilia and Burkholderia cepacia. J Antimicrob Chemother
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