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Antimicrobial Agents and Chemotherapy, February 1999, p. 287-291, Vol. 43, No. 2
Laboratoire de Bactériologie,
Hôpital Jean Minjoz, F-25030 Besançon,
France,1 and
Department of Genetics
and Microbiology, Centre Médical Universitaire, CH-1211
Geneva 4, Switzerland2
Received 17 July 1998/Returned for modification 24 August
1998/Accepted 12 November 1998
During a 6-month period, 21 pairs of Pseudomonas
aeruginosa isolates susceptible (pretherapy) and resistant
(posttherapy) to antipseudomonal Active efflux pump systems
have recently been recognized to play a major role in the intrinsic and
acquired resistances of Pseudomonas aeruginosa to
antimicrobial drugs. Because of its wide substrate specificity and its
constitutive expression in wild-type P. aeruginosa
cells, the efflux pump system encoded by the mexA-mexB-oprM
operon contributes significantly to the elevated resistance that this
opportunistic pathogen naturally displays to a variety of antibiotics
(24). Substrates for MexA-MexB-OprM include compounds as
structurally diverse as Evidence for the involvement of the MexA-MexB-OprM system in the
multidrug resistance of P. aeruginosa has essentially
been obtained with laboratory mutants selected by quinolones,
By comparing posttherapy isolates resistant to Bacterial strains and culture conditions.
Clinical strains
of P. aeruginosa were recovered from urine
(n = 3), transtracheal aspirate (n = 4), or stool (n = 1) specimens or from surgical
wounds (n = 3) from patients hospitalized at the
University Hospital of Besançon, Besançon, France, during a
6-month survey in 1996. The isolates were identified by conventional methods (2). The other P. aeruginosa strains
used in the study were PAO1 (a wild-type strain obtained from B. W. Holloway), 4098 (a mutant of PAO1 producing basal, noninducible
levels of AmpC Identification of susceptible-resistant bacterial pairs.
Repetitive isolates of P. aeruginosa showing different
levels of susceptibility to DNA methods and PCR.
The mexR-coding regions of
the P. aeruginosa isolates were amplified with primers
M1 (5'-GGTTTACTCGGCCAAACC-3') and M2
(5'-CTTCGAAAAGAATGTTC-3'), which anneal upstream of and
downstream of mexR, respectively. In some strains, an
additional set of primers (M3, 5'-CATAGCGTTGTCCTCATG-3'; M4, 5'-GTTCGTCGATAAGCTTCA-3') was used to amplify the
intergenic region between mexA and mexR that
contains the putative Complementation with the mexR gene.
The
mexR gene of PAO1 was cloned on a 4.2-kbp
KpnI-BamHI fragment derived from cosmid pOM1
(11) in vector pUC19, yielding plasmid pMXA13
(10a). A 2.1-kbp SalI DNA fragment containing mexR was subsequently recloned into the broad-host-range
vector pAK1900 which codes for amoxicillin-carbenicillin resistance
(26). One recombinant clone in which the mexR
gene was transcribed in the same orientation as the lac
promoter of the plasmid was named pAKR4. Strains of P. aeruginosa were transformed by electroporation (34)
with pAKR4 or pAK1900 (purified with the Qiagen plasmid kit), and the
transformants were selected on Mueller-Hinton agar containing
ticarcillin at concentrations equal to four times the MIC for the
resistant isolates.
Analysis of outer membrane components.
The outer membrane
proteins of the P. aeruginosa strains were extracted
and were separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel
electrophoresis (PAGE) (22). Protein bands were stained with
0.1% Coomassie blue solution or were transferred as such onto
nitrocellulose membranes prior to immunoblotting (see below). Isolation
of lipopolysaccharides (LPSs) from bacterial envelopes was carried out
by following the method of Hitchcock and Brown (9). The LPS
preparations were separated on standard SDS-15% (wt/vol)
polyacrylamide gels and were visualized by silver staining as reported
by Tsai and Frasch (37).
Production of an anti-OprM polyclonal antiserum.
To
facilitate the purification of OprM, the oprM gene
previously cloned on the recombinant plasmid pOMC1 (11) was
recloned on a 1.5-kbp SphI-NruI DNA fragment into
the expression vector pMMB207, which codes for chloramphenicol
resistance (23). The resultant construct, named pOMI5, was
then transferred from E. coli INF Western blotting (immunoblotting).
