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Antimicrobial Agents and Chemotherapy, February 1999, p. 400-402, Vol. 43, No. 2
0066-4804/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Interplay between Chromosomal 
-Lactamase and the
MexAB-OprM Efflux System in Intrinsic Resistance to -Lactams in
Pseudomonas aeruginosa
Nobuhisa
Masuda,1,*
Naomasa
Gotoh,2
Chie
Ishii,1
Eiko
Sakagawa,1
Satoshi
Ohya,1 and
Takeshi
Nishino2
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 29 July 1998/Returned for modification 10 September
1998/Accepted 9 November 1998
 |
ABSTRACT |
We investigated the role of chromosomal 
-lactamase and the
MexAB-OprM efflux system in intrinsic resistance to -lactams in
Pseudomonas aeruginosa. Determination of the
susceptibilities of a series of isogenic mutants with impaired
production of the -lactamase and the efflux system to 16 -lactams
including penicillins, cephems, oxacephems, carbapenems, and a
monobactam demonstrated that the intrinsic resistance of P. aeruginosa to most of the -lactams is due to the interplay of
both factors.
| |
TEXT |
Pseudomonas aeruginosa is
a clinically significant pathogen with intrinsic resistance to various
antimicrobial agents. Although this organism has an outer membrane with
low permeability (1, 26), this alone does not adequately
explain its intrinsic resistance (19, 20). An additional
mechanism that interferes with access of the agents to their targets is
suspected to exist. MexA-MexB-OprM (3, 14, 24),
MexC-MexD-OprJ (23), and MexE-MexF-OprN (12) are
multidrug efflux systems of P. aeruginosa. Each of these
systems consists of three components. The MexAB-OprM system expressed
in wild-type strains (5) contributes to the intrinsic resistance of P. aeruginosa to most -lactams and many
other structurally unrelated antimicrobial agents by actively extruding
them out of the cells (21, 24). In fact, inactivation of
this system by an artificial mutation causes hypersusceptibility to the
agents in this bacterium (2, 6, 24). The chromosomal AmpC
-lactamase can also contribute to the intrinsic resistance of
P. aeruginosa to -lactams by inactivating the agents via
hydrolysis (19, 20). Although the respective contributions
of the efflux system and -lactamase have been well characterized,
there is little information on the interplay between the two.
Therefore, we determined the alterations in the susceptibility of
P. aeruginosa that accompany defects in the production of
AmpC -lactamase and the MexAB-OprM system using isogenic mutants
from a laboratory strain, PAO1, and we discuss the interplay between
the two resistance factors.
After constructing KG2504 (ampC::
Smr [22]) from PAO1 by an allelic exchange
technique (8), KG2507 (
mexRAB-oprM
ampC:: Smr [22]) was
constructed from KG2504 by the same technique and was used as an
ampC and mexRAB-oprM double mutant. KG2239
(mexRAB-oprM [7]) was also used as a
MexAB-OprM-deficient mutant from PAO1. The AmpC -lactamase activity
in crude cell extracts was examined by spectrophotometric assay with
100 µM cephaloridine as a substrate as described previously
(16). No production of AmpC -lactamase was detected in
the cultures of any of the strains tested. When the cells were treated
with cefmetazole as an inducer at a concentration of one-fourth the MIC
for 1 h, PAO1 and KG2239 produced 0.3 to 0.6 U of -lactamase
per mg of protein, respectively, whereas KG2504 and KG2507 produced
amounts of -lactamase that were smaller than the detection limit.
The production of MexAB-OprM was tested by an immunoblotting assay with
TM001 (4), a murine monoclonal antibody specific to OprM
encoded on the third gene of this operon. No OprM production was
detected in KG2239 or KG2507 in the assay, although its production was
apparent in PAO1 and KG2504 (data not shown). Thus, disruption of the
ampC gene and/or deletion of the mexAB-oprM
operon in KG2239, KG2504, and KG2507 were confirmed.
MICs were determined by the usual twofold agar dilution technique with
Mueller-Hinton II agar (Becton Dickinson Microbiology Systems,
Cockeysville, Md.) with an inoculum of 104 cells.
Cefpodoxime, panipenem, and S-4661 (10) were synthesized at
Sankyo Co., Ltd., Tokyo, Japan. The other antimicrobial agents used in
this study were obtained from commercial sources. Table 1 presents the MICs of 16 -lactams for
PAO1, KG2239, KG2504, and KG2507. The -lactams included penicillins
(carbenicillin, piperacillin, and amoxicillin), cephems (cefoperazone,
cefsulodin, cefmetazole, cefpodoxime, cefuroxime, and ceftriaxone),
oxacephems (flomoxef and moxalactam), carbapenems (panipenem, imipenem,
meropenem, and S-4661), and a monobactam (aztreonam). All the
-lactams tested had potent activities against the AmpC- and
MexAB-OprM-deficient double mutant KG2507, suggesting that they have
high affinities for the penicillin-binding proteins of P. aeruginosa. In fact, although P. aeruginosa exhibits a
higher level of resistance to carbenicillin, cefpodoxime, and
moxalactam than Escherichia coli, the penicillin-binding
proteins of P. aeruginosa, like those of E. coli,
are inhibited by these agents at low concentrations (13, 25).
