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Antimicrobial Agents and Chemotherapy, May 2000, p. 1223-1228, Vol. 44, No. 5
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Evidence for Active Efflux as the Primary Mechanism of Resistance
to Ciprofloxacin in Salmonella enterica Serovar
Typhimurium
Etienne
Giraud,
Axel
Cloeckaert,
Dominique
Kerboeuf, and
Elisabeth
Chaslus-Dancla*
Station de Pathologie Aviaire et de
Parasitologie, Institut National de la Recherche Agronomique,
Centre de Recherche de Tours-Nouzilly, 37380 Monnaie, France
Received 30 July 1999/Returned for modification 3 November
1999/Accepted 31 January 2000
 |
ABSTRACT |
The occurrence of active efflux and cell wall modifications were
studied in Salmonella enterica serovar Typhimurium mutants that were selected with enrofloxacin and whose phenotypes of resistance to fluoroquinolones could not be explained only by mutations in the
genes coding for gyrase or topoisomerase IV. Mutant BN18/21 exhibited a
decreased susceptibility to ciprofloxacin (MIC = 0.125 µg/ml)
but did not have a mutation in the gyrA gene. Mutants
BN18/41 and BN18/71 had the same substitution, Gly81Cys in GyrA, but
exhibited different levels of resistance to ciprofloxacin (MICs = 2 and 8 µg/ml, respectively). None of the mutants had mutations in
the parC gene. Evidence for active efflux was provided by a
classical fluorimetric method, which revealed a three- to fourfold
decrease in ciprofloxacin accumulation in the three mutants compared to that in the parent strain, which was annuled by addition of the efflux
pump inhibitor carbonyl cyanide m-chlorophenylhydrazone. In
mutant BN18/71, a second fluorimetric method also showed a 50%
reduction in the level of accumulation of ethidium bromide, a known
efflux pump substrate. Immunoblotting and enzyme-linked immunosorbent
assay experiments with an anti-AcrA antibody revealed that the
resistance phenotype was strongly correlated with the expression level
of the AcrAB efflux pump and suggested that decreased susceptibility to
ciprofloxacin due to active efflux probably related to overproduction
of this pump could occur before that due to gyrA mutations.
Alterations were also found in the outer membrane protein and
lipopolysaccharide profiles of the mutants, and these alterations were
possibly responsible for the decrease in the permeability of the outer
membrane that was observed in the mutants and that could act
synergistically with active efflux to decrease the level of
ciprofloxacin accumulation.
| |
INTRODUCTION |
Fluoroquinolones are often the
treatment of choice in the cases of life-threatening salmonellosis due
to multidrug-resistant strains (4, 27).
Salmonella sp. strains that exhibit treatment-compromising resistance to fluoroquinolones are uncommon, but the increasing incidence of strains with decreased susceptibility is a matter of
concern (12, 28). In other gram-negative bacteria, such as
Escherichia coli, Neisseria gonorrhoeae, or
Klebsiella pneumoniae, high-level fluoroquinolone resistance
is always associated with the presence of multiple mutations in the
quinolone resistance-determining regions (QRDRs) of the genes that code
for the intracellular targets of these antibiotics, gyrase
(gyrA and gyrB) and topoisomerase IV
(parC and parE) (2, 7, 11). For
Salmonella enterica serovar Typhimurium, however, we showed
in a previous study (9) that fluoroquinolone resistance is
not well correlated with the presence of such mutations: highly
fluoroquinolone-resistant mutants selected in vitro had no mutations in
the genes that code for topoisomerase IV, and some had only one
mutation in the gyrA gene, whereas E. coli
isolates that exhibited the same level of resistance harbored at least
two mutations in gyrA and one in parC
(11).
