![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
Previous Article | Next Article 
Antimicrobial Agents and Chemotherapy, February 2001, p. 480-484, Vol. 45, No. 2
Division of Infectious Disease, Department of Internal
Medicine,1 and Department of
Microbiology,3 College of Medicine,
University of Dankook, Chonan, Department of Clinical
Pathology, Sungkyunkwan University College of
Medicine,2 and Department of
Internal Medicine, Seoul National University College of
Medicine,4 Seoul, Korea, and
Department of Microbiology, Kyoto Pharmaceutical University,
Kyoto, Japan5
Received 31 March 2000/Returned for modification 8 August
2000/Accepted 10 November 2000
In order to define the contributions of the mechanisms for
carbapenem resistance in clinical strains of Pseudomonas
aeruginosa, we investigated the presence of OprD, the expressions
of the MexAB-OprM and MexEF-OprN systems, and the production of the
Pseudomonas aeruginosa is
a clinically important pathogen with intrinsic resistance to various
antimicrobial agents. This intrinsic multidrug resistance results from
the synergy between broadly specific drug efflux pumps and a low degree
of outer membrane permeability. For the carbapenem antimicrobials, the
resistance is mostly mediated by OprD loss, which primarily confers a
resistance to imipenem but also confers a low grade resistance
to meropenem (12, 16). But the multidrug efflux systems
which mediate the resistance to quinolone, chloramphenicol, and many
other antimicrobial agents, also contribute to the carbapenem
resistance. The strains which overexpress the MexAB-OprM system or
express the MexEF-OprN system exhibit the carbapenem resistance by
pumping the drug out or repressing the transcription of
oprD, respectively (13, 20, 25). On the other
hand, the NfxB mutants which expressed MexCE-OprJ became more
susceptible to imipenem, bipenem, and some Based on the carbapenem susceptibility patterns, the clinical isolates
of carbapenem-resistant P. aeruginosa could be divided into
three groups as the imipenem-resistant and meropenem-sensitive group, the imipenem-sensitive and meropenem-resistant group,
and the imipenem-resistant and meropenem-resistant group
(2, 4, 17). This suggests that carbapenem resistance
occurs by several mechanisms in concert for the clinical isolates.
Therefore, in order to evaluate the contributions of the carbapenem
resistance mechanisms in clinical strains of imipenem-resistant
P. aeruginosa, we investigated the presence of OprD,
expressions of the MexAB-OprM and the MexEF-OprN systems, and the
production of the Bacterial strains.
A total of 117 strains of
imipenem-resistant P. aeruginosa were collected from
four hospitals in Korea during 1997: 80 strains from Hallym University
Hospital (HUH), 17 strains from Dankook University Hospital, 14 strains
from Asan Medical Center, and 6 strains from Samsung Medical Center.
The 80 strains from HUH showed only two patterns of antibiogram which
differed in their susceptibilities to amikacin and ciprofloxacin. To
examine a clonal spread, we performed ribotyping for 30 of 80 isolates
from HUH. Because the ribotyping of 30 isolates showed two patterns
which matched well with the antibiogram, we randomly selected seven isolates from 80 strains and included them in this study. Thus, 44 P. aeruginosa isolates were studied: 7 strains from HUH and 37 strains from other hospitals.
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.2.480-484.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Carbapenem Resistance Mechanisms in Pseudomonas
aeruginosa Clinical Isolates
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
-lactamases for 44 clinical strains. All of the carbapenem-resistant
isolates showed the loss of or decreased levels of OprD. Three strains overexpressed the MexAB-OprM efflux system by carrying mutations in
mexR. These three strains had the amino acid substitution
in MexR protein, Arg (CGG) 
Gln (CAG), at the position of amino acid
70. None of the isolates, however, expressed the MexEF-OprN efflux
system. For the characterization of -lactamases, at least 13 isolates were the depressed mutants, and 12 strains produced secondary
-lactamases. Based on the above resistance mechanisms, the MICs of
carbapenem for the isolates were analyzed. The MICs of carbapenem were
mostly determined by the expression of OprD. The MICs of meropenem were
two- to four-fold increased for the isolates which overexpressed
MexAB-OprM in the background of OprD loss. However, the elevated MICs
of meropenem for some individual isolates could not be explained. These
findings suggested that other resistance mechanisms would play a role
in meropenem resistance in clinical isolates of P. aeruginosa.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
-lactams (22). In addition to the OprD loss or drug efflux pumps,
chromosomal AmpC -lactamase plays an important role in carbapenem
resistance in P. aeruginosa (16, 21).
