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Antimicrobial Agents and Chemotherapy, August 1998, p. 2089-2094, Vol. 42, No. 8
Center for Adaptation Genetics and Drug
Resistance1 and
Departments of Molecular
Biology and Microbiology2 and
of
Medicine,4
Tufts University School of
Medicine, Boston, Massachusetts 02111, and Section of Infectious
Diseases and Clinical Immunology, University Hospital and Medical
Center, Ulm, Germany3
Received 26 September 1997/Returned for modification 22 January
1998/Accepted 29 April 1998
The contribution of regulatory genes to fluoroquinolone resistance
was studied with clinical Escherichia coli strains bearing mutations in gyrA and parC and with different
levels of fluoroquinolone resistance. Expression of marA
and soxS was evaluated by Northern blot analysis of
isolates that demonstrated increased organic solvent tolerance, a
phenotype that has been linked to overexpression of marA,
soxS, and rob. Among 25 cyclohexane-tolerant
strains detected by a screen for increased organic solvent tolerance
(M. Oethinger, W. V. Kern, J. D. Goldman, and S. B. Levy, J. Antimicrob. Chemother. 41:111-114, 1998), we found 5 Mar
mutants and 4 Sox mutants. A further Mar mutant was detected among 11 fluoroquinolone-resistant, cyclohexane-susceptible E. coli
strains used as controls. Comparison of the marOR sequences
of clinical Mar mutants with that of E. coli K-12 (GenBank
accession no. M96235) revealed point mutations in marR in
all mutants which correlated with loss of repressor function as
detected in a marO::lacZ
transcriptional assay. We found four other amino acid changes in MarR
that did not lead to loss of function. Two of these changes, present in
20 of the 35 sequenced marOR fragments, identified a
variant genotype of marOR. Isolates with the same
gyrA and parC mutations showed increased fluoroquinolone resistance when the mutations were accompanied by
overexpression of marA or soxS. These data
support the hypothesis that high-level fluoroquinolone resistance
involves mutations at several chromosomal loci, comprising structural
and regulatory genes.
A large number of studies of
fluoroquinolone resistance among clinical Escherichia coli
isolates has shown that mutations in the structural genes
gyrA and parC are important mechanisms of
resistance (13, 14, 21, 29, 37, 42). However, it has also
become evident that additional mutations, such as mutations in one of
the regulatory genes marRAB (10, 12, 16),
soxRS (2, 44), and robA (4)
and in other yet unidentified genes, potentially contribute to the
resistance phenotype (15, 25). These regulatory genes, when
overexpressed, confer low-level resistance to a number of structurally
unrelated compounds, including quinolones (4, 12, 18).
The mar operon in E. coli consists of two
divergently positioned transcriptional units that flank the operator
marO (for a review, see reference 1).
Transcriptional unit 2 comprises marRAB, which encodes the
Mar repressor MarR, the activator MarA, and a putative small protein,
MarB, of unknown function (10). In the absence of an
inducer, MarR represses transcription of marRAB by binding
to marO, thus negatively controlling expression of other
genes on the chromosome by the activator MarA (1, 40). Upon
induction of the marRAB operon by a variety of compounds, including tetracycline, chloramphenicol, and salicylate, MarR repression of marRAB is alleviated. Constitutive expression
of marRAB also occurs when the repressor is rendered
inactive by marR mutations (1). Mar mutants exist
among clinical, fluoroquinolone-resistant isolates of E. coli (25).
In this study, we investigated the relative contributions of regulatory
gene and structural gene changes to fluoroquinolone resistance in
E. coli, using a large number of well-characterized clinical
isolates with different levels of susceptibility to fluoroquinolones and with known mutations in the regions that determine quinolone resistance in gyrA (13) and parC
(14).
(Part of this study was presented at the 97th General Meeting of the
American Society for Microbiology, Miami Beach, Fla., 4 to 8 May 1997 [34].)
Bacterial strains, plasmids, and media.
A total of 138 independently obtained clinical E. coli isolates were
studied (33). The fluoroquinolone-resistant isolates (ofloxacin MICs,
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Overexpression of the marA or soxS
Regulatory Gene in Clinical Topoisomerase Mutants of
Escherichia coli
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
8 µg/ml) comprised 21 bloodstream isolates from different cancer centers across Europe and the Middle East (E strains),
17 bloodstream isolates cultured from hematologic-oncologic patients at
Ulm University Hospital, Ulm, Germany (HO strains), and 19 strains
which comprised predominantly urinary tract isolates obtained from
nonhematologic patients admitted to surgical services of Ulm University
Hospital (NH strains). These strains differed by pulsed-field gel
electrophoresis and PCR-randomly amplified polymorphic DNA analysis
(32) and were gyrA double mutants with either one
or two additional mutations in parC (13, 14). DNA sequencing of gyrA and parC provided information
from both strands for the regions from nucleotides 123 to 366 (Leu41
through Tyr122) of gyrA (35) and from nucleotides
145 to 492 (Lys39 through Gln138) of parC (23).