In addition to this mechanism of target modification that is strictly
specific to quinolones, gram-negative bacteria can face the
presence of toxic compounds, including antibiotics, by excluding them
from the cell. Changes in the cell envelope, including loss of
outer membrane porins or alterations of the lipopolysaccharide (LPS),
can be partially responsible for decreased susceptibility to a wide
range of unrelated antibiotics (5, 26). Active efflux
systems that act synergistically with the outer membrane could have a
high level of participation in the intrinsic and the acquired
antibiotic resistance of gram-negative bacteria (16, 22).
Some of the multidrug efflux pumps, which belong to several families,
exhibit a low specificity and thus confer decreased susceptibility or
even clinically significant resistance to several classes of
antibiotics when they are overexpressed. It was recently shown that an
S. enterica serovar Typhimurium mutant that overproduces the
AcrAB efflux pump (initially identified in E. coli as a
close homolog of the MexAB efflux system of Pseudomonas
aeruginosa) was more resistant than the parent strain to a wide
variety of compounds such as fusidic acid, chloramphenicol,
tetracycline, norfloxacin, and penicillin G (23).
The goal of the present study was to investigate if additional
resistance mechanisms such as decreased cell envelope permeability or
active efflux could explain the phenotype of S. enterica
serovar Typhimurium mutants that were selected in vitro and that
reached different levels of resistance to fluoroquinolone but that
harbored not more than one gyrA mutation (9).
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MATERIALS AND METHODS |
Bacterial strains and selection of fluoroquinolone-resistant
mutants.
Fluoroquinolone-susceptible S. enterica
serovar Typhimurium strain BN18 was isolated from the liver of a
septicemic pigeon. Mutants BN18/21, BN18/41, and BN18/71 derived from
this parent strain were obtained after, respectively, two, four, and
seven selection steps on Mueller-Hinton agar plates supplemented with increasing concentrations of enrofloxacin (9). Our previous study showed that the QRDR sequences of gyrA,
gyrB, parC, and parE of mutant BN18/21
were identical to those of parent strain BN18 (9). Mutants
BN18/41 and BN18/71 were shown to have a single mutation in the QRDR of
gyrA (Gly81Cys) but none in the QRDRs of gyrB,
parC, and parE.
S. enterica serovar Typhimurium strain SH5014 and the
corresponding multidrug-resistant mutant that overproduces the Acr
pump, strain HN891, were used as controls for MIC determinations and in
the immunoblotting experiments (23).
Susceptibility determinations.
MICs were determined by the
standard agar doubling dilution method on Mueller-Hinton medium with
inocula of 104 CFU per spot. MICs were determined after
18 h of incubation at 37°C. The antibiotics were purchased from
the indicated manufacturers: nalidixic acid, penicillin G,
carbenicillin, and cefoxitin, Sigma, Steinheim, Germany; ciprofloxacin,
Bayer AG; and tetracycline and chloramphenicol, Boehringer Mannheim,
Mannheim, Germany.
Accumulation studies. (i) Accumulation of ciprofloxacin.
Ciprofloxacin uptake was assayed by the method of Mortimer and Piddock
(20), with some modifications. Bacteria were grown to the
late logarithmic phase at 37°C in Luria-Bertani (LB) medium, harvested by centrifugation, washed in 50 mM sodium phosphate buffer
(pH 7.0), and resuspended in the same buffer to an
A600 of 12.0. The cells were equilibrated for 10 min at 37°C. Ciprofloxacin was then added to a final concentration of
10 µg/ml. After addition of ciprofloxacin, 0.5-ml samples were
removed at different time intervals. Five minutes after addition of
ciprofloxacin, the efflux pump inhibitor carbonyl cyanide
m-chlorophenylhydrazone (CCCP) was added to the reaction
mixture (final concentration, 100 µM). The samples were immediately
diluted in 1 ml of ice-cold sodium phosphate buffer and were then
centrifuged for 5 min at 5,600 × g. The pellet was
washed once with 1 ml of ice-cold buffer and resuspended in 1 ml of 0.1 M glycine hydrochloride (pH 3.0) for at least 15 h at room
temperature. The samples were then centrifuged at 5,600 × g for 10 min. The fluorescence of the supernatant was measured
with a Quanta Master C-60 spectrofluorimeter (Photon Technology
International, Monmouth Junction, N.J.) at excitation and emission
wavelengths of 279 and 447 nm, respectively (3). The
concentration of ciprofloxacin in the supernatant was calculated by
comparison with a standard curve for ciprofloxacin (0.02 to 2.5 µg/ml) in 0.1 M glycine hydrochloride (pH 3.0). The results were
expressed as nanograms of ciprofloxacin incorporated per milligram (dry
weight) of bacteria. The experiments were performed at least three
times to make sure of the reproducibility.