Although the contributions of the OprD loss, the efflux systems, and
-lactamase in the carbapenem resistance have been well characterized
in the laboratory strains, little data is available for how such
factors play together in the clinical isolates of P. aeruginosa (2, 4, 31).
-lactamases.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
)
mutant (2297 -con D2
), and nfxC
mutant PAO4222nfxC were used as standard strains (16, 28, 13). For secondary -lactamases, P. aeruginosa
carrying the genes encoding -lactamases OXA-10 (R151), OXA-2
(pUD11), and Escherichia coli carrying PSE-1 (pMON811)
served as the standards (5, 10, 11). For evaluation of the
IMP-1 enzyme, Serratia marcescens TN9106 was used as the
standard (26).
Antibiotics. Antimicrobials tested were imipenem (Choongwae Pharma Co., Seoul, Korea); meropenem, piperacillin, and cloxacillin (Yuhan Co., Seoul, Korea); ceftazidime (Glaxo Korea Co., Seoul, Korea); aztreonam (Dong-A Biotech Co., Seoul, Korea); clavulanic acid (II-Sung Pharmaceuticals, Seoul, Korea); amikacin (Young Jin Pharmaceutical Co., Seoul, Korea); ciprofloxacin (Bayer Korea, Ltd., Seoul, Korea); and tetracycline and carbenicillin (Chong Kun Dang Co., Seoul, Korea).
Susceptibility tests. MICs of antibiotics were determined by the agar dilution method on Mueller-Hinton agar (Difco, Detroit, Mich.) with a Steers multiple inoculator according to National Committee for Clinical Laboratory Standards (24). E. coli ATCC 25922 and P. aeruginosa ATCC 27853 were used as quality control strains.
Isolation of total membranes, SDS-PAGE, and immunoblot assay. The total membranes of the cells were isolated as described previously (8), electrophoresed using 10% (wt/vol) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels, and electrophoretically transferred to a nitrocellulose membrane (0.45-µm pore size; Bio-Rad, Hercules, Calif.). For the detection of OprD, OprM, or OprN, anti-OprD antibody YD010 (25), anti-OprM antibody TM002 (8), or anti-OprN antibody TN005 (8, 13) was used as primary antibodies, and alkaline phosphatase-conjugated goat antibodies to mouse immunoglobulin G (Cappel) were used as the secondary antibodies. For color development, AP Conjugate Substrate Kit (Bio-Rad) was used (8).
PCR and sequencing of the mexR gene. The mexR-coding regions of the P. aeruginosa isolates were amplified with primers M1 (5'-ACC AAT GAA CTA CCC CGT GA-3') and M2 (5'-AAT GTT CTT AAA TAT CCT CAA-3'), which corresponded to nucleotides 273 to 292 and nucleotides 707 to 727 of the mexR gene, respectively. The PCR amplification mixture (total volume, 100 µl) contained 10 mM Tris-HCl (pH 7.4), 2.5 mM MgCl2, 50 mM KCl, 0.1% Triton X-100, 200 µM concentrations of each deoxynucleoside triphosphate, 0.1 pmol of primers M1 and M2, 2.5 U of Taq polymerase (AmpliTaq; Perkin-Elmer Cetus, Norwalk, Conn.), and 10 µl of template DNA. The mixture was denatured at 95°C for 30 s and annealed at 50°C for 1.5 min, and then the chain was extended at 72°C for 1 min in a thermal cycler (Perkin-Elmer Cetus). This cycle was repeated 35 times. For sequencing, M1 and M3 (5'-TCG CGG CGG ACC AGG TTT-3'), corresponding to nucleotides 510 to 527 of the downstream mexR gene, were used. The amplified PCR products were purified by using a Qiaex gel extraction kit (Qiagen, Chatsworth, Calif.) and were used as templates for sequencing by a dideoxy termination cycle sequencing kit (Perkin-Elmer Cetus).