Ten-microgram fractions
of outer membrane proteins were separated by SDS-PAGE and transferred
electrophoretically to nitrocellulose filters. These filters were
subsequently blocked with 3% (wt/vol) gelatin and hybridized for
1 h with the whole anti-OprM serum diluted 1:5,000 in
phosphate-buffered saline. Development of membranes was carried out
with alkaline phosphatase conjugated to an anti-rabbit secondary
antibody by using the AP color reagent kit from Bio-Rad. The
specificity of the anti-OprM antibody was established as a result of
the following immunoblotting results: detection of a protein band of
ca. 50 kDa in the outer membrane extracts of strains PAO1, 4098, and
4098E (the band was overexpressed in the last strain) and loss of the
band in strains 4098ET, ERYRT, and PT75 (data not shown). These data
demonstrated the absence of cross-reactivity of the anti-OprM antibody
with OprJ and OprN, the outer membrane proteins of the multidrug efflux
systems MexC-MexD-OprJ and MexE-MexF-OprN, respectively.
Susceptibility tests.
The MICs were determined by the
standard serial twofold dilution technique in agar (2) with
a Steers replicator that inoculated 104 CFU per spot. The
following antibiotic powders were kindly provided by the indicated
companies: ceftazidime and trimethoprim, Glaxo Wellcome; aztreonam and
cefepime, Bristol-Myers Squibb; ticarcillin and cloxacillin, SmithKline
Beecham; piperacillin, Wyeth Lederle; imipenem, Merck Sharp & Dohme;
meropenem, Zeneca Pharma; ciprofloxacin, Bayer Pharma; tetracycline,
Roussel Diamant; and sulfadiazine, Doms Chibret. Nalidixic acid and
chloramphenicol were obtained from Sigma Chemical Co. Routine
determination of bacterial susceptibilities to Detection of Emergence of multidrug-resistant mutants in vivo.
During a
period of 6 months in 1996, 835 consecutive isolates of P. aeruginosa were collected from patients hospitalized at the
University Hospital of Besançon. A survey of the susceptibility patterns of the repetitive isolates allowed us to identify 21 pairs of
strains susceptible and then resistant to at least one of the eight
antipseudomonal
0066-4804/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
In Vivo Emergence of Multidrug-Resistant Mutants of
Pseudomonas aeruginosa Overexpressing the Active Efflux
System MexA-MexB-OprM
ABSTRACT
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
-lactam antibiotics were isolated
from hospitalized patients. In vivo emergence of -lactam
resistance was associated with the overexpression of AmpC -lactamase
in 10 patients. In the other 11 patients, the posttherapy isolates
produced only low, basal levels of -lactamase and had increased
levels of resistance to a variety of non--lactam antibiotics (e.g.,
quinolones, tetracyclines, and trimethoprim) compared with the
levels of -lactamase production and resistance of their pretherapy
counterparts. These data suggested the involvement of the
MexA-MexB-OprM active efflux system in the multidrug resistance
phenotype of the posttherapy strains. Immunoblotting of the outer
membrane proteins of these 11 bacterial pairs with a specific
polyclonal antibody raised against OprM demonstrated the overexpression
of OprM in all the posttherapy isolates. To determine whether mutations
in mexR, the regulator gene of the
mexA-mexB-oprM efflux operon, could account for the overproduction of the efflux system, sequencing experiments were carried out with the 11 bacterial pairs. Eight posttherapy isolates were found to contain insertions or deletions that led to frameshifts in the coding sequences of mexR. Two resistant strains had
point mutations in mexR that yielded single amino acid
changes in the protein MexR, while another strain did not show any
mutation in mexR or in the promoter region upstream of
mexR. Introduction of a plasmid-encoded wild-type
mexR gene into five posttherapy isolates partially
restored the susceptibility of the bacteria to selected antibiotics.
These results indicate that in the course of antimicrobial therapy
multidrug-resistant active efflux mutants overexpressing the
MexA-MexB-OprM system may emerge as a result of mutations in the
mexR gene.
INTRODUCTION
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
-lactams, -lactamase inhibitors,
quinolones, tetracyclines, trimethoprim, chloramphenicol, macrolides,
and novobiocin (11, 16, 17, 19, 25, 28). When
overproduced, the efflux system allows bacterial cells to reach
higher levels of resistance to the substrate antibiotics listed above.
In the nalB multidrug-resistant mutant OCR1, overexpression of mexA-mexB-oprM has been found to be associated with a
point mutation in mexR, the regulator gene of the efflux
operon, leading to a predicted substitution of Trp for Arg at position
69 in the encoded peptide, MexR (26). Such an amino acid
change has been proposed to alter the function of MexR in vivo. In
addition, inactivation of mexR in several knockout mutants
was found to result in overexpression of mexA-mexB-oprM and
elevated multidrug resistance, although to a lower extent than in the
nalB strain OCR1 (26). Altogether these
results highlighted the key role of MexR in controlling the expression
of the efflux system but also raised questions about the presence of a
second mutation in the nalB mutant OCR1.
