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Previous Article | Next Article 
Antimicrobial Agents and Chemotherapy, May 2001, p. 1467-1472, Vol. 45, No. 5
Section of Infectious Diseases and Clinical
Immunology, Department of Medicine, University Hospital and Medical
Center, D-89070 Ulm, Germany
Received 16 August 2000/Returned for modification 18 November
2000/Accepted 23 February 2001
The development of fluoroquinolone resistance in Escherichia
coli may be associated with mutations in regulatory gene loci such as marRAB that lead to increased multidrug efflux,
presumably through activation of expression of the AcrAB multidrug
efflux pump. We found that multidrug-resistant (MDR) phenotypes with enhanced efflux can also be selected by fluoroquinolones from marRAB- or acrAB-inactivated E. coli K-12 strains having a single mutation in the
quinolone-resistance-determining region of gyrA. Mutant
3-AG100MKX, obtained from a mar knockout strain after
two selection steps, showed enhanced expression of acrB in
a reverse transcriptase PCR associated with insertion of
IS186 into the AcrAB repressor gene acrR. In
vitro selection experiments with acrAB knockout strains
yielded MDR mutants after a single step. Enhanced efflux in these
mutants was due to increased expression of acrEF and
associated with insertion of IS2 into the upstream region of
acrEF, presumably creating a hybrid promoter. These observations confirm the importance of efflux-associated nontarget gene
mutations and indicate that transposition of genetic elements may have
a role in the development of fluoroquinolone resistance in E. coli.
High-level fluoroquinolone
resistance in Escherichia coli is associated with mutations
in the genes coding for the target proteins DNA gyrase
(gyrA) and topoisomerase IV (parC) (3, 33,
39). Mutations in regulatory loci such as mar (stands for multiple antibiotic resistance) and sox (stands for
superoxide stress response) may play an important role during
resistance development (19, 25, 36). Such mutations lead
to overexpression of MarA, a transcriptional activator negatively
regulated by MarR (9, 28), or of SoxS, the activator of
the superoxide SoxRS regulon (1, 28), and thereby confer
increased resistance to chemically unrelated antibiotics by activating
or depressing a number of genetic loci in E. coli that
contribute in a synergistic way to the multiple-antibiotic resistance
or multidrug resistance (MDR) phenotype. After fluoroquinolone exposure
in vitro, E. coli mutants selected from DNA gyrase single
mutants typically exhibit a MDR phenotype with enhanced multidrug
efflux but without additional target gene mutations (19).
Previous studies have shown that MarA decreases expression of the OmpF
porin and influences the expression of the E. coli multidrug
efflux pump AcrAB (29, 32). AcrAB is a multicomponent
efflux pump of the resistance-nodulation-division family that functions
in association with the outer membrane protein TolC (12).
Knockout experiments have shown that the AcrAB pump is critical for the
resistance of E. coli to bile salts (40) and a
number of antibiotics and that mar mutations are effective only in the presence of this pump (32). Upon deletion or
inactivation of AcrAB, E. coli cells without topoisomerase
mutations become hypersusceptible and resistance to fluoroquinolones
can be reversed in topoisomerase mutants (31). We wondered
whether efflux-associated mutations would be selectable in E. coli strains in which regulatory genes or the acrAB
locus was inactivated. In initial experiments with a mar
knockout parental strain, MDR phenotypes could be selected (19). The present report extends these observations,
summarizes results obtained with acrAB knockout strains, and
examines the mechanisms behind the enhanced active efflux measurable in
the mutants.
Bacterial strains.