Analytical IEF and enzyme inhibition assay. Isoelectric focusing (IEF) was performed using sonicated extracts by the method of Matthew et al. (23), by using an LKB Multiphor apparatus on prepared PAGplates (pH 3.5 to 9.5; Pharmacia Biotech Asia Pacific, Hong Kong, Republic of China) or the Mini-IEF cell system (Bio-Rad). Enzyme activities were detected by overlaying the gel with 0.5 mM nitrocefin and comparing active bands with reference enzymes.
An inhibition assay was performed by overlaying the gels with 0.5 mM nitrocefin with or without 0.3 mM cloxacillin or 0.3 mM clavulanic acid in 0.1 M phosphate buffer (pH 7.0) (6).Assays of -lactamase activity.
Cultures were grown
overnight at 37°C in tryptic soy broth, transferred with a 10%
inoculum, and incubated for 4 h with shaking. The cells were
subsequently harvested by centrifugation, resuspended in 1 ml of 0.1 M
phosphate buffer (pH 7.0), and sonicated. The supernatants were assayed
against 0.1 mM nitrocefin in 0.1 mM phosphate-buffer (pH 7.0). The
activity was standardized against the protein concentration and assayed
by the method of Lowry et al. (18).
Characterization of secondary -lactamases.
To amplify
PSE-related genes from clinical isolates, the following oligonucleotide
primers were synthesized and used for PCR: P1 (5'-TTA TTG GCA TTT
TCG CTT TTA-3'), upstream 21-mer from nucleotide 155; and P2
(5'-CGC ATC ATT TCG CTC TG-3'), downstream 17-mer from
nucleotide 942 of the PSE gene, respectively (10). For OXA-10-specific PCR, primers ABD1 (5'-TAT CGC GTG TCT TTC GAG TA-3') and ABD4 (5'-TTA GCC ACC AAT GAT GCC C-3') were
synthesized, and the PCR amplification was carried out as described
previously (6). For the sequencing of the OXA-related
gene, ABD2 (5'-CGG AAA GCC AAG AGC C-3') was used
(6). For IMP-1-specific PCR, primers IMP1 (5'-CGC AGC
AGG GCA GTC-3') and IMP2 (5'-CAA AAG CGC AAC TTA CAA AC-3')
were synthesized and used (26).
Ribotyping. The chromosomal DNA of the organisms was extracted as described earlier and digested with PvuII (Promega, Madison, Wis.) (9, 19). After electrophoresis, the digests of chromosomal DNA were transferred to a nylon membrane by the vacuum transfer method and then hybridized with the digoxigenin-labeled cDNA copy of E. coli rRNA (Boehringer Mannheim), manufactured by reverse transcription using AMV-RT (Promega).
| |
RESULTS AND DISCUSSION |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
|
|---|
Expressions of OprD.
The presence of OprD was examined by
Western analysis using an anti-OprD antibody. The result unambiguously
showed that 5 isolates expressed OprD, 4 isolates showed decreased
OprD, and 35 isolates showed OprD loss (Table
1).
Five isolates (group 5 in Table 1) which expressed OprD were all
sensitive to carbapenem. The isolates (isolates 1, 6, 71, and 85) with
decreased OprD were similarly resistant against carbapenem compared to
those with OprD loss. Therefore, all carbapenem-resistant isolates
expressed the loss of or decreased levels of OprD.
|
Expressions of MexAB-OprM and MexEF-OprN efflux system.