-lactams, or tetracyclines (13, 14, 19, 22, 35). The
relevance of the efflux mechanism in clinical strains showing a
multidrug resistance phenotype, however, has not been clearly
established, mainly because of the lack of an accurate method for
measuring the efflux rates of antibiotics in intact bacteria. Some data strongly suggest that at least a part of the strains commonly designated "intrinsically resistant" to carbenicillin (because they
produce only low, basal amounts of -lactamase) may consist of
MexA-MexB-OprM efflux mutants (15, 19). These strains, which
represent up to 69 to 74% of the -lactam-resistant isolates found
in British hospitals (3, 38), show typical cross-resistance to a variety of structurally unrelated antimicrobial agents
(18).
-lactams to their
pretherapy susceptible counterparts, we establish here the role of the
MexA-MexB-OprM pump system as a multiple-drug resistance mechanism in
clinical strains. Furthermore, we demonstrate that the emergence of
resistance associated with MexA-MexB-OprM in vivo is mostly due to
mutations affecting the regulator gene mexR.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
-lactamase) (15), 4098E (a single
step-mutant of 4098 overexpressing the MexA-MexB-OprM efflux system)
(16), 4098ET (an oprM::
-Hg transductant of 4098E) (22), PT75 (a mutant of 4098ET
overproducing the MexE-MexF-OprN efflux system) (12), and
ERYRT (a mutant of oprM::-Hg PAO1
overexpressing the MexC-MexD-OprJ pump system) (22).
Escherichia coli INF
was used as a host for DNA cloning and plasmid preparation. All bacterial strains were routinely cultured
at 37°C on Mueller-Hinton agar medium (Becton Dickinson) or grown in
brain heart infusion broth (Difco Laboratories), unless otherwise stated.
-lactams (designated pre- and
posttherapy strains) were first typed by O agglutination, according to
the Habs classification scheme, with specific antisera provided by Sanofi Diagnostics Pasteur, Marnes La Coquette, France. Bacterial pairs
were further characterized by analyzing the restriction banding
patterns obtained by pulsed-field gel electrophoresis (PFGE) (CHEF
apparatus; Bio-Rad Laboratories) of chromosomal DNA cleaved with the
endonuclease DraI (Boehringer) (7). According to
commonly accepted guidelines, two isolates are considered clonally related if their DNA fingerprints differ by less than three bands (36). Banding patterns were found to be strictly identical
between the pre- and posttherapy isolates except for isolates 12 and
14, which differed by two bands.
10 and 35 promoter sequences (26).
Approximately 100 ng of chromosomal DNA served as a template for PCRs.
The PCR mixtures contained 1 µM each primer, each
deoxynucleoside triphosphate at a concentration of 250 µM in 1× PCR
buffer, and 1.25 U of Taq polymerase (Perkin-Elmer) in a
final volume of 150 µl. The reaction mixtures were subjected to a 6 min of initial denaturation at 92°C, followed by 35 cycles of 1 min
at 92°C, 1 min at 50°C, and 1 min at 72°C and a 10-min final
extension at 72°C. PCR products were run on 0.7% agarose gels,
purified with the Wizard PCR Preps kit (Promega), and sequenced with an
ABI 373A automatic sequencer (Perkin-Elmer Division, Applied Biosystems) at the Institut d'Etude et de Transfert de Gènes of
Besançon. Both strands were sequenced, and the reactions were repeated at least once to confirm the sequencing data. Nucleotide and
deduced amino acid sequences were analyzed with the BLAST software
package (1).
to P. aeruginosa 4098ET by triparental mating by using E. coli HB101 (pRK2013) for plasmid mobilization (4).
Overnight cultures of 4098ET(pOMI5) diluted 1:100 in Luria-Bertani
broth containing 100 µg of chloramphenicol ml
1 and
1 mM (final concentration)
isopropyl--D-galactopyranoside were incubated at 37°C
up to an A650 of 0.6 to 0.7. The cell envelopes were then extracted and subjected to SDS-PAGE (22). The
electrophoretic bands corresponding to OprM (ca. 50 µg of protein per
gel) were excised thoroughly and were used to raise specific polyclonal antibodies in a rabbit (Eurogentec).