The present study also included 24 E. coli isolates with
intermediate levels of fluoroquinolone susceptibility (M strains;
ofloxacin MICs, 0.5 to 4 µg/ml) which were obtained from
nonneutropenic patients at Ulm University Hospital and comprised predominantly urinary tract isolates. Finally, 57 fluoroquinolone-susceptible isolates (S strains; ofloxacin MICs, 
0.25
µg/ml) from cancer patients at Ulm University Hospital, 18 of which
were bloodstream isolates, served as a control group. The laboratory
strains and plasmids used in this study are described in Table
1. Stock cultures were kept frozen at
80°C in the Microbank system (Mast, Germany) or in 20% glycerol.
TABLE 1.
Laboratory strains and plasmids
Susceptibility testing. The MICs of selected antimicrobial agents were determined by a standard broth microdilution procedure with cation-adjusted Mueller-Hinton broth and a final inoculum of 5 × 105 CFU/ml according to National Committee for Clinical Laboratory Standards performance and interpretive guidelines (30). Microtiter plates were purchased from Merlin Diagnostics (Bornheim, Germany). Control strains included E. coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853.
RNA extraction and Northern blot analysis.
Overnight
cultures were diluted 100-fold in fresh Luria-Bertani (LB) broth (per
liter, 10 g of tryptone, 5 g of yeast extract, and 10 g
of NaCl) and grown to the mid-logarithmic phase at 30°C with shaking.
In order to induce the marRAB operon, sodium salicylate (final concentration, 5 mM) was added to half the culture for 45 min
before the cells were harvested by centrifugation (11). Similarly, the sox regulon was induced by the
superoxide-generating agent paraquat (final concentration, 1.3 mM;
Sigma, St. Louis, Mo.) in the presence of oxygen (18). Total
RNA was extracted from a 50-ml culture by a cesium chloride (CsCl)
method (6), with some modifications. Cells were washed in TE
buffer (50 mM Tris-HCl, 50 mM EDTA [pH 8]) and lysed in the same
buffer with 3.4% sodium dodecyl sulfate (SDS). CsCl (Cabot, Revere,
Pa.) was added to 67% (wt/vol), and the preparation was centrifuged
for 10 min at 14,000 × g. The supernatant was loaded
on a cushion of 5.7 M CsCl-100 mM EDTA and centrifuged overnight at
150,000 × g at 20°C. The RNA pellet was treated with
acid-phenol-chloroform, ethanol precipitated, resuspended in water,
and stored at 80°C. The concentration of total RNA in the samples
was determined spectrophotometrically by a ribonucleotide assay
(39) based on the orcinol reaction (Sigma). Hybridization of
radiolabelled DNA probes to the membrane-bound RNA (20 µg/lane) was
performed at 65°C overnight according to the specifications of the
membrane manufacturer (Amersham, Arlington Heights, Ill.).
-32P]dCTP by using a Boehringer Mannheim
(Indianapolis, Ind.) random primer labeling kit. RNA blots were washed
twice with 2× SSPE (0.36 M NaCl, 0.02 M sodium phosphate, 0.002 M EDTA
[pH 7.7]-0.1% SDS at room temperature and twice with 1×
SSPE-0.1% SDS at 65°C. Washed membranes were air dried and exposed
on a PhosphorImager screen (Molecular Dynamics, Sunnyvale, Calif.), and
signals were visualized with ImageQuant software (Molecular Dynamics).
Northern blot analysis was performed on RNAs from at least two
independent extractions.
DNA manipulations. Total chromosomal DNA was prepared as described previously (9). The marOR region was amplified from bp 1311 to 1858 (10) in a DNA thermocycler (model 480; Perkin-Elmer Cetus, Norwalk, Conn.) with the primer pair ORAB2 and RK3, which included PstI and EcoRI restriction sites, respectively, to allow directional cloning of the PCR fragment into plasmid pSPOK (25). After purification, both DNA strands were cycle sequenced by the Tufts University DNA Sequencing Facility with the same primers. In special cases, sequencing was repeated with a different PCR DNA batch to check for errors introduced during PCR. Recombinant DNA techniques, transformation, and restriction enzyme digestions were performed by standard techniques (38). Transformation of pMAK-TU1&TU2 (Table 1) into the clinical isolate NH10 was performed by electroporation with a gene pulser apparatus (Bio-Rad, Richmond, Calif.).