(ii) Accumulation of ethidium bromide.
A previously
described fluorimetric method (1) was used, with slight
modifications, to measure the accumulation of ethidium bromide.
Briefly, cells were grown overnight, pelleted, and resuspended to an
A600 of 0.2 in sodium phosphate buffer (pH 7.0).
For demonstration of active efflux, ethidium bromide was added to the
suspension at a final concentration of 2 µg/ml. Fluorescence was used
as a measure of the amount of ethidium bromide incorporated by the cells and was recorded with a spectrofluorimeter at excitation and
emission wavelengths of 530 and 600 nm, respectively. CCCP was added to
the suspension (final concentration, 100 µM) after 400 s of
incubation. The natural fluorescence of the cells was subtracted, and
the fluorescence intensity was expressed in arbitrary units. For
demonstration of differences in outer membrane permeability, cells were
incubated with CCCP for 3 min before addition of ethidium bromide.
Results were expressed as the fluorescence intensity of the cells
incubated with ethidium bromide alone subtracted from that of the cells
incubated with CCCP and ethidium bromide.
Acr pump expression analysis by immunoblotting and ELISA.
Bacteria were grown overnight at 37°C in LB medium, harvested by
centrifugation, and resuspended at an A600 of
10.0. Cells were diluted one-half in the sample buffer of Laemmli
(15) and were heated for 10 min at 100°C. Whole-cell
proteins were separated by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) and were transferred at 0.8 mA/cm2 to a nitrocellulose membrane. The membrane was
washed three times with Tris-buffered saline (TBS; 0.15% NaCl, 10 mM
Tris-HCl [pH 7.5]), saturated for 30 min at room temperature with TBS
containing 1% skim milk, and incubated overnight at room temperature
with an anti-AcrA polyclonal antibody (23) diluted 1/2,000
in TBS containing 0.05% Tween 20 (TBS-T) and 0.33% skim milk. After
three washes in TBS-T, the membrane was incubated for 1 h with
peroxidase conjugated to protein A diluted 1/1,000 in TBS-T (Sigma, St.
Louis, Mo.). Finally, after three washes in TBS-T, the blot was
developed by incubation at room temperature in a solution of TBS
containing 0.06% 4-chloro-1-naphthol and 5 mM
H2O2. The reaction was stopped by washes in
distilled water.
For enzyme-linked immunosorbent assay (ELISA), bacteria resuspended at
an
A600 of 1.0 in phosphate-buffered saline
(PBS; pH
7.2) were sonicated. A total of 100 µl of the sonicates was
used
to coat each well of 96-well polystyrene plates for 18 h at
room
temperature. The anti-AcrA antibody serially diluted from 1/500
to
1/256,000 in PBS containing 0.05% Tween 20 (PBS-T) was incubated
on
the plates for 2 h at 37°C. Binding of the anti-AcrA antibody
was detected by a further incubation with the peroxidase-protein
A
conjugate diluted 1/1,000 in PBS-T. After incubation for 1 h
at
room temperature, the plates were filled with a substrate solution
containing 4 mM H
2O
2 and 1 mM
2,2-azino-di-3-ethylbenzthiazoline-sulfonic
acid. Excess
reagents between the different incubations were removed
by five washes
in 0.9% NaCl solution containing 0.05% Tween 20.