In
order to observe the expression of OprM, the transferred membranes were
hybridized with an anti-OprM antibody. Although most isolates expressed
OprM, it was difficult to quantitate the expression of OprM by Western
blot analysis, especially without comparison to isogenic
carbapenem-susceptible parents. But among 44 isolates, at least two
strains (isolates 59 and 60) clearly showed elevated amounts of OprM.
Thus, in an attempt to support the results of the Western blot
analysis, we sequenced the mexR genes, which were known to
control the mexA-mexB-OprM operon
negatively. The amplified products were 455 bp sized, a length which
contained the entire coding sequences. The nucleotide sequences and
deduced amino acid sequence analysis revealed that three isolates
(isolates 53, 59, and 60) had the amino acid substitution of Arg (CGG)
to Gln (CAG) at the position of amino acid 70 and a Val (GAG) Glu (GTG) change at amino acid 126. Amino acid 70 was the same position as
the amino acid that was substituted in the MexR protein of the
nalB strain OCR1 previously studied (28),
although the changed amino acid was different (Arg [CGG] Trp
[TGG]). With the mexR gene sequences and the Western
analyses, we concluded that the above three isolates were the mutants
overexpressing the MexAB-OprM efflux systems from the mutations in
mexR.
Assays of -lactamase activity.
The -lactamases were
characterized. Of 44 isolates, 12 isolates produced secondary
-lactamases, and 32 isolates produced only AmpC chromosomal enzymes.
The production of the AmpC enzymes was measured for the 32 isolates as
described previously (4). When the hydrolysis activities
of -lactamases were compared to that of PAO1 standard strain (35 U/mg of protein), 32 isolates could be divided into three groups based
on the activities of the -lactamases. Eleven isolates (isolates 42, 72, 78, and 151 in group 5 and all isolates in group 2 of Table 1)
showed a similar hydrolysis activity to that of PAO1 strain, eight
isolates (isolates 59 and 60 in group 1 and all isolates in group 3 of
Table 1) produced two- to five-fold-higher activity, and thirteen
isolates (isolate 53 in group 1, isolate 57 in group 5, and all
isolates belonging to group 4 of Table 1) showed more than
fivefold-higher activity than the PAO1 strain. We defined the
derepressed mutant as the isolate with a -lactamase activity more
than fivefold higher than that of the PAO1 strain (4).
Based on this definition, we concluded at least 13 isolates were
derepressed mutants.
-lactamase production on the MICs of
-lactams for the strains included in groups 2, 3, and 4 (Table 1).
The MIC50 values of piperacillin, ceftazidime, and aztreonam for seven isolates which showed a similar hydrolysis activity
to that of PAO1 (group 2) were 16, 4, and 4 µg/ml, respectively. For
six strains which produced two- to fivefold-higher levels of
-lactamase than PAO1 (group 3), the MIC50s for
piperacillin, ceftazidime, and aztreonam were 256, 32, and 16 µg/ml,
respectively. The corresponding values for 11 derepressed mutants
(group 4 in Table 1) were 256, 32, and 16 µg/ml, respectively. These
data clearly showed that the MICs of piperacillin, ceftazidime, and aztreonam for the derepressed mutans were significantly higher than
those of the isolates in group 2 (Mann-Whitney U test, P < 0.001). However, there were no significant differences in the MICs
of -lactams between the derepressed mutants and the isolates in
group 3. We speculated that other mechanisms of resistance against
-lactams, such as permeability change, might work in concert in
these strains.
Susceptibility patterns of carbapenem based on the resistance
mechanisms in the isolates.
The isolates could be divided into six
groups based on the results of the expression of OprD, MexAB-OprM
efflux system, and -lactamases (Table 1). A total of 3 isolates
showed the loss of OprD, hyperproduced AmpC enzymes, and overexpressed
MexAB-OprM efflux system (group 1); 7 isolates showed only the loss of
OprD (group 2); 6 isolates lost the OprD and produced AmpC enzymes two-
to fivefold higher than PAO1 strain (group 3); 11 isolates lost the
OprD and were derepressed (group 4); 5 strains expressed the OprD and
produced variable amounts of -lactamases (group 5); and 12 isolates
showed the loss of OprD, and produced secondary -lactamases (group 6).