Test for MarR function with a
marO::lacZ fusion.
Overnight
cultures of ASS121 strains bearing pSPOK, with or without different
cloned marOR sequences, were diluted 1:100 in fresh LB broth
with 100 µg of ampicillin per ml, 50 µg of kanamycin per ml, and
0.5 mM isopropyl-
-D-thiogalactopyranoside (IPTG; Alexis
Corporation, Läufelfingen, Switzerland). Following growth at
30°C to late logarithmic phase, the cells were assayed in triplicate for -galactosidase activity with a chemiluminescence assay kit (Tropix, Bedford, Mass.). Cells were solubilized with chloroform-SDS and diluted 103-fold. Twenty microliters of each dilution
was added to 200 µl of reaction buffer containing the substrate
Galacton (Tropix) and incubated for 60 min at room temperature. After
the reaction was terminated by the addition of Emerald enhancer, the
chemiluminescent signal was measured in a OptocompI luminometer (MGM
Instruments, Hamden, Conn.). Data were expressed as relative light
units/A530 and referred to the chemiluminescence
of ASS121 bearing SPOK without the insert as a percentage of the
control.
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RESULTS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Identification of point mutations in marR that are causes for marA overexpression in clinical isolates of E. coli. Since all known Mar mutants studied thus far have been shown to be cyclohexane tolerant (5, 43), we screened our collection of 138 clinical E. coli isolates for cyclohexane tolerance. We detected increased organic solvent tolerance in 1 of 57 fluoroquinolone-susceptible, in 3 of 24 low-level fluoroquinolone-resistant, and in 21 of 57 high-level fluoroquinolone-resistant E. coli clinical isolates (33). Northern blot analysis of RNAs harvested from these 25 cyclohexane-tolerant and 11 cyclohexane-susceptible, fluoroquinolone-resistant strains (some of the latter strains carried mutations in gyrA and parC which were identical to mutations in certain cyclohexane-tolerant strains) showed that six strains constitutively expressed marA (Fig. 1). One Mar mutant (NH52) was an exception in that it was cyclohexane susceptible (Table 2). Sequencing data were compared with the marOR sequence derived from AG100 (accession no. M96235 [10]) and showed six missense mutations (three transversions and three transitions) and two nonsense mutations among the six clinical E. coli isolates overexpressing marA (Table 2). The latter mutations were insertion of an early stop codon in strain M19 and a frameshift mutation in strain NH52. Since the sequences of NH52, HO99, and E22 differed by only one nucleotide from that of wild-type marOR, we repeated PCRs from the original frozen stocks of these strains and obtained identical results upon resequencing.
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Leu mutation in gyrA, and strain NH52 had
a Ser83Leu and an Asp87Asn mutation in
gyrA and a Ser80Ile mutation in parC.
Likewise, strains HO17, HO99, and E22 were gyrA double
mutants and had an additional parC mutation (13)
(see Table 4).
We cloned the marOR fragments of three of the Mar mutants
(HO17, NH52, and M19) in an IPTG-inducible vector and tested the inhibitory function of their gene products on a transcriptional marO::lacZ fusion in the
mar strain ASS121 (Table 1). The control strain (ASS121
with pSPOK without the insert) showed high -galactosidase activity, which was defined as 100% activity (Fig.
2, bar 1). Introduction of
wild-type marOR, derived from AG100, into ASS121 inhibited -galactosidase activity almost completely (5%
residual activity) (Fig. 2, bar 2). In contrast, suppression by
marOR fragments from clinical Mar mutants was weak and
yielded between 57 and 70% residual -galactosidase activity
(Fig. 2, bars 3 through 5), confirming low repressor activity. Of
importance, the comparison of HO4 with HO17 showed that the R94H
mutation decreased the function of MarR but that the mutations at amino
acids (aa) 103 and 137 did not.
|
High frequency of strains with nucleotide sequences of
marOR divergent from sequences in the GenBank
database.
Twelve of 35 sequenced strains had a marOR
nucleotide sequence identical to that of the E. coli
K-12 strain AG100 (Table 3). In
contrast, 20 of the 35 strains, including the three Mar mutants S20,
M19, and HO17 (Table 2), had nucleotide changes which always occurred in combination: 1332AC in marO and
1751GA (Gly103Ser) and 1853TC (Tyr137His) in
marR (Tables 2 and 3). These mutations by themselves did not
interfere with the wild-type function of the repressor MarR, since
marA overexpression was not seen by Northern blot analysis
in several strains carrying the mutations. When the function of the
divergent MarR (Gly103Ser and Tyr137His) was studied in the
above-described reporter gene assay, the gene products of two
representative strains (HO4 and E1) were able to suppress
-galactosidase activity to a residual level of about 5% (e.g., Fig.
