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Antimicrobial Agents and Chemotherapy, April 2001, p. 1284-1286, Vol. 45, No. 4
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 20 July 2000/Returned for modification 27 November
2000/Accepted 12 January 2001
The Pseudomonas aeruginosa nfxB mutant lacking
mexAB-oprM showed hypersusceptibility to 9 out of 24 A series of multicomponent efflux
systems, each made up of three components, play important roles in the
intrinsic and acquired resistance of gram-negative bacteria (20,
21, 22). Four of these efflux systems, MexAB-OprM (11,
24), MexCD-OprJ (25), MexEF-OprN (9),
and MexXY-OprM (1, 19, 26), have been characterized for
Pseudomonas aeruginosa. MexAB-OprM and MexXY-OprM contribute
to both intrinsic and acquired resistance, whereas MexCD-OprJ and
MexEF-OprN contribute to only acquired resistance. MexAB-OprM is
slightly expressed in wild-type strains, and a nalB mutation
causes an overexpression of the efflux system. The expression of
MexCD-OprJ and MexEF-OprN is strictly suppressed in wild-type strains,
and mutations in nfxB and nfxC cause
overexpression of MexCD-OprJ and MexEF-OprN, respectively. The
expression of MexXY, which is not detectable in the wild-type strain,
is induced by several antimicrobial agents such as tetracycline,
erythromycin, and gentamicin (17). MexXY is associated
with OprM and contributes to the intrinsic resistance to these agents.
While nfxB mutants show resistance to quinolones,
tetracycline, erythromycin, chloramphenicol, and expanded-spectrum
cephems such as cefpirome, they show hypersusceptibility to
penicillins, carbapenems, and aminoglycosides (15). A
characterization of mutants lacking the mexAB-oprM region
demonstrated that the hypersusceptibility to To investigate whether the hypersusceptibility of the nfxB
mutants to
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.4.1284-1286.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Hypersusceptibility of the Pseudomonas
aeruginosa nfxB Mutant to
-Lactams Due to Reduced Expression
of the AmpC -Lactamase
ABSTRACT
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Abstract
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References
-lactams tested. This hypersusceptibility was found for the
nfxB mutant lacking mexAB-oprM-mexXY (N108) but not for the nfxB mutant lacking both
mexAB-oprM-mexXY and ampC. The level of
the AmpC -lactamase induction was reduced in N108. Thus, the
reduced AmpC induction must be the cause of the hypersusceptibility.
TEXT
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Abstract
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-lactams such as
carbenicillin and aztreonam is caused by the reduced expression of
MexAB-OprM in the nfxB mutants (4).
-lactams is generally attributable to this mechanism, in
this study, we compared the susceptibilities of the isogenic mutants,
i.e., the MexCD-OprJ-producing KG2259 (
MexAB-OprM of COR6
[4]) and the non-MexCD-OprJ-producing KG2239
(MexAB-OprM of PAO1 [4]), to the 24 -lactams.
Table 1 shows the MICs determined by the
usual twofold agar dilution technique with Mueller-Hinton II agar
(Becton Dickinson Microbiology Systems, Cockeysville, Md.). The results
indicated that these -lactams can be classified into three groups.
The first group, which consists of piperacillin, cloxacillin,
nafcillin, cefpirome, cefepime, cefozopran, and cefoselis, showed 8- to
64-fold-lower activities against KG2259 than against KG2239. The second
group, which consists of penicillin G, cefuroxime, cefmetazole,
ceftazidime, cefsulodin, meropenem, S-4661 (7), and
aztreonam, showed almost the same activities against KG2239 and KG2259.
The third group, which consists of sulbenicillin, cefpodoxime,
ceftriaxone, moxalactam, flomoxef, imipenem, panipenem, biapenem, and
R-95867 (an active form of a new oral carbapenem, CS-834
[3]), showed 4- to 32-fold-higher activities when
MexCD-OprJ was expressed and MexAB-OprM was not. These results suggest
that there is at least one other mechanism responsible for the
hypersusceptibility to the third group of -lactams independent of
the decreased expression of MexAB-OprM with accompanying expression of
MexCD-OprJ.
TABLE 1.
Susceptibilities of isogenic MexAB-OprM mutants with or
without MexCD-OprJ expression
The deletion of mexXY from KG2239 (N103 [17])
and KG2259 (N108 [18]) did not affect their
susceptibilities to the third group of -lactams, whereas the
deletion of mexCD-oprJ from N108 (KG4507 [N. Gotoh,
unpublished data]) eliminated these hypersusceptibilities (Table
2). These results suggest that MexCD-OprJ
expression is directly related to hypersusceptibility.