Secondary -lactamases produced by P. aeruginosa.
Among 44 isolates, 12 isolates produced
secondary -lactamases (Table 1). On IEF, six isolates produced the
-lactamases with a pI value of 6.1, and four isolates produced
-lactamases with a pI of 5.6. The enzyme with a pI of 5.6 was
inhibited by 0.3 mM clavulanic acid but not by 0.3 mM cloxacillin on
the gel overlaying inhibition test, and the pI 6.1 -lactamase was
not inhibited by clavulanic acid or cloxacillin. On the basis of the MIC profiles, the pI values of the enzymes, and the inhibition profiles, we suspected that the enzyme with a pI value of 5.6 was
PSE-1-like and that the pI 6.1 -lactamase was OXA-10-like. Thus,
PCRs with a PSE-specific primers and OXA-10-specific primers were
performed with these isolates. All four isolates which produced pI 5.6 enzyme showed the amplified PCR products with PSE-specific PCR.
Southern hybridization of the PCR products from four isolates revealed
that they were PSE-1-like enzymes. In a PCR with the OXA-10-specific
primers, five of six isolates which produced the -lactamases with a
pI value of 6.1 showed a positive amplified product (except isolate
44). Among five isolates which harbored blaOXA-10-like genes, the amplified product from
isolate 152 was sequenced, which revealed that the nucleotide sequences
matched exactly with the known sequences of OXA-10 (11).
-lactamase, we performed the PCR with
IMP-1-specific primers. But no amplified product was detected from any
isolates, which indicated that there was no IMP-1-producing organism in
our isolates.
Ribotyping. We performed the ribotyping for the isolates. The ribotyping experiment showed 18 different patterns in the isolates (Table 1). Because we performed DNA restriction with PvuII only in this experiment, it is possible that the ribotyping could not differentiate some organisms.
The resistance against imipenem is usually mediated by OprD loss, and the imipenem does not select for the multidrug efflux pumps. However, because imipenem was the only carbapenem used in this country until recent times, we selected the isolates based on imipenem resistance for looking at the resistance mechanisms for carbapenems. In the laboratory strains, the organism which lost OprD showed an MIC of meropenem a fourfold lower than that of imipenem (12, 16). However, the MIC values for our clinical strains showed that 25 of 39 strains (excluding five strains which were sensitive to imipenem) were also resistant to meropenem at 4 µg/ml and most of the clinical isolates (33 of 44 isolates) were resistant to ciprofloxacin. Thus, we suspected that other resistance mechanisms such as efflux systems might play a role for the carbapenem resistance in these isolates. However, our strains showed only three isolates overexpressing the MexAB-OprM system and none of the nfxC-type mutant. For the quinolone resistance, we speculate that MexCD-OprJ might play a main role although we did not study the OprJ expression in these isolates. Actually, ofloxacin and ciprofloxacin were the most commonly used quinolones, but enoxacin or sparfloxacin was not much used in Korea. The reason why we could not find any nfxC mutant among our imipenem-resistant isolates might be the selection of different efflux pumps by the different quinolones (14). Recently, there have been several reports which proposed alternative ports of entry for meropenem (12, 27). Moreover, analysis of preliminary sequence data from the P. aeruginosa genome suggested the existence of 14 open reading frames sharing significant homology with either OprD or OprE porins (12). Our results also suggested that other mechanisms of meropenem other than MexAB-OprM might exist in the clinical isolates of P. aeruginosa.| |
ACKNOWLEDGMENTS |
|---|
This work was supported by the Korean Research Foundation made in the program of 1997.