2, bar 6). Hence, we conclude that the observed marOR
sequences represent a variant genotype of E. coli
without loss of MarR function.
|
Ser,
Glu31stop codon, and Arg94His, respectively, were responsible for
the loss of MarR function. In contrast, two other amino
acid changes, Ser3Asn and Ala53Glu, were seen in strains with
wild-type levels of expression of marA (Table 3).
Clinical isolates of E. coli overexpressing
soxS.
Expression of soxS mRNA in the presence of
oxygen by vigorous shaking was investigated for all 25 cyclohexane-resistant strains and for the 11 cyclohexane-susceptible
strains. RNA from the soxRS-deleted strain DJ901 and from
the soxS-overexpressing strain JTG1078 served as negative
and positive controls, respectively (Table 1; Fig. 3). Northern blot analysis demonstrated
overexpression of soxS in four of the clinical isolates
relative to expression in the control strains (Fig. 3). All Sox
mutants were cyclohexane resistant. For three strains, ofloxacin MICs
were high (64 µg/ml). gyrA and parC data are
shown in Table 4 for strains E3 and E19.
Strain E17 was a gyrA double mutant (Ser83Leu,
Asp87Asn) with a Ser80Arg mutation in parC. Strain M1,
which is intermediately resistant to fluoroquinolone (ofloxacin MIC, 4 µg/ml), carried a Ser80Leu mutation in gyrA.
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Expression of marA or soxS among fluoroquinolone-resistant strains with identical mutations in gyrA and parC. For strains with the same mutations in gyrA and parC, oflaxacin MICs were nevertheless quite different (Table 4). For five strains overexpressing marA (HO17, HO99, and E22) or soxS (E3 and E19), ofloxacin MICs were four- to eightfold higher than those for strains matched for their gyrA and parC mutations but with wild-type levels of expression of these regulatory genes (NH1 and HO12). Three strains (E10, HO13, and E7) showed increased fluoroquinolone resistance without overexpressing marA or soxS. Two of these strains, E10 and HO13, were also cyclohexane susceptible. The basis of the increased fluoroquinolone resistance in these three strains is still unknown. The contribution of marA and soxS to the higher level of fluoroquinolone resistance in the topoisomerase mutants was not directly testable because of a lack of available antibiotic resistance markers for inactivation of the genes in these strains. However, transformation of the clinical topoisomerase mutant, non-Mar strain NH10 with pMAK-TU1&TU2, which specifies marA overexpression in trans, resulted in a twofold increase in the MIC of ofloxacin (16 versus 8 µg/ml) and a fourfold increase in the MIC of pefloxacin (64 versus 16 µg/ml) in conjunction with a newly observed cyclohexane tolerance. Thus, overexpression of marA can result in two- to fourfold increased resistance to fluoroquinolone compared with that mediated by topoisomerase mutations.
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Resistance to the fluoroquinolones in E. coli is principally caused by mutations in the structural genes for topoisomerase II (gyrA and gyrB) and topoisomerase IV (parC and parE) (13, 14, 19, 21, 29, 37, 42) and by mutations affecting regulatory genes, namely, marA (10, 12), soxS (2, 44), and rob (4). In a previous study of 46 ofloxacin-resistant clinical E. coli strains, we noted a wide range of ofloxacin MICs for strains with identical mutations in gyrA and parC (13, 14). Similarly, among another 36 E. coli strains recently described, as many as 22 (61%) had higher levels of fluoroquinolone resistance than was expected from the mutations in the topoisomerase genes alone (15).
We adopted a screening approach to investigate the possible involvement of the regulatory genes marA and soxS in fluoroquinolone resistance in clinical E. coli isolates. Since overexpression of these loci has been linked to increased organic solvent tolerance as well as fluoroquinolone resistance (5, 27, 28, 43), we tested the strains for cyclohexane tolerance. This phenotype was significantly more frequent among the fluoroquinolone-resistant strains than among the fluoroquinolone-susceptible controls and was also associated with a higher level of resistance (33). Among 25 cyclohexane-tolerant strains detected by the screen (33) and 11 cyclohexane-susceptible strains used as controls, we found 6 Mar mutants and 4 Sox mutants in which the level of fluoroquinolone resistance was higher than was attributable to mutations in the structural genes gyrA and parC. We infer that overexpression of these regulatory genes enhanced fluoroquinolone resistance. However, other possibilities include mutations in the structural genes for the second subunits of gyrase, gyrB (29, 36, 45), and topoisomerase V, parE (7), although these appear to be rare events and would not explain the organic solvent tolerance.