E. coli K-12 strain AG100 has
been previously described (13). The mar
deletion mutant AG100MK was constructed by P1 transduction from
AG100/Kan as the donor strain into AG100. AG100/Kan was constructed by
replacement of a chromosomal 1.24-kb BspHI fragment of the mar locus in AG100 by homologous recombination with the
kanamycin resistance (Kan) cassette from pKMN33 (25). The
mutant strain 1-AG100MKX was selected from strain AG100MK with 0.25 µg of ofloxacin per ml. This strain acquired a point mutation in the
gyrA gene at codon 83 (serine
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.5.1467-1472.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Enhanced Expression of the Multidrug Efflux Pumps AcrAB and
AcrEF Associated with Insertion Element Transposition in
Escherichia coli Mutants Selected with a
Fluoroquinolone
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
leucine), leading to an
eightfold increase in the MICs of ofloxacin and ciprofloxacin
(19).
acrAB::Tn903Kanr
sequence from strain JZM120 (27) into the gyrA
single mutant 1-AG100. The AG100 acrAB knockout mutant
1-DC14PS was a gyrA single mutant obtained in a single step
(selecting concentration, 0.03 µg of ofloxacin per ml) from DC14,
which, in turn, had been constructed by inactivating acrAB
with a Kanr cassette by homologous recombination. Plasmid
pCAB30 used for this replacement is a derivative of pCVD442
(10), which contains part of the acrAB gene
cluster from AG100 and a Kanr cassette inserted in
acrAB, which caused the deletion of part of acrA
and acrB.
Chemicals and media. Ofloxacin was obtained from Hoechst, Frankfurt, Germany. Sodium salicylate and carbonyl cyanide m-chlorophenylhydrazone (CCCP) were purchased from Sigma Chemicals, St. Louis, Mo. Mueller-Hinton broth and agar were from Oxoid, Basingstoke, England. Luria-Bertani (LB) broth and agar were prepared by standard protocols.
Selection of mutants. Strains were grown overnight in LB medium. Inocula of ~1010 CFU were plated on LB agar containing inhibitory concentrations (2 to 16 times the MIC) of ofloxacin. Plates were incubated for 24 to 48 h at 37°C. Three to six single colonies from each selecting concentration, if available, were purified on ofloxacin-supplemented plates and examined for antimicrobial susceptibility.
Susceptibility testing. MICs of selected antimicrobial agents were determined by E-test (Viva Diagnostika, Hürth, Germany) or by a standard microdilution procedure with an inoculum of 5 × 105 CFU/ml according to NCCLS performance and inoculum guidelines (29) using commercially available microtiter plates from Merlin Diagnostics GmbH, Bornheim, Germany.
Fluoroquinolone uptake. Cells were grown to logarithmic phase in LB broth at 37°C, washed twice in 50 mM sodium phosphate buffer (pH 7.0), and resuspended in the same buffer containing 0.2% glucose. After an incubation of 10 min at 37°C, ofloxacin was added to a final concentration of 10 µg/ml. At timed intervals, 1-ml samples were removed and centrifuged immediately through silicone oil, and the pellet was resuspended in 0.1 M glycine hydrochloride (pH 3.0). After overnight incubation at room temperature, samples were centrifuged and the amount of released ofloxacin was determined spectrofluorometrically (excitation wavelength, 292 nm; emission wavelength, 496 nm) (8). The intracellular drug accumulation by the mutant strain was expressed as a percentage of the uptake measured in the corresponding parental strain. Experiments were done in triplicate and included repeated measurements after CCCP (200 µM) was added as an inhibitor of the proton-motive force.
DNA sequencing. The quinolone-resistance-determining regions (QRDRs) of gyrA (nucleotides 123 to 366), parC (nucleotides 145 to 492), and marOR were amplified by PCR and purified by use of Quiaquick spin columns (Qiagen, Hilden, Germany) as previously described (30). Direct cycle sequencing was performed in an automatic model 373 A DNA sequencer (Applied Biosystems).
RNA extraction and Northern blot analysis. Overnight cultures were diluted 1:100 in LB broth and grown to the mid-logarithmic phase at 37°C with shaking. Cultures were split, and sodium salicylate (5 mM final concentration) was added to one-half of the cultures for induction. RNA was isolated using an RNeasy Kit (Qiagen) according to the manufacterer's instructions. Northern blot analysis was performed by using 1.5% agarose-formaldehyde gels as described previously (2). As probes, a 387-bp marA fragment and a 432-bp soxRS fragment were used. Hybridization was performed with an ECL detection kit (Amersham Pharmacia Biotech, Freiburg, Germany).
