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Antimicrobial Agents and Chemotherapy, December 2000, p. 3322-3327, Vol. 44, No. 12
Biological Research Laboratories, Sankyo Co.,
Ltd., Shinagawa-ku, Tokyo 140-8710,1 and
Department of Microbiology, Kyoto Pharmaceutical University,
Yamashina, Kyoto 607-8414,2 Japan
Received 28 March 2000/Returned for modification 27 June
2000/Accepted 11 September 2000
To find the exact substrate specificities of three species of
tripartite efflux systems of Pseudomonas aeruginosa,
MexAB-OprM, MexCD-OprJ, and MexXY-OprM, we constructed a series of
isogenic mutants, each of which constitutively overproduced one of the three efflux systems and lacked the other two, and their isogenic mutants, which lacked all these systems. Comparison of the
susceptibilities of the constructed mutants to 52 antimicrobial agents
belonging to various groups suggested the following substrate
specificities. All of the efflux systems extrude a wide variety of
antimicrobial agent groups, i.e., quinolones, macrolides,
tetracyclines, lincomycin, chloramphenicol, most penicillins (all but
carbenicillin and sulbenicillin), most cephems (all but cefsulodin and
ceftazidime), meropenem, and S-4661, but none of them extrude polymyxin
B or imipenem. Extrusion of aminoglycosides is specific to MexXY-OprM,
and extrusion of a group of the Several tripartite efflux systems
coded on the chromosome play important roles in the intrinsic and
acquired resistance in Pseudomonas aeruginosa. Each system
consists of a cytoplasmic membrane component of the
resistance-nodulation-division family presumed to function as a
transporter, an outer membrane component presumed to form channels, and
a membrane fusion protein presumed to link the two membrane proteins
for reviews, see references (13, 14, and 15). The
MexA-MexB-OprM efflux system (7, 16) contributes to both
intrinsic and acquired resistance in P. aeruginosa, while
the MexC-MexD-OprJ (17) and MexE-MexF-OprN (6)
efflux systems contribute only to the acquired resistance in this
bacterium. Studies with mutants that overproduce or lack MexAB-OprM
demonstrated that this efflux system extrudes quinolones, macrolides,
tetracycline, chloramphenicol, novobiocin, and most Simultaneous expression of plural efflux systems can cause
misunderstanding of the substrate specificity of each multidrug efflux
system. In this study, to find the exact substrate specificities of the
MexAB-OprM, MexCD-OprJ, and MexXY-OprM efflux systems, we constructed
and compared the susceptibilities of a series of isogenic mutants, each
of which constitutively overproduced one of the three efflux systems
and lacked the other two, and their isogenic mutants, which lacked all
these systems.
Bacterial strains, plasmids, and media.
The bacterial
strains and plasmids used in this study are listed in Table
1. Bacterial cells were grown on
Mueller-Hinton II agar (MHA; Becton Dickinson Microbiology Systems,
Cockeysville, Md.) or in Mueller-Hinton broth (MHB; Becton Dickinson
Microbiology Systems) at 37°C. Minimal agar medium (2) was
used for selection of P. aeruginosa. The following
antibiotics were added to the media at the indicated concentrations:
carbenicillin, 100 µg/ml for Escherichia coli and 200 µg/ml for P. aeruginosa; streptomycin, 30 µg/ml for
E. coli and 100 µg/ml for P. aeruginosa; and
chloramphenicol, 30 µg/ml for E. coli and
MexAB-OprM-deficient P. aeruginosa and 300 µg/ml for
MexAB-OprM-producing P. aeruginosa. MHA was supplemented with 10% (wt/vol) sucrose, as required.
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Substrate Specificities of MexAB-OprM, MexCD-OprJ,
and MexXY-OprM Efflux Pumps in Pseudomonas
aeruginosa
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
-lactams, i.e., carbenicillin,
sulbenicillin, ceftazidime, moxalactam, and aztreonam, is specific to
MexAB-OprM. Moreover, MexAB-OprM and MexCD-OprJ extrude novobiocin,
cefsulodin, and flomoxef, while MexXY-OprM does not. These substrate
specificities are distinct from those reported previously.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
-lactams but not
imipenem (7, 8, 22). Studies with mutants overproducing
MexCD-OprJ demonstrated that this efflux system extrudes quinolones,
erythromycin, tetracycline, chloramphenicol, and expanded-spectrum
cephems such as cefpirome (9, 10). Furthermore,
characterization of mutants lacking the mexA-mexB-oprM region demonstrated that the MexCD-OprJ efflux system extrudes ceftazidime and cefoperazone as well as cefpirome, while it does not
extrude carbenicillin and aztreonam (3). Recently, Aires et
al. (1) and our group (12) showed that MexX-MexY
extrudes aminoglycosides, tetracycline, and erythromycin in cooperation with spontaneously expressed OprM, thereby contributing to the intrinsic resistance to these agents in P. aeruginosa. Our
group also showed that no expression of MexXY is observed in wild-type strains but that the expression can be induced by subinhibitory concentrations of its substrates such as tetracycline and gentamicin (12). However, the precise substrate specificity of the
MexXY-OprM efflux system is unclear.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
TABLE 1.
Bacterial strains and plasmids used in this study
Susceptibility testing. MICs were determined by the usual twofold agar dilution technique with MHA with an inoculum size of 104 cells. S-4661 (5) was synthesized at Sankyo Co., Ltd., Tokyo, Japan. Ciprofloxacin, tosufloxacin, and sparfloxacin were synthesized at Ube Industries, Ltd., Tokyo, Japan. The other antimicrobial agents used in this study were obtained from commercial sources.