Unexpectedly, one of the Mar mutants was found by its cyclohexane tolerance among clinical fluoroquinolone-susceptible E. coli strains. Although the strain was classified as fluoroquinolone-susceptible E. coli, the ofloxacin MIC of 0.25 µg/ml for this strain was already beyond the MIC at which 90% of fluoroquinolone-susceptible strains are inhibited and its antibiotic profile was characteristic of a Mar mutant, such as AG102 (16). Sequencing of gyrA and parC revealed no mutations in the quinolone-resistance-determining regions. We conclude that the small increase in fluoroquinolone resistance was due to overexpression of marA, as has been shown for the laboratory strain AG102 (12). Further studies are needed to determine whether the observed frequency of about 2% Mar mutants among apparent fluoroquinolone-susceptible E. coli strains cultured from hospital inpatients corresponds to the background level of Mar mutants.
Overexpression of marA in the clinical Mar mutants was due
exclusively to point mutations in marR. Our previous study
(25) described deletions in all three Mar mutants,
accompanied by single amino acid changes in two of the mutants. Of
interest, the point mutations found in the present study affected amino
acids at highly conserved positions (aa 49 and 78) or a completely
conserved position (aa 94) in the newly recognized family of MarR
homologs (1, 26). In addition to the mutations that
alleviated marR repression, we identified a small deletion
in marO (from nucleotides 1369 to 1373) and four amino acid
changes in marR without loss of repressor activity
(Ser3Asn, Ala53Glu, Gly103Ser, and Tyr137His). Two of the
last mutations, Gly103Ser and Ser3Asn, had previously been
reported for two clinical Mar mutants (KM-D and J28, respectively) along with nucleotide deletions (25). It now appears that
the nucleotide deletions and not the single amino acid changes are the
cause of the Mar phenotype in these strains. The high frequency of
the amino acid changes Gly103Ser and Tyr137His in the clinical strains tested suggests that the underlying nucleotide changes are
genotypic variations without a change of phenotype.
The two-component soxRS regulatory system is involved in the adaptive response of E. coli to superoxide stress (2, 18, 41, 44). SoxR acts as the sensor and transcriptional activator of SoxS, which in turn activates a number of superoxide stress as well as antibiotic resistance genes (2, 31, 44). Four clinical E. coli strains displayed high constitutive expression of soxS, which was increased even further by paraquat induction (Fig. 3). Since constitutive expression of the sox locus follows mutations in soxR (2, 41, 44), we suspect that overexpression of soxS in the clinical strains is linked to mutations in soxR. That none of the clinical strains were both Mar and Sox mutants may relate to the fact that both regulatory systems control the expression of overlapping sets of target genes (3, 26).
Recent data suggest that a double mutation in gyrA plus a mutation at parC confers a ciprofloxacin MIC of 8 µg/ml (14, 15, 42). This finding indicates that additional mechanisms contribute to the fluoroquinolone resistance phenotype in about a third of fluoroquinolone-resistant E. coli isolates (14, 15, 42). In our study, half of the strains for which fluoroquinolone MICs were unexplainably high were Mar or Sox mutants (Table 4). The results of previous studies (22, 25) and the increase in fluoroquinolone MICs by marA overexpression in trans in the clinical strain NH10 are consistent with the hypothesis that removal of marA decreases fluoroquinolone MICs for the Mar strains to the lower level seen in the topoisomerase mutants which have a wild-type marRAB. Our conclusion is corroborated by recent work of Heisig and Wiedemann (20), who investigated the quinolone-resistance-determining region of KM-F, one of our previously reported Mar mutants (25). They found one mutation each in gyrA, gyrB, and parC (20). Upon deletion of mar by a kanamycin cassette (25), the ciprofloxacin MIC dropped by two dilutional steps, from 32 to 8 µg/ml (20). However, since the clinical strains of the present study were not isogenic (32), one cannot exclude the possibility that other differences between strains contributed to the level of fluoroquinolone resistance. Mutations in acrAB (24, 43) or rob (4), for instance, would affect both drug and organic solvent resistance and may account for the increased fluoroquinolone resistance in some of the other strains.
The Mar mutant NH52 deserves attention since it was cyclohexane susceptible despite overexpressing marA. The strain may be defective in one of the structural genes, such as acrAB, that is involved in mar-mediated cyclohexane tolerance (43). Alternatively, its wild-type tolerance to organic solvents, without overexpressing marA, might have been low for other reasons. Two of the 138 strains studied here were E. coli strains which do not grow with hexane (33). Both hypotheses would fit the observation that strain NH52 has wild-type levels of susceptibility to chloramphenicol and tetracycline.