-lactam antibiotics
was performed by the agar diffusion method (33) with disks
from Sanofi Diagnostics Pasteur. Posttherapy isolates selected for
further investigation were resistant to one or more of the following
drugs at the indicated concentrations: ticarcillin, 16 µg
ml1; ticarcillin with clavulanic acid, 16 and 2.13 µg
ml1, respectively; piperacillin, 16 µg
ml1; piperacillin with tazobactam, 16 and 2.13 µg
ml1, respectively; cefsulodin, 8 µg ml1;
ceftazidime, 4 µg ml1; cefepime, 4 µg
ml1; and aztreonam, 4 µg ml1.
-lactamases in the clinical strains.
Pre-
and posttherapy isolates of P. aeruginosa were first
screened for -lactamase production by the nitrocefin (BBL,
Cockeysville, Md.) method performed with EDTA-treated cells reported by
Williams et al. (38). The -lactamase contents of strains
yielding a negative or a very weak positive reaction by this method
were subsequently reexamined by isoelectric focusing on pH 3.5 to 9.5 Ampholine PAGplate gels (Pharmacia Biotech) with crude cell lysates (21). Bands of -lactamases were detected by overlaying
the gels with a 0.05% (wt/vol) solution of nitrocefin in 0.1 M
phosphate buffer (pH 7.0).
RESULTS
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
-lactams to which susceptibility is routinely tested
by the disk diffusion method (listed in Materials and Methods). The
identity of the pre- and posttherapy isolates of each pair was first
established by O serotyping and was then confirmed by comparing the
restriction banding patterns obtained by PFGE after cleavage of
total DNA with endonuclease DraI (data not shown).
Analysis of the -lactamase contents of the clonally related bacteria
(i) by isoelectric focusing with cell lysates and (ii) by a
nitrocefin test performed with EDTA-treated cells (38)
demonstrated that 10 of the 21 -lactam-resistant strains overproduced the chromosomally encoded AmpC -lactamase. The other 11 resistant strains, like their susceptible counterparts, produced only
barely detectable amounts of -lactamase. Table
1 shows that these minimal
-lactamase producers not only were more resistant to -lactams
than the respective pretherapy isolates (two- to eightfold
increased MICs) but were also more resistant to a variety of
structurally unrelated antibiotics. Interestingly, the mutants displayed higher levels of resistance to meropenem but not to imipenem
compared with the susceptible pretherapy strains (except isolate 283, which was more resistant to imipenem than strain 284). Altogether,
these data suggested that the posttherapy mutants might overexpress the
MexA-MexB-OprM active efflux pump system.
TABLE 1.
Relative antibiotic susceptibilities of pre- and
posttherapy strains of P. aeruginosa
Overexpression of OprM.
The involvement of the MexA-MexB-OprM
system in the resistance phenotype of the minimal
-lactamase-producing strains was studied by comparing the amounts of
OprM, the outer membrane component of the system, in the
susceptible-resistant clonally related bacteria. Outer membrane
proteins were separated by SDS-PAGE, transferred to nitrocellulose
films, and hybridized with a polyclonal rabbit antibody raised against
OprM. The Western blots unambiguously showed that all the posttherapy
isolates contained greater amounts of OprM in their outer membranes
than their respective pretherapy counterparts (Fig.
1). In an attempt to see if some other
alterations were present in the resistant mutants, we also examined the
electrophoretic profiles of outer membrane proteins stained with
Coomassie blue and the banding patterns of the LPSs for every bacterial
pair. However, no marked difference was noticed between the pre- and posttherapy strains except for the expression of the ca. 50-kDa protein
band corresponding to OprM (as identified by Western blotting).
|
Mutations in mexR.
MexR, the product of the regulator
gene mexR, has been reported to negatively control the
transcription of the mexA-mexB-oprM operon in reference
strain PAO1 (26). We carried out sequencing experiments to
determine whether overexpression of the MexA-MexB-OprM pump system in
the posttherapy resistant strains was associated with mutations in
mexR. A 480-bp DNA region encompassing the mexR gene was amplified by PCR as described in Materials and Methods. Direct
sequencing of the PCR products from the 11 susceptible strains showed
that 6 of them had a mexR sequence strictly identical to
that published by Poole et al. (26), while a Val
Glu
substitution at codon 126 was present in the remaining five strains due
to a TA transversion at position 377 (starting with position 1 at the A of the start codon of mexR). Comparison of the
sequences of post- and pretherapy-derived amplicons revealed the
presence of deletions (6 of 11 strains) or insertions (2 of 11 strains) of one or more nucleotides in the mexR gene of the resistant
isolates, resulting in the production of altered peptides (Table
2). While the deletions were confined to
two distinct regions of mexR (around nucleotide 47 and
between positions 366 to 388), the two insertions occurred in a small
region between nucleotides 65 to 70. Two posttherapy strains were
found to possess base pair substitutions in mexR leading to
single amino acid changes in the encoded protein. These mutations
(Asp8Glu and Ala66Val) were different from the Arg69Trp substitution reported for nalB strain OCR1 (26).