Molecular biology techniques. Chromosomal DNA and plasmids were isolated with a DNeasy tissue kit and a QIAfilter plasmid kit (Qiagen K.K., Tokyo, Japan), respectively. PCRs were performed with a Perkin-Elmer 480 thermal cycler with PfuTurb DNA polymerase (Stratagene, La Jolla, Calif.). Restriction endonucleases, alkaline phosphatase, and the DNA-ligation kit were obtained from Takara Shuzo Co., Ltd., Kyoto, Japan. Restriction fragments were isolated, as required, from agarose gels with TaKaRa RECOCHIP (Takara, Shuzo Co., Ltd.). All molecular biology techniques were carried out according to the manufacturer's instructions or as described by Sambrook et al. (19). Transformation of E. coli with plasmid DNA was performed as described previously (3).
Deletion of mexAB by gene replacement. To construct isogenic mutants lacking the mexA-mexB region, PCR primers for amplification of the mexA-mexB region and its flanking regions were synthesized on the basis of the nucleotide sequences of the Pseudomonas genome sequencing project database (http://www.pseudomonas.com/). After amplification of a 0.9-kb region downstream of mexB on PAO1 chromosomal DNA as a template with AB3 (5'-TTTCTCGAGCTGGCGATCTTCTGGGTACC-3') and AB4 (5'-TTTAAGCTTACTTCGGTCAGCAGGGTCTG-3'), a primer pair containing a newly added cutting site (underlined) for restriction nucleases, the region was ligated into the XhoI-HindIII site in a multicloning site of pMT5059 (23) to yield pNS004. Next, a 1.0-kb region upstream of the mexA gene amplified by PCR with the primer pair AB1 (5'-TTTGAATTCGGTGATCAGTGCCTTGTCGC-3') and AB2 (5'-TTACTAGTCGACAGCACCTTGGTGTAGC-3') was ligated into the EcoRI-SpeI site of pNS004 to yield pNS005. (A 12-bp sequence derived from a multicloning site of pMT5059 was still left between the two DNA fragments inserted on pNS005.) After addition of a NotI-flanked Mob cassette from pMT5071 (24), the resulting plasmid, pNS006, was mobilized from the E. coli strain S17-1 (21), to the P. aeruginosa strains to introduce deletion of the mexA-mexB region into the recipient chromosomes by allelic exchange, as described previously (12). Deletion of mexAB was confirmed by PCR with the primer pair AB5 (5'-CTCATGAGGACAACGCTATGCAACGAACG-3') and AB6 (5'-TGGGTCAGGTCGAAACTCTTCTGGTAGGTG-3'). The sizes of the amplified DNA fragments obtained with these primers were 4.9 kb for the wild-type strain and 1.2 kb for the mexAB-deficient strains (data not shown).
Insertional deletion of ampC by gene replacement. Plasmid pKMB004 residing in S17-1 was conjugationally mobilized to P. aeruginosa cells. After mating on MHA at 37°C overnight, the cell mixture was suspended in saline. Aliquots of the suspensions were spread on minimal agar plates supplemented with streptomycin, and the plates were incubated at 30°C for 2 days. The transconjugants were plated onto MHA supplemented with 10% sucrose and streptomycin. Clones showing hypersusceptibilities to amoxicillin were used in subsequent experiments.
Production of polyclonal antisera specific to MexA, MexC, and MexX. To obtain antibodies specific to MexA, MexC, and MexX, the oligopeptides (C)YQIDPATYEADYQSA (amino acids 92 to 106 of MexA), (C)AQARVRRYEPLVKIQ (amino acids 120 to 134 of MexC), and (C)EDSPTPLTRVEQID (amino acids 197 to 210 of MexX) were synthesized and conjugated to keyhole limpet hemocyanin by Chiron Technologies Pty., Ltd. (Clayton, Victoria, Australia). A cysteine residue was added to each N terminus for conjugation. Rabbit antiserum raised against each antigen was prepared by Takara Shuzo Co., Ltd.
Isolation of total and outer membranes, SDS-PAGE, and immunoblot assay. Exponentially growing cells in MHB were harvested as described previously (9). MHB was supplemented with tetracycline, as required. Total membranes (3) and outer membranes (9) were prepared as described previously. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrophoretic transfer were performed as described previously (10). Production levels of MexA, MexC, and MexX were tested by immunoblot assay with rabbit polyclonal antisera specific for MexA, MexC, and MexX, respectively (see above), and production levels of OprM and OprJ were tested with murine monoclonal antibodies specific for OprM (TM001 [3]) and OprJ (HJ001 [4]), respectively. Binding of the primary antibodies was detected as described previously (3), with alkaline phosphatase-conjugated goat antibodies to rabbit or mouse immunoglobulins (Biosource International, Camarillo, Calif.) used as the secondary antibodies and an Alkaline Phosphatase Conjugate Substrate kit (Bio-Rad Laboratories, Hercules, Calif.) used for color development.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Construction of mutants overproducing one of three efflux
systems.
To confirm the precise substrate specificity of the
MexXY-OprM efflux system, we constructed a mutant that overproduced
MexXY-OprM. mexXY and oprM are located at
different places on the chromosome, and their expressions are regulated
independently. Our first step in constructing the
MexXY-OprM-overproducing mutant was to perform an in-frame deletion of
mexAB from MexAB-OprM-overproducing strain OCR1. To avoid
affecting the expression of OprM in the strain obtained (designated
strain N126), we conserved the point mutation in mexR in
OCR1 (18), together with the presumed second promoter responsible for OprM expression identified upstream of oprM
(27). The amount of OprM produced in N126 was lower than
that produced in OCR1 but was almost the same as that produced in PAO1
(Fig. 1, lanes 1 and 5). The reduction in
the level of OprM expression in N126 might be a polar effect caused by
deletion of mexAB. Next, we isolated a MexXY-overproducing
mutant, N127, from N126 by selection on a plate containing 4 µg of
gentamicin and 0.5 µg of ofloxacin per ml (Fig. 1, lane 6).