The exact incidence of Mar or Sox mutants among clinical isolates of E. coli remains unknown. We may have missed some mutants like NH52 in the organic solvent screen, although this number would be very small. It appears, however, that mutations in the regulatory genes marA and soxS play a role in about 10 to 15% of clinical fluoroquinolone-resistant E. coli strains, which is in line with the results of our previous study (25). Mar and Sox mutants which have a higher level of fluoroquinolone resistance than expected from mutations in the structural genes gyrA and parC will likely be found among other strains. Our data support the hypothesis that chromosomal loci other than gyrA and parC contribute to fluoroquinolone resistance in a substantial number of clinical E. coli isolates.
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ACKNOWLEDGMENTS |
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This study was supported in part by research grants from the U.S. Public Health Service (GM 51661 to S.B.L.), the Deutsche Forschungsgemeinschaft (Oe 195/1-1 to M.O.), and the University of Ulm (P172/1994 to M.O. and W.V.K.).
We thank Bruce Demple for helpful discussion of results and Laura McMurry and Patrick McDermott for invaluable comments during the preparation of the manuscript. The expert help of A. S. Ritter is greatly appreciated.
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FOOTNOTES |
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* Corresponding author. Mailing address: Center for Adaptation Genetics and Drug Resistance, Tufts University School of Medicine, 136 Harrison Ave., Boston, MA 02111. Phone: (617) 636-6764. Fax: (617) 636-0458. E-mail: slevy{at}opal.tufts.edu.
Present address: Herz- und Diabeteszentrum NRW, 32 545 Bad
Oeynhausen, Germany.
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REFERENCES |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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| 1. | Alekshun, M. N., and S. B. Levy. 1997. Regulation of chromosomally mediated multiple antibiotic resistance: the mar regulon. Antimicrob. Agents Chemother. 41:2067-2075[Medline]. |
| 2. |
Amábile-Cuevas, C. F., and B. Demple.
1991.
Molecular characterization of the soxRS genes of Escherichia coli: two genes control a superoxide stress regulon.
Nucleic Acids Res.
19:4479-4484 |
| 3. |
Ariza, R. R.,
S. P. Cohen,
N. Bachhawat,
S. B. Levy, and B. Demple.
1994.
Repressor mutations in the marRAB operon that activate oxidative stress genes and multiple antibiotic resistance in Escherichia coli.
J. Bacteriol.
176:143-148 |
| 4. |
Ariza, R. R.,
Z. Li,
N. Ringstad, and B. Demple.
1995.
Activation of multiple antibiotic resistance and binding of stress-inducible promoters by Escherichia coli Rob protein.
J. Bacteriol.
177:1655-1661 |
| 5. | Asako, H., H. Nakajima, K. Kobayashi, M. Kobayashi, and R. Aono. 1997. Organic solvent tolerance and antibiotic resistance increased by overexpression of marA in Escherichia coli. Appl. Environ. Microbiol. 63:1428-1433[Abstract]. |
| 6. | Ausubel, R. M., R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl. 1996. Current protocols in molecular biology, p. 4.4.1-4.4.7. John Wiley & Sons, New York, N.Y. |
| 7. | Breines, D. M., S. Ouabdesselam, E. Y. Ng, J. Tankovic, S. Shah, C. J. Soussy, and D. C. Hooper. 1997. Quinolone resistance locus nfxD of Escherichia coli is a mutant allele of the parE gene encoding a subunit of topoisomerase IV. Antimicrob. Agents Chemother. 41:175-179[Abstract]. |
| 8. | Carlioz, A., and D. Touati. 1986. Isolation of superoxide dismutase mutants in Escherichia coli: is superoxide dismutase necessary for aerobic life? EMBO J. 5:623-630[Medline]. |
| 9. |
Chen, W., and T. Kuo.
1993.
A simple and rapid method for the preparation of gram-negative genomic DNA.
Nucleic Acids Res.
21:2260 |
| 10. |
Cohen, S. P.,
H. Hächler, and S. B. Levy.
1993.
Genetic and functional analysis of the multiple antibiotic resistance (mar) locus in Escherichia coli.
J. Bacteriol.
175:1484-1492 |
| 11. |
Cohen, S. P.,
S. B. Levy,
J. Foulds, and J. L. Rosner.
1993.
Salicylate induction of antibiotic resistance in Escherichia coli: activation of the mar operon and a mar-independent pathway.
J. Bacteriol.
175:7856-7862 |
| 12. |
Cohen, S. P.,
L. M. McMurry,
D. C. Hooper,
J. S. Wolfson, and S. B. Levy.
1989.