The plates were
shaken for 1 h at room temperature, and optical
density values
were recorded with an automatic ELISA reader (Bio-Tek
EL 312; Packard
Instrument, Rungis, France). The levels of AcrA
in the samples prepared
from mutants were deduced from the differences
in antibody titers
between the mutants and the parent
strain.
OMPs and LPS preparations.
Outer membrane proteins (OMPs)
were extracted by the method of Jerse and Kaper (14), with
slight modifications. Briefly, bacteria were grown overnight, harvested
by centrifugation, washed once in 10 mM Tris-HCl (pH 8.0), and
resuspended in the same buffer. Crude extracts prepared by sonication
were centrifuged at 3,800 × g for 10 min. The
supernatant was incubated with 1% Triton X-100 for 30 min at 37°C
and ultracentrifuged at 100,000 × g for 1 h. The
pellet was resuspended in 10 mM Tris (pH 8.0). Samples were prepared
for SDS-PAGE by mixing them with an equal volume of the sample buffer
of Laemmli (15) and heating the mixture for 10 min at
100°C. Protein gels were stained with Coomassie blue.
LPS was isolated by proteinase K treatment of whole cells as described
by Hitchcock and Brown (
13). LPS preparations tested
by
SDS-PAGE were stained with
silver.
| |
RESULTS |
S. enterica serovar Typhimurium mutants BN18/21,
BN18/41, and BN18/71, all derived from parent strain BN18, were chosen
for this study because their phenotype of resistance to
fluoroquinolones could not be totally explained only by the mutation
identified in the gyrase (9). Mutant BN18/21 was resistant
to nalidixic acid and exhibited decreased susceptibility to
ciprofloxacin, but it had no mutations in the QRDRs of the genes
gyrA, gyrB, parC, and parE.
Mutant BN18/41 had a single mutation, Gly81Cys, in the
gyrA gene but none in the other genes. Mutant BN18/71 had the same single mutation as BN18/41 but required fourfold more ciprofloxacin for inhibition. These observations led us to investigate the involvement of additional resistance mechanisms, that is, active
efflux, decreased outer membrane permeability, and cell wall alterations.
Susceptibilities to unrelated antibiotics.
The mutants
selected with enrofloxacin exhibited higher levels of resistance than
the parent strain not only to antibiotics of the same family (i.e.,
quinolones) but also to structurally unrelated compounds (Table
1). The MICs of tetracyclines and chloramphenicol were increased fourfold and eightfold in mutants BN18/21 and BN18/71, respectively. The MICs of 
-lactams were increased fourfold (penicillin G and carbenicillin) and eightfold (cefoxitin) for mutant BN18/21. For mutants BN18/41 and BN18/71, the
MICs of the three -lactams were increased 8-fold for penicillin G
and carbenicillin and 16-fold for cefoxitin. These results strongly suggested that the serial passages on fluoroquinolone had resulted in
the selection of an MDR (multiple drug resistance) phenotype, classically characterized by an enhanced activity of a
broad-specificity drug efflux pump (22).
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TABLE 1.
Susceptibilities to quinolones and structurally unrelated
antibiotics, GyrA substitutions, and AcrA expression levels of
control strains, parent strain BN18, and mutants derived from BN18
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Evidence for active efflux. (i) Ciprofloxacin accumulation.
Ciprofloxacin uptake appeared to be drastically reduced in the three
mutants compared to that in the parent strain (Fig.
1). At steady state, reached within 1 min
following addition of ciprofloxacin, all three mutants accumulated
about three- to fourfold less ciprofloxacin than the parent strain. The
lack of correlation between the MICs of the antibiotics for the mutants
and the level of accumulation of ciprofloxacin could be due to the
technical limits of this fluorometric method. Addition of CCCP, a
proton motive force uncoupler, induced a very rapid and significant
increase in cell-associated ciprofloxacin in the three mutants. As a
consequence, at steady state following addition of CCCP, the
accumulation of ciprofloxacin was nearly identical in all the strains.