Furthermore, we reexamined the substrate specificities of MexAB-OprM
and MexCD-OprJ to compare them with the substrate specificity of
MexXY-OprM. Chimeric MexAB-OprJ and MexCD-OprM pumps can function
in the efflux of antimicrobial agents (4, 22, 26). Given the
presence of the mexX-mexY operon in the mutants constructed
previously, the functioning of these chimeric pumps might suggest that
MexXY influences the previously observed specificities of MexAB-OprM
and MexCD-OprJ. For instance, MexXY may be associated with OprJ and may
affect the susceptibility even in KG2259, a mutant that produces large
amounts of MexCD-OprJ and lacks mexAB-oprM. Therefore, we
isolated mutants lacking the mexXY region from OCR1 and
KG2259 using pNS003, as described previously (12), and
designated these mutants N107 and N108, respectively. The
overproduction or lack of each efflux pump component in N107, N108, and
N127 was confirmed by immunoblot assay (Fig. 1). We have reported that
no production of MexX is observed in wild-type strains but that the
MexX production is induced by antimicrobial agents such as tetracycline
(12). Thus, loss of MexX in N107 and N108 was confirmed by
culture with subinhibitory concentrations of tetracycline. Moreover, we
deleted each overexpressed efflux system from N107, N108, and N127 by
gene replacement and designated them N148, N150, and N128,
respectively. Insertional deletion of mexD was performed
with pKMJ075, as described previously (4). Deletions of
mexAB and mexXY were performed as described
above. Disruption of each gene was confirmed by PCR (data not shown).
|
Substrate specificities of three efflux systems to non--lactam
agents.
Table 2 presents the MICs of
all of the antimicrobial agents tested except the -lactams for N107,
N108, N127, and the respective efflux system-deficient mutants, N148,
N150, and N128. N127 showed lower levels of susceptibility to
quinolones, macrolides, tetracyclines, lincomycin, chloramphenicol, and
aminoglycosides than N128, suggesting that MexXY-OprM extrudes these
agents. In contrast, N107 and N108 showed lower levels of
susceptibility to quinolones, macrolides, tetracyclines, lincomycin,
chloramphenicol, and novobiocin than N148 and N150, respectively,
suggesting that MexAB-OprM and MexCD-OprJ extrude these agents.
|
Substrate specificities of three efflux systems to
-lactams.
The presence of chromosomal AmpC -lactamase makes
it quite difficult to interpret data on MICs because some -lactams
are removed by both the -lactamase and the efflux system(s)
(11). Thus, we introduced ampC::
into the
chromosomes of N107, N108, and N127 by gene replacement and designated
them N116, N119, and N133, respectively. Deletion of AmpC was confirmed
by spectrophotometric assay, as described previously (8).
Moreover, we deleted each overexpressed efflux system from N116, N119,
and N133 by gene replacement as described above and designated them
N151, N153, and N154, respectively. Table
3 presents the MICs of various -lactams for N116, N119, N133, and the respective efflux
system-deficient mutants, N151, N153, and N154. First, N133 was less
susceptible than N154 to meropenem, S-4661, all of the penicillins
except carbenicillin and sulbenicillin, and all of the cephems except cefsulodin and ceftazidime, suggesting that MexXY-OprM also extrudes these -lactams. Next, N119 was less susceptible than N153 to flomoxef, meropenem, S-4661, all of the penicillins except
carbenicillin and sulbenicillin, and all of the cephems except
ceftazidime, suggesting that MexCD-OprJ extrudes not only cephems
(except for ceftazidime) but also flomoxef, meropenem, S-4661, and all
of the penicillins except carbenicillin and sulbenicillin. Finally, N116 was less susceptible than N151 to all of the -lactams tested except imipenem, suggesting that MexAB-OprM extrudes all of the -lactams except imipenem.
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DISCUSSION |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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In this study, we found that MexXY-OprM also extrudes several
-lactams. However, deletion of MexXY from PAO1 produced no significant change in susceptibility to any of the four substrates of
MexXY-OprM, cefuroxime, cefpirome, cefepime, and cefozopran (unpublished data). In addition, exposure to cefpirome did not induce
MexX in PAO1 (unpublished data). These results suggest that MexXY-OprM
does not contribute to the intrinsic resistance to -lactams, despite
the potency of MexXY-OprM in the extrusion of several -lactams. This
is reminiscent of our previous result (12), which showed
that while ofloxacin is a substrate of MexXY-OprM, it induces no
production of MexXY in the wild-type strain, thereby confirming that
MexXY-OprM does not contribute to the intrinsic resistance to
ofloxacin. Furthermore, cefpirome induced production of MexX in a
mutant lacking both MexAB and AmpC (unpublished data), just as
ofloxacin was shown to induce production of MexX in the N126 lacking
MexAB in our earlier study (12). Thus, MexAB-OprM and AmpC
are primary systems for removal of cefpirome in the wild-type strain,
whereas MexXY-OprM is a compensatory system for removal of cefpirome in
the mutant lacking the primary systems.
In the present study, we elucidate the exact substrate specificities of
MexAB-OprM, MexCD-OprJ, and MexXY-OprM (listed in Table
4). Although most antimicrobial agents
are substrates of all efflux systems, the three systems have slight but
significant differences in substrate specificities.
MexAB-OprM-overproducing mutants such as OCR1 show no significant
change in susceptibility to gentamicin (8, 9), whereas
MexAB-OprM-deficient mutants show lower levels of susceptibility to
gentamicin than the wild-type strains (20, 25). This
discrepancy is expected since MexXY-OprM contributes to the resistance
to gentamicin, while MexAB-OprM does not. MexAB-OprM extrudes the
broadest kinds of -lactams. MexCD-OprJ and MexXY-OprM have substrate
specificities similar to those of -lactams, the exception being that
the former extrudes cefsulodin and flomoxef, while the latter does not.