Cross-resistance to fluoroquinolones in multiple-antibiotic-resistant (Mar) Escherichia coli selected by tetracycline or chloramphenicol: decreased drug accumulation associated with membrane changes in addition to OmpF reduction.
Antimicrob. Agents Chemother.
33:1318-1325 |
| 13. |
Conrad, S.,
M. Oethinger,
K. Kaifel,
G. Klotz,
R. Marre, and W. V. Kern.
1996.
gyrA mutations in high-level fluoroquinolone-resistant Escherichia coli clinical isolates.
J. Antimicrob. Chemother.
38:443-455 |
| 14. | Conrad, S., L. Scheit, M. Oethinger, G. Klotz, R. Marre, and W. V. Kern. 1996. gyrA and parC mutations in high-level fluoroquinolone-resistant Escherichia coli clinical isolates, abstr. C9, p. 35. In Abstracts of the 36th Interscience Conference of Antimicrobial Agents and Chemotherapy. American Society for Microbiology, Washington, D.C. |
| 15. | Everett, M. J., Y. F. Jin, V. Ricci, and L. V. Piddock. 1996. Contributions of individual mechanisms to fluoroquinolone resistance in 36 Escherichia coli strains isolated from humans and animals. Antimicrob. Agents Chemother. 40:2380-2386[Abstract]. |
| 16. |
George, A. M., and S. B. Levy.
1983.
Amplifiable resistance to tetracycline, chloramphenicol, and other antibiotics in Escherichia coli: involvement of a non-plasmid-determined efflux of tetracycline.
J. Bacteriol.
155:531-540 |
| 17. |
Greenberg, J. T.,
J. H. Chou,
P. A. Monach, and B. Demple.
1991.
Activation of oxidative stress genes by mutations at the soxQ/cfxB/marA locus in Escherichia coli.
J. Bacteriol.
173:4433-4439 |
| 18. |
Greenberg, J. T.,
P. Monach,
J. H. Chou,
P. D. Josephy, and B. Demple.
1990.
Positive control of a global antioxidant defense regulon activated by superoxide-generating agents in Escherichia coli.
Proc. Natl. Acad. Sci. USA
87:6181-6185 |
| 19. | Heisig, P. 1996. Genetic evidence for a role of parC mutations in development of high-level fluoroquinolone resistance in Escherichia coli. Antimicrob. Agents Chemother. 40:879-885[Abstract]. |
| 20. | Heisig, P., and B. Wiedemann. 1997. In vitro activity of the new quinolone Bay 12-8039 against defined mutants of Escherichia coli and Staphylococcus aureus, abstr. F-140, p. 169. In Abstracts of the 37th Interscience Conference on Antimicrobial Agents and Chemotherapy. American Society for Microbiology, Washington, D.C. |
| 21. | Hooper, D. C., J. S. Wolfson, E. Y. Ng, and M. N. Swartz. 1987. Mechanisms of action of and resistance to ciprofloxacin. Am. J. Med. 82:12-20[Medline]. |
| 22. | Hüllen, V., P. Heisig, and B. Wiedemann. 1997. Bedeutung des marR-Genes für die klinische Resistenz von E. coli gegenüber fluorierten Chinolonen, abstr. Sy 9.2. Chemotherapie J. 15:12. |
| 23. | Kato, J.-I., Y. Nishimura, R. Imamura, H. Niki, S. Hiraga, and H. Suzuki. 1990. New topoisomerase essential for chromosome segregation in E. coli. Cell 63:393-404[Medline]. |
| 24. | Ma, D., D. N. Cook, M. Alberti, N. G. Pon, H. Nikaido, and J. E. Hearst. 1995. Genes acrA and acrB encode a stress-induced efflux system of Escherichia coli. Mol. Microbiol. 16:45-55[Medline]. |
| 25. | Maneewannakul, K., and S. B. Levy. 1996. Identification of mar mutants among quinolone-resistant clinical isolates of Escherichia coli. Antimicrob. Agents Chemother. 40:1695-1698[Abstract]. |
| 26. | Miller, P. F., and M. C. Sulavik. 1996. Overlaps and parallels in the regulation of intrinsic multiple-antibiotic resistance in Escherichia coli. Mol. Microbiol. 21:441-448[Medline]. |
| 27. | Nakajima, H., K. Kobayashi, M. Kobayashi, H. Asako, and R. Aono. 1995. Overexpression of the robA gene increases organic solvent tolerance and multiple antibiotic and heavy metal ion resistance in Escherichia coli. Appl. Environ. Microbiol. 61:2302-2307[Abstract]. |
| 28. | Nakajima, H., M. Kobayashi, T. Negishi, and R. Aono. 1995. soxRS gene increased the level of organic solvent tolerance in Escherichia coli. Biosci. Biotechnol. Biochem. 59:1323-1325[Medline]. |
| 29. |
Nakamura, S.,
M. Nakamura,
T. Kojima, and H. Yoshida.
1989.
gyrA and gyrB mutations in quinolone-resistant strains of Escherichia coli.