These results indicated that an active efflux process limits the
accumulation of ciprofloxacin by the cells in a much more efficient way
in the mutants than in the parent strain.
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FIG. 1.
Accumulation of ciprofloxacin (Cip) by parent strain
BN18 ( ) and mutants BN18/21 ( ), BN18/41 ( ), and BN18/71 ( ).
CCCP (100 µM) was added at the time indicated by the arrow.
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(ii) Ethidium bromide accumulation.
We used a fluorimetric
method to investigate if the suspected active efflux could also
decrease the level of incorporation in the mutants of ethidium
bromide, a toxic hydrophobic cation known to be a substrate of efflux
pumps (25). The levels of accumulation of ethidium
bromide by parent strain BN18 and by the most resistant mutant, mutant
BN18/71, are compared in Fig. 2.
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FIG. 2.
Accumulation of ethidium bromide by parent strain
BN/18 and mutant strain BN18/71. CCCP (100 µM) was added at the time
indicated by the arrow. The inset shows the accumulation before
addition of CCCP on an enlarged scale.
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After 400 s the accumulation of ethidium bromide
in BN18/71 was approximately 50% of that observed in BN18. In
contrast to
the results obtained with ciprofloxacin accumulation,
addition
of CCCP induced a drastic increase in ethidium bromide
uptake
in both strains. However, the entry of ethidium bromide
appeared
to be faster in BN18 than in BN18/71. These results confirmed
that a proton motive force-dependent process was able to limit
the
incorporation of a toxic compound structurally unrelated to
fluoroquinolones. This process appeared to be much more efficient
in
the fluoroquinolone-selected mutants than in the parent strain.
Moreover, the apparently slower penetration of ethidium bromide
in
the mutant strain after addition of CCCP also suggested a lower
permeability of the cell
envelope.
(iii) Acr pump expression analysis.
Immunoblotting experiments
revealed that AcrA, the periplasmic component of the AcrAB efflux
system, faintly detected in strain BN18, was highly
overproduced in all the derived mutants (Fig. 3). The AcrA expression levels appeared
to correlate with the ciprofloxacin MICs for these mutants. This
correlation was confirmed by an enzyme immunoassay (ELISA): AcrA
production increased gradually with the number of selection steps on
enrofloxacin, reaching a 10-fold increase in mutant BN18/71 (Table 1).
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FIG. 3.
Immunoblot analysis of AcrA proteins of the parent
strain and the three mutants. SH5014 (parent strain) and HN891 (the
corresponding AcrA-overproducing mutant) are control strains described
in Materials and Methods. K, kilodaltons.
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Evidence for decreased outer membrane permeability.
In order
to assess the alteration of outer membrane permeability in the
resistant mutants, the level of accumulation of ethidium bromide was measured after inhibition of active efflux by CCCP. The rate of accumulation of ethidium bromide was slower for
the first mutant, BN18/21, and further decreased for mutants BN18/41 and BN18/71 (Fig. 4). This result
indicated a decreased outer membrane permeability in the mutants.
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FIG. 4.
Rate of accumulation of ethidium bromide by parent
strain BN18 and three mutants after inhibition of active efflux by
CCCP.
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SDS-PAGE analysis of the OMPs revealed no major alteration in the
expression of porins (Fig.
5). Expression
of a protein band
that migrated at 40 to 45 kDa was strongly repressed
in mutant
strains BN18/41 and BN18/71. The same observation was made
with
other fluoroquinolone-resistant mutants obtained independently
in
another selection line (data not shown). Slighter modifications
were
observed with bands with smaller molecular masses. In particular,
bands
that migrated at approximately at 25, 28, and 32 kDa appeared
to be
more strongly expressed in the mutants than in the parent
strain.
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FIG. 5.
OMP profiles prepared from parent strain BN18 and three
mutants. A total of 20 µg of OMPs was loaded per lane. Arrows on the
right side indicate the protein bands discussed in the text. Molecular
masses in kilodaltons are indicated on the left.