MexCD-OprJ and MexXY-OprM extrude most penicillins tested but not
carbenicillin and sulbenicillin. Carbenicillin and sulbenicillin
possess a negatively charged substitution at position C-6 that the
other penicillins tested lack. This may explain the differences in the
substrate specificities to penicillins.
|
N150 was more susceptible to macrolides, tetracyclines, some of the
quinolones, and lincomycin than N148 and N128, although all three
strains lack the three efflux systems (Table 2). N153 was also more
susceptible to cloxacillin, nafcillin, cefamandole, cefuroxime,
cefpirome, cefoselis, and cefoxitin than N151 and N154 (Table 3). N103
(12), a strain which lacks MexAB-OprM and MexXY, showed
lower levels of susceptibility than N150 and higher levels of
susceptibility than N148 and N128 (data not shown). N106, an
ampC:: strain of N103, also showed a lower level of
susceptibility than N153 and a higher level of susceptibility than N151
and N154 (data not shown). The reason for the discrepancy in
susceptibilities is unclear, but there are several possibilities. The
first is that the OprM overexpressed in N148, N128, N151, and N154 is
associated with unknown periplasmic and inner membrane components and
contributes to the resistance to these agents. A second possibility is
that the mutation of nfxB suppresses an efflux system(s)
other than MexAB-OprM and MexXY-OprM. A third possibility, that OprJ
enhances the permeability of the P. aeruginosa outer
membrane to these agents as a porin, cannot be excluded immediately,
but we can rule it out by the finding that the susceptibilities of N103
and N106 to the agents were not affected by plasmid-bone OprJ
expression (data not shown).
Bacterial genome sequencing projects have elucidated that each bacterium has several transporter genes. Multiple-knockout experiments such as those performed in this study might be necessary to elucidate the actual substrate specificities of other efflux pumps.
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ACKNOWLEDGMENTS |
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This research was partially supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan and by a grant from the Ministry of Health and Welfare of Japan.
We are grateful to K. Okamoto for providing pKMB004.
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FOOTNOTES |
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* Corresponding author. Present address: Department of Genetics, Warren Alpert Building, Room 513, Harvard Medical School, 200 Longwood Ave., Boston, MA 02115. Phone: (617) 432-7561. Fax: (617) 432-7266. E-mail: nmasud{at}shina.sankyo.co.jp.
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REFERENCES |
|---|
|
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
| 1. |
Aires, J. R.,
T. Köhler,
H. Nikaido, and P. Plésiat.
1999.
Involvement of an active efflux system in the natural resistance of Pseudomonas aeruginosa to aminoglycosides.
Antimicrob. Agents Chemother.
43:2624-2628 |
| 2. |
Fukuoka, T.,
N. Masuda,
T. Takenouchi,
N. Sekine,
M. Iijima, and S. Ohya.
1991.
Increase in susceptibility of Pseudomonas aeruginosa to carbapenem antibiotics in low-amino-acid media.
Antimicrob. Agents Chemother.
35:529-532 |
| 3. |
Gotoh, N.,
H. Tsujimoto,
M. Tsuda,
K. Okamoto,
A. Nomura,
T. Wada,
M. Nakahashi, and T. Nishino.
1998.
Characterization of the MexC-MexD-OprJ multidrug efflux system in  mexA-mexB-oprM mutants of Pseudomonas aeruginosa.
Antimicrob. Agents Chemother.
42:1938-1943 |
| 4. | Gotoh, N., H. Tsujimoto, A. Nomura, K. Okamoto, M. Tsuda, and T. Nishino. 1998. Functional replacement of OprJ by OprM in the MexCD-OprJ multidrug efflux system of Pseudomonas aeruginosa. FEMS Microbiol. Lett. 165:21-27[Medline]. |
| 5. | Iso, Y., T. Irie, Y. Nishino, K. Motokawa, and Y. Nishitani. 1996. A novel 1 beta-methylcarbapenem antibiotic, S-4661. Synthesis and structure-activity relationships of 2-(5-substituted pyrrolidin-3-ylthio)-1-beta-methylcarbapenems. J. Antibiot. (Tokyo) 49:199-209[Medline]. |
| 6. | Köhler, T., M. Michéa-Hamzepour, U. Henze, N. Gotoh, L. K. Curty, and J.-C. Pechère. 1997. Characterization of MexE-MexF-OprN, a positively regulated multidrug efflux system of Pseudomonas aeruginosa. Mol. Microbiol. 23:345-354[CrossRef][Medline]. |
| 7. | Li, X.-Z., H. Nikaido, and K. Poole. 1995. Role of MexA-MexB-OprM in antibiotic efflux in Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 39:1948-1953[Abstract]. |
| 8. |
Masuda, N., and S. Ohya.
1992.
Cross-resistance to meropenem, cephems, and quinolones in Pseudomonas aeruginosa.
Antimicrob. Agents Chemother.
36:1847-1851 |
| 9. | Masuda, N., E. Sakagawa, and S. Ohya. 1995. Outer membrane proteins responsible for multiple drug resistance in Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 39:645-649[Abstract]. |
| 10. | Masuda, N., N. Gotoh, S. Ohya, and T. Nishino. 1996. Quantitative correlation between susceptibility and OprJ production in NfxB mutants of Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 40:909-913[Abstract]. |
| 11. |
Masuda, N.,
N. Gotoh,
C. Ishii,
E. Sakagawa,
S. Ohya, and T. Nishino.
1999.
Interplay between chromosomal -lactamase and the MexAB-OprM efflux system in intrinsic resistance to -lactams in Pseudomonas aeruginosa.
Antimicrob. Agents Chemother.
43:400-402 |
| 12. |
Masuda, N.,
E. Sakagawa,
S. Ohya,
N. Gotoh,
H. Tsujimoto, and T. Nishino.
2000.
Contribution of the MexX-MexY-OprM system to intrinsic resistance in Pseudomonas aeruginosa.
Antimicrob. Agents Chemother.
44:2242-2246 |
| 13. |
Nikaido, H.
1996.
Multidrug efflux pumps of gram-negative bacteria.