We are very grateful to G. A. Jacoby, who provided the strains
carrying the plasmids encoding PSE-1, OXA-10, and OXA-2 -lactamases; to D. M. Livermore, who provided OprD() mutant 2297 -con D2; and to K. Poole for giving us the strain PAO1 and the nalB
mutant OCR1. We thank E. Osano for giving us the strain harboring IMP-1 -lactamase. We also thank H. Choy and J. Tran for their critical review of the manuscript.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Division of Infectious Disease, Department of Internal Medicine, College of Medicine, University of Dankook, San 29, Anseo-dong, Chonan, Chungnam, 330-715, Korea. Phone: 82-41-550-3918. Fax: 82-41-556-3256. E-mail: paihj{at}unitel.co.kr.
| |
REFERENCES |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
|
|---|
| 1. |
Bauerfeind, A.,
I. Stemplinger,
R. Jungwirth,
P. Mangold,
S. Amann,
E. Akalin,
O. Ang,
C. Bal, and J. M. Casellas.
1996.
Characterization of -lactamase gene blaper-2, which encodes an extended-spectrum class A -lactamase.
Antimicrob. Agents Chemother.
40:616-620[Abstract].
|
| 2. |
Bonfiglio, G.,
Y. Laksai,
L. Granchino,
G. Amicosante, and G. Nicoletti.
1998.
Mechanisms of -lactam resistance amongst Pseudomonas aeruginosa isolated in an Italian survey.
J. Antimicrob. Chemother.
42:697-702 |
| 3. |
Bush, K.,
G. A. Jacoby, and A. A. Medeiros.
1995.
A functional classification scheme for -lactamases and its correlation with molecular structure.
Antimicrob. Agents Chemother.
39:1211-1233[Medline].
|
| 4. |
Chen, H. Y.,
M. Yuan, and D. M. Livermore.
1995.
Mechanisms of resistance to -lactam antibiotics amongst Pseudomonas aeruginosa isolates collected in the UK in 1993.
J. Med. Microbiol.
43:300-309[Abstract].
|
| 5. |
Dale, J. W.,
D. Godwin,
D. Mossakowska,
P. Stephenson, and S. Wall.
1985.
Sequence of the OXA-2 -lactamase; comparison with other penicillin-reactive enzymes.
FEBS Lett.
191:39-44[CrossRef][Medline].
|
| 6. | Danel, F., L. M. C. Hall, D. Gur, and D. M. Livermore. OXA-14, another extended-spectrum variant of OXA-10
(PSE-2) -lactamase from Pseudomonas aeruginosa.
Antimicrob. Agents Chemother. 39:1881-1884.
|
| 7. | Gotoh, N., N. Itoh, H. Tsujimoto, J. Yamagishi, Y. Oyamada, and T. Nishino. 1994. Isolation of OprM-deficient mutants of Pseudomonas aeruginosa by transposon insertion mutagenesis; evidence of involvement in multiple antibiotic resistance. FEMS Microbiol. Lett. 122:267-274[CrossRef][Medline]. |
| 8. |
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 |
| 9. | Grattard, F., B. Pozzetto, A. Ros, and O. G. Gaudin. 1994. Differentiation of Pseudomonas aeruginosa strains by ribotyping: high discriminatory power by using a single restriction endonuclease. J. Med. Microbiol. 40:275-281[Abstract]. |
| 10. |
Huovinen, P., and G. A. Jacoby.
1991.
Sequence of the PSE-1 -lactamase gene.
Antimicrob. Agents Chemother.
35:2428-2430 |
| 11. | Huovinen, P., S. Huovinen, and G. A. Jacoby.
Sequence of PSE-2 -lactamase. Antimicrob. Agents Chemother.
32:134-136.
|
| 12. |
Kohler, T.,
M. Michea-Hamzehpour,
F. E. Simone, and J. C. Pechere.
1999.
Carbapenem activities against Pseudomonas aeruginosa: respective contribution of OprD and efflux systems.
Antimicrob. Agents Chemother.