Reverse transcription of total RNA and PCR of cDNA. Two hundred nanograms of total RNA (20 ng/µl) was mixed with 1 µl of pd(N)6 random hexamers (100 ng/µl; Pharmacia) and incubated for 5 min at 65°C. The RNA was then immediately cooled on ice. Nine microliters of a reverse transcription reaction mixture containing 5× Superscript buffer, 2 µl of 10 mM dithiothreitol, 1 µl of deoxynucleoside triphosphates (10 mM), 10 U of RNasin (Promega), and 50 U of Supercript II reverse transcriptase (RT) (Gibco BRL) was added to the RNA-hexamer mix, and the solution was incubated at 42°C for 30 min.
Two microliters of the cDNA was used for the amplification of the specific cDNA species in a standard PCR. Expression of gapA (coding for D-glyceraldehyde-3-phosphate-dehydrogenase) (6) in the same cDNA preparation was used as a standard. The PCR was performed in a thermocycler (Biometra) using the following program: once for 5 min at 94°C; 28 cycles of 1 min at 94°C, 1 min at 51.5 to 55°C (depending on the primers used), and 1 min at 72°C; and finally once for 5 min at 72°C. The resulting PCR products were detected on a 1.5% agarose gel containing ethidium bromide. The bands were analyzed densitrometrically (Image Master 1D; Pharmacia), and results were normalized for the gapA band density.| |
RESULTS AND DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
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Selection of MDR phenotypes from mar and
acrAB knockout strains.
Mutants 2-AG100MKX (selecting
concentration, 1 µg of ofloxacin per ml) and 3-AG100MKX (selecting
concentration, 4 µg of ofloxacin per ml) were selected in a stepwise
manner from the mar knockout strain 1-AG100MKX (single
gyrA mutation). For both mutants increased MICs of the
fluoroquinolones ofloxacin, ciprofloxacin (Table
1), and moxifloxacin (data not shown)
were seen, but no additional mutations in the QRDRs of the target genes
gyrA and parC were detected.
|
10 and ~5 × 1010, respectively. All individual colonies of both
2-AG100AKX and 2-DC14PS had similar phenotypes, and for these strains
increased MICs of fluoroquinolones and of chemically unrelated agents
such as tetracycline, chloramphenicol, and others were seen (Table 1),
results resembling the phenotype seen with mutants overexpressing the
mar or sox locus. However, both 2-AG100AKX and
2-DC14PS had neither additional target gene mutations nor mutations in
mar or sox that would lead to an overexpression
of these loci (Table 1).
Mutant 3-AG100AKX was obtained from 2-AG100AKX in an additional
selection step (ofloxacin concentration, 1 µg/ml; frequency, ~3 × 109). For mutant 3-AG100AKX, we found an
increase in the MIC of chloramphenicol (from 8 to 32 µg/ml) and very
modest increases in the MICs of the other agents, including the
fluoroquinolones, but again, no additional mutations in the QRDRs of
target genes or in the regulatory loci analyzed were detected (Table
1). The failure of selecting a mutation leading to constitutive
expression of marA in acrAB knockout strains
supports previous results which showed that the effects of MarA
overexpression are mediated mainly by AcrAB (32).
Measurement of intracellular ofloxacin concentration with and without
CCCP as an inhibitor of the proton-motive force confirmed that enhanced
drug efflux was the mechanism of the MDR of mutants 3-AG100MKX and
2-AG100AKX. 3-AG100MKX showed a clear reduction of the intracellular
ofloxacin concentration (59% of that of the parental strain),
suggesting that AcrAB or another efflux pump was activated (Fig.