J. Bacteriol.
178:5853-5859 |
| 14. | Nikaido, H. 1998. Antibiotic resistance caused by gram-negative multidrug efflux pumps. Clin. Infect. Dis. 27:S32-S41. |
| 15. | Nikaido, H. 1998. Multiple antibiotic resistance and efflux. Curr. Opin. Microbiol. 1:516-523[CrossRef][Medline]. |
| 16. |
Poole, K.,
K. Krebes,
C. McNally, and S. Neshat.
1993.
Multiple antibiotic resistance in Pseudomonas aeruginosa: evidence for involvement of an efflux operon.
J. Bacteriol.
175:7363-7372 |
| 17. | Poole, K., N. Gotoh, H. Tsujimoto, Q. Zhao, A. Wada, T. Yamasaki, S. Neshat, J. Yamagishi, X.-Z. Li, and T. Nishino. 1996. Overexpression of the mexC-mexD-oprJ efflux operon in nfxB-type multidrug-resistant strains of Pseudomonas aeruginosa. Mol. Microbiol. 21:713-724[CrossRef][Medline]. |
| 18. | Poole, K., K. Tetro, Q. Zhao, S. Neshat, D. Heinrichs, and N. Bianco. 1996. Expression of the multidrug resistance operon mexA-mexB-oprM in Pseudomonas aeruginosa: mexR encodes a regulator of operon expression. Antimicrob. Agents Chemother. 40:2021-2028[Abstract]. |
| 19. | 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. |
| 20. |
Schweizer, H. P.
1998.
Intrinsic resistance to inhibitors of fatty acid biosynthesis in Pseudomonas aeruginosa is due to efflux: application of a novel technique for generation of unmarked chromosomal mutations for the study of efflux systems.
Antimicrob. Agents Chemother.
42:394-398 |
| 21. | Simon, R., U. Priefer, and A. Pühler. 1983. A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in gram-negative bacteria. Bio/Technology 1:784-790[CrossRef]. |
| 22. |
Srikumar, R.,
X.-Z. Li, and K. Poole.
1997.
Inner membrane efflux components are responsible for -lactam specificity of multidrug efflux pumps in Pseudomonas aeruginosa.
J. Bacteriol.
179:7875-7881 |
| 23. |
Tsuda, M.,
H. Miyazaki, and T. Nakazawa.
1995.
Genetic and physical mapping of genes involved in pyoverdine production in Pseudomonas aeruginosa PAO.
J. Bacteriol.
177:423-431 |
| 24. | Tsuda, M. 1998. Use of a transposon-encoded site-specific resolution system for construction of large and defined deletion mutations in bacterial chromosome. Gene 207:33-41[CrossRef][Medline]. |
| 25. | Yoneyama, H., A. Ocaktan, M. Tsuda, and T. Nakae. 1997. The role of the mex-gene products in antibiotic extrusion in Pseudomonas aeruginosa. Biochem. Biophys. Res. Commun. 233:611-618[CrossRef][Medline]. |
| 26. | Yoneyama, H., A. Ocaktan, N. Gotoh, T. Nishino, and T. Nakae. 1998. Subunit swapping in the mex-extrusion pumps in Pseudomonas aeruginosa. Biochem. Biophys. Res. Commun. 244:898-902[CrossRef][Medline]. |
| 27. |
Zhao, Q.,
X.-Z. Li,
R. Srikumar, and K. Poole.
1998.
Contribution of outer membrane efflux protein OprM to antibiotic resistance in Pseudomonas aeruginosa independent of MexAB.
Antimicrob. Agents Chemother.
42:1682-1688 |
Antimicrobial Agents and Chemotherapy, December 2000, p. 3322-3327, Vol. 44, No. 12
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Copyright © 2000, American Society for Microbiology. All rights reserved.
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155: 3312-3321
[Abstract] [Full Text] -
Strateva, T., Yordanov, D.
(2009). Pseudomonas aeruginosa - a phenomenon of bacterial resistance. J Med Microbiol
58: 1133-1148
[Abstract] [Full Text] -
Pena, C., Suarez, C., Tubau, F., Juan, C., Moya, B., Dominguez, M. A., Oliver, A., Pujol, M., Ariza, J.
(2009). Nosocomial Outbreak of a Non-Cefepime-Susceptible Ceftazidime-Susceptible Pseudomonas aeruginosa Strain Overexpressing MexXY-OprM and Producing an Integron-Borne PSE-1 ss-Lactamase. J. Clin. Microbiol.
47: 2381-2387
[Abstract] [Full Text] -
Baum, E. Z., Crespo-Carbone, S. M., Morrow, B. J., Davies, T. A., Foleno, B. D., He, W., Queenan, A. M., Bush, K.
(2009). Effect of MexXY Overexpression on Ceftobiprole Susceptibility in Pseudomonas aeruginosa. Antimicrob. Agents Chemother.
53: 2785-2790
[Abstract] [Full Text] -
Vettoretti, L., Plesiat, P., Muller, C., El Garch, F., Phan, G., Attree, I., Ducruix, A., Llanes, C.
(2009). Efflux Unbalance in Pseudomonas aeruginosa Isolates from Cystic Fibrosis Patients. Antimicrob. Agents Chemother.
53: 1987-1997
[Abstract] [Full Text] -
Bulitta, J. B., Ly, N. S., Yang, J. C., Forrest, A., Jusko, W. J., Tsuji, B. T.
(2009). Development and Qualification of a Pharmacodynamic Model for the Pronounced Inoculum Effect of Ceftazidime against Pseudomonas aeruginosa. Antimicrob. Agents Chemother.
53: 46-56
[Abstract] [Full Text] -
Jeannot, K., Elsen, S., Kohler, T., Attree, I., van Delden, C., Plesiat, P.
(2008). Resistance and Virulence of Pseudomonas aeruginosa Clinical Strains Overproducing the MexCD-OprJ Efflux Pump. Antimicrob. Agents Chemother.
52: 2455-2462
[Abstract] [Full Text] -
Zhang, L., Mah, T.-F.