Antimicrob. Agents Chemother.
33:254-255 |
| 30. | National Committee for Clinical Laboratory Standards. 1994. Performance standards for antimicrobial susceptibility testing; fifth informational supplement. M100-S5. National Committee for Clinical Laboratory Standards, Villanova, Pa. |
| 31. |
Nunoshiba, T.,
E. Hidalgo,
C. F. Amábile-Cuevas, and B. Demple.
1992.
Two-stage control of an oxidative stress regulon: the Escherichia coli SoxR protein triggers redox-inducible expression of the soxS regulatory gene.
J. Bacteriol.
174:6054-6060 |
| 32. | Oethinger, M., S. Conrad, K. Kaifel, A. Cometta, J. Bille, G. Klotz, M. P. Glauser, R. Marre, the International Antimicrobial Therapy Cooperative Group of the European Organization for Research and Treatment of Cancer, and W. V. Kern. 1996. Molecular epidemiology of fluoroquinolone-resistant Escherichia coli bloodstream isolates from patients admitted to European cancer centers. Antimicrob. Agents Chemother. 40:387-392[Abstract]. |
| 33. |
Oethinger, M.,
W. V. Kern,
J. D. Goldman, and S. B. Levy.
1998.
Association of organic solvent tolerance and fluoroquinolone resistance in clinical isolates of Escherichia coli.
J. Antimicrob. Chemother.
41:111-114 |
| 34. | Oethinger, M., W. V. Kern, I. Podglajen, and S. B. Levy. 1997. The multiple antibiotic resistance (mar) locus in fluoroquinolone-resistant blood isolates of E. coli, abstr. A-71, p. 13. In Abstracts of the 97th General Meeting of the American Society for Microbiology 1997. American Society for Microbiology, Washington, D.C. |
| 35. |
Oram, M., and L. M. Fisher.
1991.
4-Quinolone resistance mutations in the DNA gyrase of Escherichia coli clinical isolates identified by using the polymerase chain reaction.
Antimicrob. Agents Chemother.
35:387-389 |
| 36. | Ouabdesselam, S., D. C. Hooper, J. Tankovic, and C. J. Soussy. 1995. Detection of gyrA and gyrB mutations in quinolone-resistant clinical isolates of Escherichia coli by single-strand conformational polymorphism analysis and determination of levels of resistance conferred by two different single gyrA mutations. Antimicrob. Agents Chemother. 39:1667-1670[Abstract]. |
| 37. | Piddock, L. J. V. 1995. Mechanisms of resistance to fluoroquinolones: state-of-the-art 1992-1994. Drugs 49:29-35. |
| 38. | Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. |
| 39. | Schneider, W. C. 1957. Determination of nucleic acids in tissues by pentose analysis. Methods Enzymol. 3:680-684. |
| 40. |
Seoane, A. S., and S. B. Levy.
1995.
Characterization of MarR, the repressor of the multiple antibiotic resistance (mar) operon in Escherichia coli.
J. Bacteriol.
177:3414-3419 |
| 41. |
Tsaneva, I. R., and B. Weiss.
1990.
soxS, a locus governing a superoxide response regulon in Escherichia coli K-12.
J. Bacteriol.
172:4197-4205 |
| 42. | Vila, J., J. Ruiz, A. Marcos, and T. Jimenez de Anta. 1996. Detection of mutations in parC in quinolone-resistant clinical isolates of Escherichia coli. Antimicrob. Agents Chemother. 40:491-493[Abstract]. |
| 43. |
White, D. G.,
J. D. Goldman,
B. Demple, and S. B. Levy.
1997.
Role of the acrAB locus in organic solvent tolerance mediated by expression of marA, soxS, and robA in Escherichia coli.
J. Bacteriol.
179:6122-6126 |
| 44. |
Wu, J., and B. Weiss.
1991.
Two divergently transcribed genes, soxR and soxS, control a superoxide response regulon of Escherichia coli.
J. Bacteriol.
173:2864-2871 |
| 45. |
Yoshida, H.,
M. Bogaki,
M. Nakamura,
L. Yamanaka, and S. Nakamura.
1991.
Quinolone resistance-determining region in the DNA gyrase gyrB gene of Escherichia coli.
Antimicrob. Agents Chemother.
35:1647-1650 |
Antimicrobial Agents and Chemotherapy, August 1998, p. 2089-2094, Vol. 42, No. 8
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