43:424-427 |
| 13. | Kohler, T., M. Michea-Hamzehpour, U. Henze, N. Gotoh, L. K. Curty, and J. C. Pechere. 1997. Characterization of Mex E-MexF-OprN, a positively regulated multidrug efflux system of Pseudomonas aeruginosa. Mol. Microbiol. 23:345-354[CrossRef][Medline]. |
| 14. | Kohler, T., M. Michea-Hamzehpour, P. Plesiat, A. Kahr, and J. Pechere. 1997. Differential selection of multidrug efflux systems by quinolones in Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 41:2540-2543[Abstract]. |
| 15. | 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]. |
| 16. |
Livermore, D. M.
1992.
Interplay of impermeability and chromosomal -lactamase activity in imipenem-resistant Pseudomonas aeruginosa.
Antimicrob. Agents Chemother.
36:2046-2048 |
| 17. |
Livermore, D. M.
1996.
Are all -lactams created equal?
Scand. J. Infect. Dis. Suppl.
101:33-43[Medline].
|
| 18. |
Lowry, O. H.,
N. J. Rosebrough,
A. L. Farr, and R. J. Randall.
1951.
Protein measurement with the Folin phenol reagent.
J. Biol. Chem.
193:265-275 |
| 19. | Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. |
| 20. |
Maseda, H.,
H. Yoneyama, and T. Nakae.
2000.
Assignment of the substrate-selective subunits of the MexEF-OprN multidrug efflux pump of Pseudomonas aeruginosa.
Antimicrob. Agents Chemother.
44:658-664 |
| 21. |
Masuda, N.,
N. Gotoh,
C. I. Ishii,
E. Sakagawa,
S. Ohya, and T. Nishino.
1999.
Interplay between chromosomal -lactamase and the MexAB-OprM efflux system in intrinsic resistance to -lactamas in Pseudomonas aeruginosa.
Antimicrob. Agents Chemother.
43:400-402 |
| 22. | 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]. |
| 23. |
Matthew, M.,
M. Harris,
M. J. Marshall, and G. W. Rose.
1975.
The use of analytical isoelectric focusing for detection and identification of -lactamases.
J. Gen. Microbiol.
88:169-178[Medline].
|
| 24. | National Committee for Clinical Laboratory Standards. 1993. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, 3rd ed. Approved standard M7-A3. National Committee for Clinical Laboratory Standards, Villanova, Pa. |
| 25. |
Ochs, M.,
M. McCusker,
M. Bains, and R. Hancock.
1999.
Negative regulation of the Pseudomonas aeruginosa outer membrane porin OprD selective for imipenem and basic amino acids.
Antimicrob. Agents Chemother.
43:1085-1090 |
| 26. |
Osano, E.,
Y. Arakawa,
R. Wacharotayankun,
M. Ohta,
T. Horii,
H. Ito,
F. Yoshimura, and N. Kato.
1994.
Molecular characterization of an enterobacterial metallo beta-lactamase found in a clinical isolate of Serratia marcescens that shows imipenem resistance.
Antimicrob. Agents Chemother.
38:71-78 |
| 27. | Perez, F. J., C. Gimeno, D. Navarro, and J. Garcia-de-Lomas. 1996. Meropenem permeation through the outer membrane of Pseudomonas aeruginosa can involve pathways other than the OprD porin channel. Chemotherapy 42:210-214[Medline]. |
| 28. | Poole, K., K. Tetro, Q. Zhao, S. Neshat, D. E. 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]. |
| 29. |
Trias, J., and H. Nikaido.
1990.
Outer membrane protein D2 catalyzes facilitated diffusion of carbapenem and penems through the outer membrane of Pseudomonas aeruginosa.
Antimicrob. Agents Chemother.
34:52-57 |
| 30. | Yoshihara, E., N. Gotoh, T. Nishino, and T. Nakae. 1996. Protein D2 porin of the Pseudomonas aeruginosa outer membrane bears the protease activity. FEBS Lett. 394:179-182[CrossRef][Medline]. |
| 31. |
Ziha-Zarifi, I.,
C. Llanes,
T. Kohler,
JC. 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 |
This article has been cited by other articles:
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Clin. Vaccine Immunol. | Clin. Microbiol. Rev. |
|---|---|
| J. Clin. Microbiol. | ALL ASM JOURNALS |