The 16 -lactams were grouped into the following three categories on
the basis of the qualitative pattern of change in susceptibilities accompanying the deficiency of AmpC and/or MexAB-OprM. Group I consists
of carbenicillin, piperacillin, cefoperazone, aztreonam, and
cefsulodin. The loss of AmpC from PAO1 caused less than 2-fold increases in susceptibility to these agents, whereas the loss of
MexAB-OprM from PAO1 caused 4- to 64-fold increases in susceptibility. Group II consists of amoxicillin, cefmetazole, flomoxef, panipenem, and
imipenem. The loss of MexAB-OprM from PAO1 caused less than 2-fold
increases in susceptibility to these agents, whereas the loss of AmpC
from PAO1 caused 4- to 512-fold increases in susceptibility. Thus, in
PAO1 the efflux system removes the group I agents from the periplasm
more effectively than the -lactamase does and the -lactamase
removes the group II agents from the periplasm more effectively than
the efflux system does. Group III consists of cefpodoxime, cefuroxime,
ceftriaxone, moxalactam, meropenem, and S-4661. The loss of either AmpC
or MexAB-OprM alone from PAO1 did not cause any substantial change in
susceptibilities to these agents, whereas the loss of both AmpC and
MexAB-OprM from PAO1 caused remarkable increases in susceptibility to
these agents. Thus, group III agents are equally removed from the
periplasm by both the -lactamase and the efflux system. The results
obtained in this study are consistent with those obtained in another
investigation with another series of OprM-deficient isogenic mutants
derived from the AmpC-deficient strain PAO4089 (met-9020 pro-9024
blaJ9111 blaP9202; H. Matsumoto, Shinshu University)
(17).
Most of the agents tested are known to be stabler against chromosomal
-lactamase of P. aeruginosa than the old cephems, e.g., cephaloridine (3, 18). However, the loss of AmpC from
MexAB-OprM-deficient strain KG2239 caused a drastic increase in
susceptibility to group II and III agents. Thus, a synergistic effect
between the slow inactivation of these agents by the -lactamase and
low level of permeability of the outer membrane for these agents might
contribute to this resistance. Moreover, a spontaneous mutant that
constitutively produced the AmpC -lactamase from PAO1 was 4 to 128 times more resistant to group I agents than its parent (17).
Thus, the absence of any effect of the loss of AmpC on the
susceptibilities to the group I agents may be due to the lower levels
of AmpC inducibility by these agents rather than their higher AmpC stabilities.
The loss of either AmpC or MexAB-OprM from PAO1 caused little change in
the susceptibilities to group III agents, suggesting that each removal
mechanism contributes little to the intrinsic resistance to these
agents individually. However, the loss of both AmpC and MexAB-OprM from
PAO1 caused remarkable increases in the susceptibilities to these
agents. These results show that either mechanism alone is almost
sufficient to provide the wild-type level of resistance to group III
agents, and both mechanisms contribute equally and powerfully to the
removal of these agents from the periplasm. Thus, P. aeruginosa possesses redundant mechanisms for the removal of these agents.
We can explain the limitation in the change in susceptibilities to
imipenem caused by the loss of AmpC and/or MexAB-OprM, although we
cannot explain the limited change in susceptibilities to cefsulodin. To
elucidate the former limitation we also isolated by the same technique
a series of OprD-deficient isogenic mutants with impaired production of
AmpC and/or MexAB-OprM (17). The loss of AmpC from PAO1 and
MexAB-OprM-deficient strain KG2239 caused only fourfold increases in
susceptibilities to imipenem, whereas the loss of AmpC from
OprD-deficient strains caused 64-fold increases. Thus, in PAO1, the
higher permeation of imipenem through the OprD outer membrane channel
contributes to the limited change in susceptibilities to imipenem by
the loss of AmpC.
Homologues of the MexAB-OprM efflux system are found in many species of
gram-negative bacteria such as Neisseria gonorrhoeae (9), E. coli (15), Pseudomonas
putida (11), Burkholderia cepacia
(2), and Haemophilus influenzae (4).
Thus, the interplay of double removal systems might be at work in a
number of gram-negative bacteria.
| |
ACKNOWLEDGMENTS |
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 strains KG2504 and KG2507.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Biological
Research Laboratories, Sankyo Co., Ltd., 2-58 Hiromachi 1-chome,
Shinagawa-ku, Tokyo 140-8710, Japan. Phone: 81-3-3492-3131. Fax:
81-3-5436-8566. E-mail: nmasud{at}shina.sankyo.co.jp.
| |
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Antimicrobial Agents and Chemotherapy, February 1999, p. 400-402, Vol. 43, No. 2
0066-4804/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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