1). A decreased intracellular ofloxacin
concentration was also clearly documented for 2-AG100AKX (42% of that
of the parental strain) (Fig. 1). The enhanced efflux in this mutant in
association with the acquired MDR phenotype was interesting. Since the
mutant had a deletion in the gene coding for AcrAB, activation of an
alternative efflux mechanism without involvement of the mar
and sox regulatory loci was the likely resistance mechanism in this mutant.
|
Induction of the efflux pump genes acrAB or acrEF. Previous work has documented the importance of AcrAB for active efflux of fluoroquinolones in E. coli (32). We therefore initially examined the level of expression of acrAB in mutant strain 3-AG100MKX. RT-PCR analysis of acrB indicated that the AcrAB message relative to those seen in 1-AG100MKX and in 2-AG100MKX was slightly increased by about 1.5-fold (estimated from densitometrical analysis of RT-PCR bands [Fig. 1]). In view of the known role of AcrAB in solvent tolerance (41), this result was consistent with the observation that 3-AG100MKX showed increased tolerance to organic solvents compared with that of 2-AG100MKX (19).
We studied the expression of other different efflux pump systems (emrAB, mdfA, acrD, and acrEF) known to transport quinolones or showing homology to acrAB (11, 22-24). No induction of either of these genes was detected in RT-PCR experiments (data not shown). Also, the expression of tolC, which codes for the outer membrane component of the AcrAB pump complex (12), was unaffected, as was the expression of tonB, a gene coding for the TonB protein, which probably functions as an energy coupler (7) between inner and outer membranes and which was shown to influence the activity of the AcrAB homologous MexAB-OprM multidrug efflux pump in Pseudomonas aeruginosa (44). We concluded from these findings that mar- and sox-independent transcriptional activation of AcrAB most likely was responsible for the enhanced efflux and the MDR phenotype seen in mutant 3-AG100MKX. The same experimental approach was used for study of the acrAB deletion mutants 2-AG100AKX and 3-AG100AKX. Expression of emrAB, mdfA, and acrD as well as of tolC and tonB was unchanged in these mutants (data not shown), but RT-PCR analysis showed that the expression of acrE was strongly induced (Fig. 1). The same result was obtained in RT-PCR experiments with 2-DC14PS, which strongly overexpressed acrE. E. coli AcrEF, which is homologous to AcrAB, is thought to be involved in indole efflux (18), and upregulation of AcrEF might compensate an AcrAB deletion (J. Xu, M. L. Nilles, and K. P. Bertrand, Abstr. 93rd Gen. Meet. Am. Soc. Microbiol. 1993, abstr. K-169, 1993). Only limited data have been reported on the substrate specificity of AcrEF (18, 23). Based on changes in the MICs for 2-AG100AKX and 2-DC14PS relative to those for their precursors, we can confirm that AcrEF has a broad substrate specificity similar to that of AcrAB and includes specificities not only for ofloxacin, ciprofloxacin, tetracyclines, and chloramphenicol but also for oxacillin, cefuroxime, trimethoprim, macrolides or azalides, clindamycin, and linezolid (Table 1), as well as quinupristin-dalfopristin (MIC change,
2-fold), mupirocine (MIC
change, 8-fold), and the newer fluoroquinolones moxifloxacin (MIC
change, 8-fold) and clinafloxacin (MIC change, 8-fold) (data not shown).
Compared with MICs for 2-AG100AKX, the MICs of tetracyclines,
chloramphenicol, trimethoprim, members of the
macrolide-azalide-streptogramin group, and linezolid for mutant
3-AG100AKX showed further increases and the MICs of fluoroquinolones
and cefuroxime showed very modest increases. No significant change in
ofloxacin uptake (Fig. 1) was measurable in this mutant, indicating
enhanced activity of a pump with limited fluoroquinolone affinity or
the presence of an additional, so far unknown, resistance-enhancing mechanism.
Despite several attempts, we were unable to construct a
acrAB-acrEF double knockout mutant from
2-AG100AKX to study in more detail the effect of pump inactivation on
the resistance level. Similar observations were described by
Kawamura-Sato et al. (18), suggesting that one of the two
homologous pumps needs to be present in the cell. This hypothesis,
however, has been challenged, given the recent description of the
successful construction of a viable acrAB-acrEF double knockout mutant (D. Cho,
D. Lofland, J. Blais, K. Tangen, D. Cotter, O. Lomovskaya, S. Chamberland, and M. N. Dudley, Abstr. 40th Intersci. Conf.
Antimicrob. Agents Chemother., abstr. 1497, 2000).