(2008). Involvement of a Novel Efflux System in Biofilm-Specific Resistance to Antibiotics. J. Bacteriol.
190: 4447-4452
[Abstract] [Full Text] -
Gutierrez, O., Juan, C., Cercenado, E., Navarro, F., Bouza, E., Coll, P., Perez, J. L., Oliver, A.
(2007). Molecular Epidemiology and Mechanisms of Carbapenem Resistance in Pseudomonas aeruginosa Isolates from Spanish Hospitals. Antimicrob. Agents Chemother.
51: 4329-4335
[Abstract] [Full Text] -
Henrichfreise, B., Wiegand, I., Pfister, W., Wiedemann, B.
(2007). Resistance Mechanisms of Multiresistant Pseudomonas aeruginosa Strains from Germany and Correlation with Hypermutation. Antimicrob. Agents Chemother.
51: 4062-4070
[Abstract] [Full Text] -
Junker, L. M., Clardy, J.
(2007). High-Throughput Screens for Small-Molecule Inhibitors of Pseudomonas aeruginosa Biofilm Development. Antimicrob. Agents Chemother.
51: 3582-3590
[Abstract] [Full Text] -
Henrichfreise, B., Wiegand, I., Luhmer-Becker, I., Wiedemann, B.
(2007). Development of Resistance in Wild-Type and Hypermutable Pseudomonas aeruginosa Strains Exposed to Clinical Pharmacokinetic Profiles of Meropenem and Ceftazidime Simulated In Vitro. Antimicrob. Agents Chemother.
51: 3642-3649
[Abstract] [Full Text] -
Walther-Rasmussen, J., Hoiby, N.
(2007). Class A carbapenemases. J Antimicrob Chemother
60: 470-482
[Abstract] [Full Text] -
Wolter, D. J., Schmidtke, A. J., Hanson, N. D., Lister, P. D.
(2007). Increased Expression of ampC in Pseudomonas aeruginosa Mutants Selected with Ciprofloxacin. Antimicrob. Agents Chemother.
51: 2997-3000
[Abstract] [Full Text] -
Plasencia, V., Borrell, N., Macia, M. D., Moya, B., Perez, J. L., Oliver, A.
(2007). Influence of High Mutation Rates on the Mechanisms and Dynamics of In Vitro and In Vivo Resistance Development to Single or Combined Antipseudomonal Agents. Antimicrob. Agents Chemother.
51: 2574-2581
[Abstract] [Full Text] -
Reinhardt, A., Kohler, T., Wood, P., Rohner, P., Dumas, J.-L., Ricou, B., van Delden, C.
(2007). Development and Persistence of Antimicrobial Resistance in Pseudomonas aeruginosa: a Longitudinal Observation in Mechanically Ventilated Patients. Antimicrob. Agents Chemother.
51: 1341-1350
[Abstract] [Full Text] -
El'Garch, F., Jeannot, K., Hocquet, D., Llanes-Barakat, C., Plesiat, P.
(2007). Cumulative Effects of Several Nonenzymatic Mechanisms on the Resistance of Pseudomonas aeruginosa to Aminoglycosides. Antimicrob. Agents Chemother.
51: 1016-1021
[Abstract] [Full Text] -
Marr, A. K., Overhage, J., Bains, M., Hancock, R. E. W.
(2007). The Lon protease of Pseudomonas aeruginosa is induced by aminoglycosides and is involved in biofilm formation and motility. Microbiology
153: 474-482
[Abstract] [Full Text] -
Rodloff, A. C., Goldstein, E. J. C., Torres, A.
(2006). Two decades of imipenem therapy. J Antimicrob Chemother
58: 916-929
[Abstract] [Full Text] -
Quale, J., Bratu, S., Gupta, J., Landman, D.
(2006). Interplay of Efflux System, ampC, and oprD Expression in Carbapenem Resistance of Pseudomonas aeruginosa Clinical Isolates.. Antimicrob. Agents Chemother.
50: 1633-1641
[Abstract] [Full Text] -
Lister, P. D., Wolter, D. J., Wickman, P. A., Reisbig, M. D.
(2006). Levofloxacin/imipenem prevents the emergence of high-level resistance among Pseudomonas aeruginosa strains already lacking susceptibility to one or both drugs. J Antimicrob Chemother
57: 999-1003
[Abstract] [Full Text] -
Piddock, L. J. V.
(2006). Clinically Relevant Chromosomally Encoded Multidrug Resistance Efflux Pumps in Bacteria. Clin. Microbiol. Rev.
19: 382-402
[Abstract] [Full Text] -
Hocquet, D., Nordmann, P., El Garch, F., Cabanne, L., Plesiat, P.
(2006). Involvement of the MexXY-OprM Efflux System in Emergence of Cefepime Resistance in Clinical Strains of Pseudomonas aeruginosa.. Antimicrob. Agents Chemother.
50: 1347-1351
[Abstract] [Full Text] -
Morita, Y., Sobel, M. L., Poole, K.
(2006). Antibiotic Inducibility of the MexXY Multidrug Efflux System of Pseudomonas aeruginosa: Involvement of the Antibiotic-Inducible PA5471 Gene Product.. J. Bacteriol.
188: 1847-1855
[Abstract] [Full Text] -
Tam, V. H., Schilling, A. N., Neshat, S., Poole, K., Melnick, D. A., Coyle, E. A.
(2005). Optimization of Meropenem Minimum Concentration/MIC Ratio To Suppress In Vitro Resistance of Pseudomonas aeruginosa. Antimicrob. Agents Chemother.
49: 4920-4927
[Abstract] [Full Text] -
Ambrose, K. D., Nisbet, R., Stephens, D. S.
(2005). Macrolide Efflux in Streptococcus pneumoniae Is Mediated by a Dual Efflux Pump (mel and mef) and Is Erythromycin Inducible. Antimicrob. Agents Chemother.
49: 4203-4209
[Abstract] [Full Text] -
Pournaras, S., Maniati, M., Spanakis, N., Ikonomidis, A., Tassios, P. T., Tsakris, A., Legakis, N. J., Maniatis, A. N.