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LPS profiles (Fig.
6) revealed
significant alterations of the LPS: mutants BN18/41 and BN18/71
exhibited LPSs with a smaller
amounts of short and intermediate-length
O-polysaccharidic chains.
As a consequence, more long O-chain LPSs were
detected in mutants
BN18/41 and BN18/71 than in the parent strain.
The cell wall alterations (OMPs and LPS) observed in the mutants
appeared to correlate with their decreased outer membrane
permeability.
| |
DISCUSSION |
Our previous study (9) had shown that genetic changes
in the genes that code for the target proteins of fluoroquinolones, i.e., gyrase and topoisomerase IV, could not totally account for the
resistance phenotypes of S. enterica serovar Typhimurium
mutants selected in vivo or in vitro. Therefore, we investigated the
possible involvement of an active efflux mechanism, which acts
synergistically or not, with alterations of the bacterial cell wall.
The observation that the fluoroquinolone-selected mutants had also
gained resistance to unrelated antibiotics such as tetracycline,
chloramphenicol, and -lactams made us suspect that we had selected
Mar (multiple antibiotic resistance) mutants. This broad-spectrum
resistance phenotype has been shown in E. coli to result
from increased active efflux (16). It was also demonstrated
in E. coli that the AcrAB multidrug efflux system plays a
major role in the drug resistance phenotype of Mar mutants
(24). In serovar Typhimurium, overproduction of the Acr pump
was also directly linked to increased rates of resistance to a wide
range of compounds including quinolones such as nalidixic acid or
norfloxacin (23). However, in the selection process that can
lead to clinical resistance, it was not clear whether (i) decreased
susceptibility to fluoroquinolones due to enhanced active efflux could
be consistently selected in a predetermined way before the occurrence
of gyrA mutations or (ii) the level of expression of the Acr
pump was correlated to the fluoroquinolone MICs for strains in which
gyrase mutations alone could not totally explain the resistance phenotype.
Time course ciprofloxacin uptake experiments were performed with parent
and mutant strains that exhibited increasing rates of resistance, one
of which had no mutation in the QRDRs of gyrA, gyrB, parC, and parE and the two
others of which had only one mutation in gyrA that led to
the Gly81Cys substitution (Table 1). A very large difference in the
amount of cell-associated ciprofloxacin, which was nearly reversed by
addition of the protonophore CCCP, was observed between the parent
strain and the three derived mutants (Fig. 1). This result provided
evidence of an active efflux process that uses the proton motive force
as an energy source. However, no differences in the levels of
accumulation could be seen for the different mutants. In another
fluorimetric assay, the monitoring of the accumulation of ethidium
bromide, a known substrate for the efflux pump (1), by the
parent strain and the most resistant mutant also gave evidence for the
existence of an active efflux process (Fig. 2). As is expected for Mar
mutants, we could confirm using an anti-AcrA antibody the
overproduction of the Acr pump in the mutants. Interestingly, this
overproduction already reached 3.5-fold that for the parent strain in
mutant BN18/21 (ciprofloxacin MIC, 0.125 µg/ml), which was not yet
mutated in the gyrA gene. In mutants BN18/41 and BN18/71,
for which the ciprofloxacin MICs reached 2 and 8 µg/ml, respectively,
AcrA expression levels were about 7- and 10-fold higher than those in
BN18. Similarly, an overexpression of AcrA was observed in clinical
mutants that were resistant to enrofloxacin and that had decreased
susceptibility to ciprofloxacin (data not shown). The AcrAB pump is
known to be partly responsible for the intrinsic level of resistance of gram-negative bacteria when it is expressed at its basic expression level (21). These results suggested that its overproduction could participate, independently from gyrA mutations, in a
first decrease in susceptibility to fluoroquinolones and in the
accession to high fluoroquinolone resistance levels.