Transposition of insertion sequence (IS) elements is responsible for induction of efflux pumps in the mutant strains. In the absence of mar and sox mutations, loss of transcriptional control of acrAB and acrEF was possibly due to alterations in the local repressor genes acrR and acrS, respectively, or to altered promoter regions in the corresponding operons. acrR mutations leading to enhanced MDR in a mar mutant strain have been described by Okusu et al. (32). Similar loss-of-function mutations in efflux pump repressor genes were previously described for P. aeruginosa and Neisseria gonorrhoeae (16, 38, 43, 45).
To further characterize the mechanism of efflux pump activation in 3-AG100MKX, the promoter-repressor gene region of acrAB was amplified. The PCR product obtained from the mar knockout mutant 3-AG100MKX (sense primer, 5'-CGACGCAGTGAACCAGAA-3'; antisense primer, 5'-CCTGAGAGCATCAGAACG-3') covering acrR and the acrAB promoter region was about 1.3 kb larger than the PCR product obtained from the parental strain. Nucleotide sequencing of the PCR product revealed that this enlargement was due to the integration of the IS element IS186 (Fig. 2). This integration at nucleotide 93 of the acrR gene is likely to cause inactivation of AcrR, which in turn leads to the increased expression of acrAB seen in the RT-PCR experiments (Fig. 1).
|
Concluding remarks. Previous in vitro selection experiments with E. coli indicated that mutations in regulatory genes follow an initial gyrA mutation and precede additional topoisomerase gene mutations (19). This finding is supported by the observations made in this study. Of note, mutations associated with enhanced efflux and selectable by fluoroquinolone exposure are not limited to the mar or sox regulon. Thus, there may be options for E. coli to acquire more than one mutation leading to enhanced fluoroquinolone and multidrug efflux. We postulate such double efflux-associated mutations for both 3-AG100MKX and 3-AG100AKX as well as for some of the mutants reported earlier which had an initial mar mutation (leading to reduced drug uptake) and showed further reduction in intracellular drug uptake after a subsequent selection step (19). In view of the large number of proven or putative efflux pump genes on the E. coli chromosome, including acrAB homologous genes (37), and their complex regulation, involvement of more than one efflux pump system seems possible, but activation of a given efflux pump by more than one mechanism is another explanation. According to a recent study of selected fluoroquinolone-resistant clinical strains of E. coli with MDR phenotypes, some had mutations in both the mar and sox regulatory genes as well as in acrR (M. A. Webber and L. J. V. Piddock, Abstr. 40th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 759, 2000).
Gene activation and inactivation by transposition of IS elements is a well-known phenomenon. In some cases, the development of resistance has been causally linked to IS transposition (5, 17, 21, 34, 36, 42). Insertional inactivation of acrR in antibiotic-resistant mutants selected by fluoroquinolones has not previously been reported, but examples of IS element transposition and associated changes in permeability exist. They include E. coli K-12 mar mutants in which IS2 integrated into marOR, resulting in MarA overexpression (14, 26). Also, porin-deficient, cefoxitin-resistant clinical isolates of Klebsiella pneumoniae, for example, with IS elements in the ompK36 gene have been reported (15). For the multidrug transporter MdfA, it was shown that the integration of an IS element creates a putative promoter that activates the expression of the corresponding gene (4), similar to the acrEF expression observed here after IS2 transposition in mutants 2-AG100AKX and 2-DC14PS. More detailed analysis of several fluoroquinolone-resistant in vitro mutants selected in this laboratory indicates that IS element transposition is more prevalent than expected. However, functional consequences for the resistance levels of the mutants are not always obvious. Also, the specific role of fluoroquinolone exposure in such changes and the prevalence and significance of such changes under in vivo conditions remain to be determined.| |
ACKNOWLEDGMENTS |
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This study was supported by Deutsche Forschungsgemeinschaft grant Ke700/1-1 and research grants P449/97 and P559/99 from the University of Ulm.