(2005). Spread of efflux pump-overexpressing, non-metallo-{beta}-lactamase-producing, meropenem-resistant but ceftazidime-susceptible Pseudomonas aeruginosa in a region with blaVIM endemicity. J Antimicrob Chemother
56: 761-764
[Abstract] [Full Text] -
Gillis, R. J., White, K. G., Choi, K.-H., Wagner, V. E., Schweizer, H. P., Iglewski, B. H.
(2005). Molecular Basis of Azithromycin-Resistant Pseudomonas aeruginosa Biofilms. Antimicrob. Agents Chemother.
49: 3858-3867
[Abstract] [Full Text] -
Jeannot, K., Sobel, M. L., El Garch, F., Poole, K., Plesiat, P.
(2005). Induction of the MexXY Efflux Pump in Pseudomonas aeruginosa Is Dependent on Drug-Ribosome Interaction. J. Bacteriol.
187: 5341-5346
[Abstract] [Full Text] -
Salunkhe, P., Smart, C. H. M., Morgan, J. A. W., Panagea, S., Walshaw, M. J., Hart, C. A., Geffers, R., Tummler, B., Winstanley, C.
(2005). A Cystic Fibrosis Epidemic Strain of Pseudomonas aeruginosa Displays Enhanced Virulence and Antimicrobial Resistance. J. Bacteriol.
187: 4908-4920
[Abstract] [Full Text] -
Ueda, O., Wexler, H. M., Hirai, K., Shibata, Y., Yoshimura, F., Fujimura, S.
(2005). Sixteen Homologs of the Mex-Type Multidrug Resistance Efflux Pump in Bacteroides fragilis. Antimicrob. Agents Chemother.
49: 2807-2815
[Abstract] [Full Text] -
Poole, K.
(2005). Efflux-mediated antimicrobial resistance. J Antimicrob Chemother
56: 20-51
[Abstract] [Full Text] -
Poole, K.
(2005). Aminoglycoside Resistance in Pseudomonas aeruginosa. Antimicrob. Agents Chemother.
49: 479-487
[Full Text] -
Kriengkauykiat, J., Porter, E., Lomovskaya, O., Wong-Beringer, A.
(2005). Use of an Efflux Pump Inhibitor To Determine the Prevalence of Efflux Pump-Mediated Fluoroquinolone Resistance and Multidrug Resistance in Pseudomonas aeruginosa. Antimicrob. Agents Chemother.
49: 565-570
[Abstract] [Full Text] -
Oliver, A., Levin, B. R., Juan, C., Baquero, F., Blazquez, J.
(2004). Hypermutation and the Preexistence of Antibiotic-Resistant Pseudomonas aeruginosa Mutants: Implications for Susceptibility Testing and Treatment of Chronic Infections. Antimicrob. Agents Chemother.
48: 4226-4233
[Abstract] [Full Text] -
Kolayli, F., Karadenizli, A., Savli, H., Ergen, K., Hatirnaz, O., Balikci, E., Budak, F., Vahaboglu, H.
(2004). Effect of carbapenems on the transcriptional expression of the oprD, oprM and oprN genes in Pseudomonas aeruginosa. J Med Microbiol
53: 915-920
[Abstract] [Full Text] -
Mushtaq, S., Ge, Y., Livermore, D. M.
(2004). Doripenem versus Pseudomonas aeruginosa In Vitro: Activity against Characterized Isolates, Mutants, and Transconjugants and Resistance Selection Potential. Antimicrob. Agents Chemother.
48: 3086-3092
[Abstract] [Full Text] -
Jones, R. N., Huynh, H. K., Biedenbach, D. J.
(2004). Activities of Doripenem (S-4661) against Drug-Resistant Clinical Pathogens. Antimicrob. Agents Chemother.
48: 3136-3140
[Abstract] [Full Text] -
Nehme, D., Li, X.-Z., Elliot, R., Poole, K.
(2004). Assembly of the MexAB-OprM Multidrug Efflux System of Pseudomonas aeruginosa: Identification and Characterization of Mutations in mexA Compromising MexA Multimerization and Interaction with MexB. J. Bacteriol.
186: 2973-2983
[Abstract] [Full Text] -
Llanes, C., Hocquet, D., Vogne, C., Benali-Baitich, D., Neuwirth, C., Plesiat, P.
(2004). Clinical Strains of Pseudomonas aeruginosa Overproducing MexAB-OprM and MexXY Efflux Pumps Simultaneously. Antimicrob. Agents Chemother.
48: 1797-1802
[Abstract] [Full Text] -
Chan, Y. Y., Tan, T. M. C., Ong, Y. M., Chua, K. L.
(2004). BpeAB-OprB, a Multidrug Efflux Pump in Burkholderia pseudomallei. Antimicrob. Agents Chemother.
48: 1128-1135
[Abstract] [Full Text] -
Mushtaq, S., Ge, Y., Livermore, D. M.
(2004). Comparative Activities of Doripenem versus Isolates, Mutants, and Transconjugants of Enterobacteriaceae and Acinetobacter spp. with Characterized {beta}-Lactamases. Antimicrob. Agents Chemother.
48: 1313-1319
[Abstract] [Full Text] -
Ramos-Aires, J., Plesiat, P., Kocjancic-Curty, L., Kohler, T.
(2004). Selection of an Antibiotic-Hypersusceptible Mutant of Pseudomonas aeruginosa: Identification of the GlmR Transcriptional Regulator. Antimicrob. Agents Chemother.
48: 843-851
[Abstract] [Full Text] -
Middlemiss, J. K., Poole, K.
(2004). Differential Impact of MexB Mutations on Substrate Selectivity of the MexAB-OprM Multidrug Efflux Pump of Pseudomonas aeruginosa. J. Bacteriol.