It has previously been demonstrated in E. coli that
the AcrAB efflux system is essentially regulated by the
global regulator loci marRAB or soxRS (17,
24), which also regulates the synthesis of the major porin OmpF
(5). Therefore, we examined whether OMP expression had been
altered in our S. enterica serovar Typhimurium mutants.
We also investigated the possible modification of the LPS, since it has
been shown in E. coli that LPS alterations could affect
the correct folding (6) and trimerization (29,
30) of porins and also since quinolone resistance has been
associated with changes in the LPSs of gram-negative bacteria such as
Burkholderia cepacia (26). Cell envelope
modifications in both the OMPs and the LPS, possibly in relation to
decreased outer membrane permeability, were found in the
fluoroquinolone-selected mutants. These modifications appeared to be
much more important in the two most resistant mutants, BN18/41 and
BN18/71, which had mutations in the gyrA gene, than in the
BN18/21 mutant, which exhibited only enhanced active efflux. In
contrast to what can be expected from Mar mutants
(22), no porin expression decrease was observed. However,
expression of several proteins was altered: one protein band at 40 to
45 kDa was strongly repressed, while other bands appeared to be
highly expressed in the resistant mutants. In the absence of
identification of these protein bands, it is difficult to
determine whether their altered expression is involved in the
resistance phenotype. As a matter of fact, mutations in a global
regulator locus such as the mar locus that affect the
resistance phenotype can also have pleiotropic effects on the
expression of genes not necessarily involved in the resistance
phenotype. Work is in progress to identify proteins whose expression is
altered in the mutants by using two-dimensional gel electrophoresis and
N-terminal amino acid microsequencing.
Examination of the LPS profile revealed an increase in the proportion
of the long O-polysaccharidic chains. The role of the composition of
the LPS on the accumulation of quinolones has been extensively studied
previously, but it remains unclear, as contradictory results have been
obtained (3, 8, 10, 18, 19). It is thought that hydrophilic
quinolones like ciprofloxacin preferentially use the porin pathway to
penetrate the cells (3). Therefore, accessibility to the
porin through the LPS is expected to be determinant. It has been
hypothesized in quinolone-selected P. aeruginosa strains that increased amounts of LPS form a permeability barrier which acts preferentially against hydrophilic quinolones
(18). The lengthening of the O chains could also result in a
lower level of accessibility to the outer membrane.
In summary, we demonstrated enhanced active efflux and gradually
increased levels of production of the AcrAB pump in mutants increasingly resistant to ciprofloxacin and other antibiotics. This
overproduction appeared to be early compared to the occurrence of a
gyrA mutation and thus could be responsible for the first decrease in susceptibility to fluoroquinolones. Our study suggests that, in the absence of multiple mutations that affect the genes that
code for gyrase and topoisomerase IV, as observed in E. coli, active efflux that possibly acts synergistically with a
decrease in outer membrane permeability could play a major role in
ciprofloxacin resistance in S. enterica serovar Typhimurium.
Although no Salmonella strains resistant to ciprofloxacin
could be isolated in the field (9), overexpression of AcrA
observed in the clinical strains with decreased susceptibilities to
ciprofloxacin suggests the generality of the efflux phenomenon.
| |
ACKNOWLEDGMENTS |
This work was supported by grants from the Conseil Régional
de la Région Centre.
We thank C. Mouline and G. Flaujac for technical assistance,
H. Nikaido (University of California, Berkeley) for
providing the anti-AcrA antibody and strains HN891 and SH5014, and
A. P. Teixeira-Gomes and I. Moriyon for helpful discussions.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Station de
Pathologie Aviaire et de Parasitologie, Institut National de la
Recherche Agronomique, Centre de Recherche de Tours-Nouzilly, 37380 Monnaie, France. Phone: 33-2-47-42-77-65. Fax: 33-2-47-42-77-74. E-mail address: chaslus{at}tours.inra.fr.
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Antimicrobial Agents and Chemotherapy, May 2000, p. 1223-1228, Vol. 44, No. 5
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Abstract
Introduction