We thank Petra Steinke for excellent technical assistance.
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FOOTNOTES |
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* Corresponding author. Mailing address: Medizinische Universitätsklinik und Poliklinik, D-89070 Ulm, Germany. Phone: 49-731-502 4423. Fax: 49-731-502 4488. E-mail: winfried.kern{at}medizin.uni-ulm.de.
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REFERENCES |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
|
|---|
| 1. |
Amabile-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 |
| 2. | Ausubel, F. M., R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl. 1987. Current protocols in molecular biology, vol. 4. John Wiley and Sons, Inc., New York, N.Y. |
| 3. |
Bagel, S.,
V. Hüllen,
B. Wiedemann, and P. Heisig.
1999.
Impact of gyrA and parC mutations on quinolone resistance, doubling time, and supercoiling degree of Escherichia coli.
Antimicrob. Agents Chemother.
43:868-875 |
| 4. |
Bohn, C., and P. Bouloc.
1998.
The Escherichia coli cmlA gene encodes the multidrug efflux pump Cmr/MdfA and is responsible for isopropyl- -D-thiogalactopyranoside exclusion and spectinomycin sensitivity.
J. Bacteriol.
180:6072-6075 |
| 5. |
Bolognese, F.,
C. Di-Lecce,
E. Galli, and P. Barbieri.
1999.
Activation and inactivation of Pseudomonas stutzeri methylbenzene catabolism pathways mediated by transposable element.
Appl. Environ. Microbiol.
65:1876-1882 |
| 6. | Branlant, G., and C. Branlant. 1985. Nucleotide sequence of the Escherichia coli gap gene. Different evolutionary behaviour of the NAD+-binding domain and of the catalytic domain of D-glyceraldehyde-3-phosphate dehydrogenase. Eur. J. Biochem. 150:61-66[Medline]. |
| 7. | Braun, V. 1995. Energy-coupled transport and signal transduction through the gram-negative outer membrane via TonB-ExbB-ExbD-dependent receptor proteins. FEMS Microbiol. Rev. 16:295-307[CrossRef][Medline]. |
| 8. |
Chapman, J. S., and N. H. Georgopapadakou.
1989.
Fluorometric assay for fleroxacin uptake by bacterial cells.
Antimicrob. Agents Chemother.
33:27-29 |
| 9. |
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 |
| 10. |
Donnenberg, M. S., and J. B. Kaper.
1991.
Construction of an eae deletion mutant of enteropthogenic Escherichia coli by using a positive-selection suicide vector.
Infect. Immun.
59:4310-4317 |
| 11. |
Edgar, R., and E. Bibi.
1997.
MdfA, an Escherichia coli multidrug resistance protein with an extraordinarily broad spectrum of drug recognition.
J. Bacteriol.
179:2274-2280 |
| 12. |
Fralick, J. A.
1996.
Evidence that TolC is required for functioning of the Mar/AcrAB efflux pump of Escherichia coli.
J. Bacteriol.
178:5803-5805 |
| 13. |
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 |
| 14. |
Hächler, H.,
S. P. Cohen, and S. B. Levy.
1991.
marA, a regulated locus which controls expression of chromosomal multiple antibiotic resistance in Escherichia coli.
J. Bacteriol.
173:5532-5538 |
| 15. |
Hernandez-Alles, S.,
V. J. Benedi,
L. Martinez-Martinez,
A. Pascual,
A. Aguilar,
J. M. Tomas, and S. Alberti.
1999.
Development of resistance during antimicrobial therapy caused by insertion sequence interruption of porin genes.
Antimicrob. Agents Chemother.
43:937-939 |
| 16. |
Jalal, S.,
O. Ciofu,
N. Hoiby,
N. Gotoh, and B. Wretlind.
2000.