186: 1258-1269
[Abstract] [Full Text] -
He, G.-X., Kuroda, T., Mima, T., Morita, Y., Mizushima, T., Tsuchiya, T.
(2004). An H+-Coupled Multidrug Efflux Pump, PmpM, a Member of the MATE Family of Transporters, from Pseudomonas aeruginosa. J. Bacteriol.
186: 262-265
[Abstract] [Full Text] -
Hearn, E. M., Dennis, J. J., Gray, M. R., Foght, J. M.
(2003). Identification and Characterization of the emhABC Efflux System for Polycyclic Aromatic Hydrocarbons in Pseudomonas fluorescens cLP6a. J. Bacteriol.
185: 6233-6240
[Abstract] [Full Text] -
Sobel, M. L., McKay, G. A., Poole, K.
(2003). Contribution of the MexXY Multidrug Transporter to Aminoglycoside Resistance in Pseudomonas aeruginosa Clinical Isolates. Antimicrob. Agents Chemother.
47: 3202-3207
[Abstract] [Full Text] -
Yomoda, S., Okubo, T., Takahashi, A., Murakami, M., Iyobe, S.
(2003). Presence of Pseudomonas putida Strains Harboring Plasmids Bearing the Metallo-{beta}-Lactamase Gene blaIMP in a Hospital in Japan. J. Clin. Microbiol.
41: 4246-4251
[Abstract] [Full Text] -
Hocquet, D., Bertrand, X., Kohler, T., Talon, D., Plesiat, P.
(2003). Genetic and Phenotypic Variations of a Resistant Pseudomonas aeruginosa Epidemic Clone. Antimicrob. Agents Chemother.
47: 1887-1894
[Abstract] [Full Text] -
Magnet, S., Smith, T.-A., Zheng, R., Nordmann, P., Blanchard, J. S.
(2003). Aminoglycoside Resistance Resulting from Tight Drug Binding to an Altered Aminoglycoside Acetyltransferase. Antimicrob. Agents Chemother.
47: 1577-1583
[Abstract] [Full Text] -
Van Bambeke, F., Glupczynski, Y., Plesiat, P., Pechere, J. C., Tulkens, P. M.
(2003). Antibiotic efflux pumps in prokaryotic cells: occurrence, impact on resistance and strategies for the future of antimicrobial therapy. J Antimicrob Chemother
51: 1055-1065
[Full Text] -
Savli, H., Karadenizli, A., Kolayli, F., Gundes, S., Ozbek, U., Vahaboglu, H.
(2003). Expression stability of six housekeeping genes: a proposal for resistance gene quantification studies of Pseudomonas aeruginosa by real-time quantitative RT-PCR. J Med Microbiol
52: 403-408
[Abstract] [Full Text] -
Hocquet, D., Vogne, C., El Garch, F., Vejux, A., Gotoh, N., Lee, A., Lomovskaya, O., Plesiat, P.
(2003). MexXY-OprM Efflux Pump Is Necessary for Adaptive Resistance of Pseudomonas aeruginosa to Aminoglycosides. Antimicrob. Agents Chemother.
47: 1371-1375
[Abstract] [Full Text] -
Jo, J. T. H., Brinkman, F. S. L., Hancock, R. E. W.
(2003). Aminoglycoside Efflux in Pseudomonas aeruginosa: Involvement of Novel Outer Membrane Proteins. Antimicrob. Agents Chemother.
47: 1101-1111
[Abstract] [Full Text] -
Eda, S., Maseda, H., Nakae, T.
(2003). An Elegant Means of Self-protection in Gram-negative Bacteria by Recognizing and Extruding Xenobiotics from the Periplasmic Space. J. Biol. Chem.
278: 2085-2088
[Abstract] [Full Text] -
Wang, Y., Ha, U., Zeng, L., Jin, S.
(2003). Regulation of Membrane Permeability by a Two-Component Regulatory System in Pseudomonas aeruginosa. Antimicrob. Agents Chemother.
47: 95-101
[Abstract] [Full Text] -
Cavallo, J. D., Plesiat, P., Couetdic, G., Leblanc, F., Fabre, R., Groupe d'Etude de la Resistance de Pseudomonas aer,
(2002). Mechanisms of {beta}-lactam resistance in Pseudomonas aeruginosa: prevalence of OprM-overproducing strains in a French multicentre study (1997). J Antimicrob Chemother
50: 1039-1043
[Abstract] [Full Text] -
Okamoto, K., Gotoh, N., Nishino, T.
(2002). Extrusion of Penem Antibiotics by Multicomponent Efflux Systems MexAB-OprM, MexCD-OprJ, and MexXY-OprM of Pseudomonas aeruginosa. Antimicrob. Agents Chemother.
46: 2696-2699
[Abstract] [Full Text] -
Lin, J., Michel, L. O., Zhang, Q.
(2002). CmeABC Functions as a Multidrug Efflux System in Campylobacter jejuni. Antimicrob. Agents Chemother.
46: 2124-2131
[Abstract] [Full Text] -
Aires, J. R., Pechere, J.-C., Van Delden, C., Kohler, T.
(2002). Amino Acid Residues Essential for Function of the MexF Efflux Pump Protein of Pseudomonas aeruginosa. Antimicrob. Agents Chemother.
46: 2169-2173
[Abstract] [Full Text] -
Magnet, S., Courvalin, P., Lambert, T.
(2001). Resistance-Nodulation-Cell Division-Type Efflux Pump Involved in Aminoglycoside Resistance in Acinetobacter baumannii Strain BM4454. Antimicrob. Agents Chemother.
45: 3375-3380
[Abstract] [Full Text] -
Masuda, N., Sakagawa, E., Ohya, S., Gotoh, N., Nishino, T.
(2001). Hypersusceptibility of the Pseudomonas aeruginosa nfxB Mutant to {beta}-Lactams Due to Reduced Expression of the AmpC {beta}-Lactamase. Antimicrob. Agents Chemother.
45: 1284-1286
[Abstract] [Full Text]
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