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Given that OprJ and other outer membrane components of the multicomponent efflux systems of P. aeruginosa are assumed to form a channel, expression of OprJ might enhance the permeability of the P. aeruginosa outer membrane to the agents. To examine this hypothesis, we introduced an OprJ expression plasmid into N103. Although we confirmed the expression of OprJ by immunoblot assay using an OprJ-specific antibody (5), the susceptibilities of these strains to the agents were not affected by this expression (data not shown).
We previously demonstrated that the chromosomal AmpC -lactamase acts
as one of the factors causing the intrinsic resistance of P. aeruginosa to -lactams via its interplay with the efflux system
(16). To examine the possibility that the AmpC
-lactamase is involved in hypersusceptibility, we compared the
susceptibilities of N119 (ampC::
of N108
[18]) and N106 (ampC:: of N103
[18]). N108 was 8- to 32-fold more susceptible than was
N103 to R-95867, cefpodoxime, ceftriaxone, and flomoxef, whereas N119
was 4- to 128-fold less susceptible than N106 to these agents (Table
2). We also evaluated the susceptibilities of N103 and N108 to R-95867 in the presence of various concentrations of Syn2190 (23),
an AmpC -lactamase inhibitor. MICs of Syn2190 were >4,096, 8, and 128 µg/ml against PAO1, N103, and N108, respectively. Figure
1 shows the effect of subinhibitory
concentrations of Syn2190 on the susceptibilities of N103 and N108. The
susceptibility of N103 to R-95867 increased as the concentration of
Syn2190 increased, while the subinhibitory concentrations of Syn2190
had little effect on the susceptibility of N108 to R-95867. Syn2190 did
not induce -lactamase activity at 0.03 to 2 µg/ml in N103 and at
0.5 to 128 µg/ml in N108, although it did induce slight -lactamase
activity at the higher concentrations in N103 (data not shown). The
presence of the intact ampC gene imparted R-95867 resistance
to N103 (compared with N106) but not to N108 (compared with N119)
(Table 2). In addition, the inhibition of AmpC -lactamase had little
effect on the susceptibility of N108, a strain that produces MexCD-OprJ (Fig. 1). Since these results suggested a defect in the AmpC expression of N108, we examined the -lactamase activity induced by R-95867 in
N103 and N108. These strains were incubated with various concentrations (0.25 to 64 µg/ml) of R-95867 for 1 h, and the induced
-lactamase was quantified by a spectrophotometric assay with 50 µM
cephaloridine used as a substrate, as described previously
(14). N108 produced a lower amount of -lactamase than
did N103 (Fig. 2), suggesting that the
decreased level of AmpC expression is the cause of the hypersusceptibility to R-95867 in the MexCD-OprJ-producing strain.
|
) and N108 (
) to R-95867. MICs were
determined by the microdilution broth method.
|
Although R-95867 is stable in response to hydrolysis by AmpC
(3), a synergistic effect between the slow inactivation of the agent by AmpC and the low level of permeability of the outer membrane might contribute to the resistance in P. aeruginosa, imparting the same mode of resistance seen when this
organism is exposed to imipenem and meropenem (12, 16).
The nfxB mutant showed hypersusceptibility to only certain
kinds of -lactams (15) (Table 2). A balance between the
reduced expression of AmpC and the extrusion of each -lactam by
MexCD-OprJ might determine the phenotype, i.e., hypersusceptibility or
resistance. The induction mechanism of AmpC -lactamase has been well
studied for Enterobacter cloacae (2, 6, 8). The
inhibition of cell wall synthesis by -lactam results in the
accumulation of precursors,
N-acetylglucosamyl-1,6-anhydromuropeptides, in the
periplasm. These precursors are transported via AmpG into the
cytoplasm, where they are converted into 1,6-anhydromuropeptides by a
cytosolic -N-acetylglucosaminidase. The
1,6-anhydromuropeptides convert the transcriptional regulator AmpR into
an activator of AmpC expression. Given that AmpG and AmpR were also
reported for P. aeruginosa (10, 13), a similar
induction mechanism must be present in P. aeruginosa.
MexCD-OprJ might extrude some of the
N-acetylglucosamyl-1,6-anhydromuropeptides or
1,6-anhydromuropeptides and reduce the -lactamase expression. In a
previous paper (15), we reported that nfxB
mutants isolated from -lactamase-deficient strains were also more
susceptible than were their parent strains to carbenicillin, imipenem,
moxalactam, and aztreonam. This discrepancy can be explained by the
MexAB-OprM expression of the -lactamase-deficient strains. The
reduced level of MexAB-OprM must have caused the hypersusceptibility of
the nfxB mutants isolated from the AmpC-deficient strains.
Our results suggest that the reduction of AmpC induction is the cause of the hypersusceptibility, although further experiments are needed to elucidate the mechanism of the reduction of AmpC induction.
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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.
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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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Antimicrobial Agents and Chemotherapy, April 2001, p. 1284-1286, Vol. 45, No. 4
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.4.1284-1286.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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