Molecular mechanism of fluoroquinolone resistance in Pseudomonas aeruginosa isolates from cystic fibrosis patients.
Antimicrob. Agents Chemother.
44:710-712 |
| 17. | Jaurin, B., and S. Normark. 1983. Insertion of IS2 creates a novel ampC promoter in Escherichia coli. Cell 32:809-816[CrossRef][Medline]. |
| 18. | Kawamura-Sato, K., K. Shibayama, T. Horii, Y. Iimuma, Y. Arakawa, and M. Ohta. 1999. Role of multiple efflux pumps in Escherichia coli in indole expulsion. FEMS Microbiol. Lett. 179:345-352[CrossRef][Medline]. |
| 19. |
Kern, W. V.,
M. Oethinger,
A. S. Jellen-Ritter, and S. B. Levy.
2000.
Non-target gene mutations in the development of fluoroquinolone resistance in Escherichia coli.
Antimicrob. Agents Chemother.
44:814-820 |
| 20. | Klein, J. R., B. Henrich, and R. Plapp. 1990. Molecular cloning of the envC gene of Escherichia coli. Curr. Microbiol. 21:341-347. |
| 21. |
Lemaitre, N.,
W. Sougakoff,
C. Truffot-Pernot, and V. Jarlier.
1999.
Characterization of new mutations in pyrazinamide-resistant strains of Mycobacterium tuberculosis and identification of conserved regions important for the catalytic activity of the pyrazinamidase PncA.
Antimicrob. Agents Chemother.
43:1761-1763 |
| 22. |
Lomovskaya, O., and K. Lewis.
1992.
emr, an Escherichia coli locus for multidrug resistance.
Proc. Natl. Acad. Sci. USA
89:8938-8942 |
| 23. |
Ma, D.,
D. N. Cook,
M. Alberti,
N. G. Pon,
H. Nikaido, and J. E. Hearst.
1993.
Molecular cloning and characterization of acrA and acrE genes of Escherichia coli.
J. Bacteriol.
175:6299-6313 |
| 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[CrossRef][Medline]. |
| 25. | Mannewannakul, 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. |
Martin, R. G.,
P. S. Nyantakyi, and J. L. Rosner.
1995.
Regulation of the multiple antibiotic resistance (mar) regulon by marORA sequences in Escherichia coli.
J. Bacteriol.
177:4176-4178 |
| 27. | McMurry, L. M., M. Oethinger, and S. B. Levy. 1998. Overexpression of marA, soxS, or acrAB produces resistance to triclosan in laboratory and clinical strains of Escherichia coli. FEMS Microbiol. Lett. 166:305-309[CrossRef][Medline]. |
| 28. | Miller, P., and M. C. Sulavik. 1996. Overlaps and parallels in the regulation of intrinsic multiple-antibiotic resistance in Escherichia coli. Mol. Microbiol. 21:441-448[CrossRef][Medline]. |
| 29. | National Committee for Clinical Laboratory Standards. 1997. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, 4th ed. Approved standard M7-A4. National Committee for Clinical Laboratory Standards, Wayne, Pa. |
| 30. |
Oethinger, M.,
I. Podglajen,
W. V. Kern, and S. B. Levy.
1998.
Overexpression of the marA or soxS regulatory gene in clinical topoisomerase mutants of Escherichia coli.
Antimicrob. Agents Chemother.
42:2089-2094 |
| 31. |
Oethinger, M.,
W. V. Kern,
A. S. Jellen-Ritter,
L. M. McMurry, and S. B. Levy.
2000.
Ineffectiveness of topoisomerase mutations in mediating clinically significant fluoroquinolone resistance in Escherichia coli in the absence of the AcrAB efflux pump.
Antimicrob. Agents Chemother.
44:10-13 |
| 32. |
Okusu, H.,
D. Ma, and H. Nikaido.
1996.
AcrAB efflux pump plays a major role in the antibiotic resistance phenotype of Escherichia coli multiple-antibiotic-resistance (Mar) mutants.
J. Bacteriol.
178:306-308 |
| 33. | Piddock, L. J. V. 1999. Mechanisms of fluoroquinolone resistance: an update 1994-1998. Drugs 58(Suppl. 2):11-18. |
| 34. | Podglajen, I., J. Breuil, and E. Collatz. 1994. Insertion of a novel DNA sequence, IS1186, upstream of the silent carbapenemase gene cfiA, promotes expression of carbapenem resistance in clinical isolates of Bacteroides fragilis. Mol. Microbiol. 12:105-114[Medline]. |
| 35. | Provence, D. L., and R. Curtiss, III. 1994. Gene transfer in gram-negative bacteria, p. 317-347. In P. Gerhardt, R. G. E. Murray, W. A. Wood, and N. R. Krieg (ed.), Methods for general and molecular bacteriology. American Society for Microbiology, Washington, D.C. |
| 36. |
Rudant, E.,
P. Courvalin, and T. Lambert.
1998.
Characterization of IS18, an element capable of activating the silent aac(6')-Ij gene of Acinetobacter sp. 13 strain BM2716 by transposition.
Antimicrob. Agents Chemother.
42:2759-2761 |
| 37. |
Saier, M. H., Jr.,
I. T. Paulsen,
M. K. Sliwinski,
S. S. Pao,
R. A. Skurray, and H. Nikaido.
1998.
Evolutionary origins of multidrug and drug-specific efflux pumps in bacteria.
FASEB J.
12:265-274 |
| 38. | Shafer, W. M., J. T. Balthazar, K. E. Hagman, and S. A. Morse. 1995. Missense mutations that alter the DNA-binding domain of the MtrR protein occur frequently in rectal isolates of Neisseria gonorrhoeae that are resistant to faecal lipids. Microbiology 141:907-911[Abstract]. |
| 39. |
Tavio, M.,
J. Vila,
J. Ruiz,
A. M. Martin-Sanchez, and M. T. de Anta.
1999.
Mechanisms involved in the development of resistance to fluoroquinolones in Escherichia coli isolates.
J. Antimicrob. Chemother.
44:735-742 |
| 40. |
Thanassi, D. G.,
L. W. Cheng, and H. Nikaido.
1997.
Active efflux of bile salts by Escherichia coli.
J. Bacteriol.
179:2512-2518 |
| 41. |
White, D. G.,
B. Demple, and S. B. Levy.
1997.
Role of the acrAB locus in organic solvent tolerance mediated by the expression of marA, soxS, or robA in Escherichia coli.
J. Bacteriol.
179:6122-6126 |
| 42. |
Whiteway, J.,
P. Koziarz,
J. Veall,
N. Sandhu,
P. Kumar,
B. Hoecher, and I. B. Lambert.
1998.
Oxygen-insensitive nitroreductases: analysis of the roles of nfsA and nfsB in the development of resistance to 5-nitrofuran derivatives in Escherichia coli.
J. Bacteriol.
180:5529-5539 |
| 43. |
Zarantonelli, L.,
G. Borthagaray,
E.-H. Lee, and W. M. Shafer.
1999.
Decreased azithromycin susceptibility of Neisseria gonorrhoeae due to mtrR mutations.
Antimicrob. Agents Chemother.
43:2468-2472 |
| 44. |
Zhao, Q.,
X. Li,
A. Mistry,
R. Srikumar,
L. Zhang,
O. Lomovskaya, and K. Poole.
1998.
Influence of the TonB energy-coupling protein on efflux-mediated multidrug resistance in Pseudomonas aeruginosa.
Antimicrob. Agents Chemother.
42:2225-2231 |
| 45. |
Ziha-Zarifi, I.,
C. Llanes,
T. Köhler,
J.-C. Pechere, and P. Plesiat.
1999.
In vivo emergence of multidrug-resistant mutants of Pseudomonas aeruginosa overexpressing the active efflux system MexA-MexB-OprM.
Antimicrob. Agents Chemother.
43:287-291 |
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