In contrast, no mutation was detected for strain 92 either in
mexR or in the 273-bp intergenic promoter region extending
between mexR and mexA (data not shown).
|
Complementation of mutants.
To further investigate whether
the mutations in mexR were solely responsible for the
observed multidrug resistance phenotype, plasmid pAKR4 carrying the
mexR gene on the broad-host-range vector pAK1900 was
constructed. Attempts at introducing these two plasmids into the
resistant strains by electroporation were successful for only 5 of the
11 posttherapy strains. The effect of the mexR gene in the
posttherapy strains was determined by measuring the MICs of aztreonam,
meropenem, nalidixic acid, chloramphenicol, and trimethoprim and was
expressed as ratios of the MICs for bacterial cells harboring pAK1900
and the MICs for those transformed with the mexR carrying
plasmid pAKR4 (Table 3). The presence of
plasmid pAKR4 decreased the MICs (ratios of 4 to 8) of all the
antibiotics listed above for isolate 128, a result that indicates that
the intact mexR gene successfully complemented the defect of
that strain. The introduction of the cloned gene into the other
posttherapy mutants was followed by a substantial decrease in the
levels of resistance to only some of the tested drugs (see data for
strains 109, 283, and 17 in Table 3). As expected, isolate 92 (for
which no mutation was found in mexR), when transformed with
pAKR4, showed unchanged susceptibilities to aztreonam and meropenem,
two substrates of the MexA-MexB-OprM efflux system (11, 15,
19), but became more susceptible to nalidixic acid (fourfold).
The levels of resistance of the reference strain PAO1 were not notably
affected by the presence of pAKR4.
|
Antibiotic treatments received by the patients.
The emergence
of bacterial resistance in vivo is known to depend on multiple and
rather complex factors (5). In an attempt to better
understand the therapeutic circumstances that may lead to the emergence
of MexA-MexB-OprM efflux mutants, we retrospectively examined the
antibiotic treatments given to the patients from whom pre- and
posttherapy bacterial pairs had been isolated. It was found that most
patients (9 of 11) had received combination therapies involving either
-lactams and fluoroquinolones (5 patients), -lactams and
aminoglycosides (3 patients), or aminoglycosides and fluoroquinolones
(1 patient), while 2 patients had been treated by monotherapy with
ticarcillin and clavulanic acid, respectively. Interestingly, in four
of four patients receiving aminoglycosides, the resistant mutants were
isolated once the aminoglycosides were no longer administered (to
avoid nephrotoxicity) during a period of single-drug therapy with a
-lactam or a fluoroquinolone. With the notable exception of three
antibiotic regimens containing amoxicillin (in combination with a
fluoroquinolone) given empirically, all other treatments included drugs
known for their antipseudomonal activities (data not shown).
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DISCUSSION |
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Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
|
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The importance of antibiotic efflux in multidrug-resistant strains of P. aeruginosa is becoming more and more recognized in the hospital setting. Previous reports have shown that, in particular, nfxB- and nfxC-type mutants, which overexpress the MexC-MexD-OprJ and MexE-MexF-OprN efflux systems, respectively, occur in clinical isolates of various origins (6, 10, 39).
In the present report, we document for the first time that
nalB-type mutants, which overexpress the
mexA-mexB-oprM efflux operon, have been selected in vivo
during the course of antibiotic therapy. Several studies have pointed
out the fact that many clinical isolates of P. aeruginosa resistant to carbenicillin actually produce only low,
basal amounts of -lactamase (3, 38). Furthermore, the
development of such resistant strains in patients receiving -lactam
antibiotics has repeatedly been reported since the early days of
antipseudomonal chemotherapy (27, 31, 32). These so-called
intrinsically resistant or -lactamase-negative strains often show
cross-resistance to a variety of unrelated drugs such as tetracyclines,
chloramphenicol, and fluoroquinolones (18), a multidrug
resistance phenotype similar or close to that of
MexA-MexB-OprM-overproducing mutants selected in vitro. Although this
similarity tends to suggest that most clinical isolates intrinsically
resistant to carbenicillin overproduce the MexA-MexB-OprM pump system,
the relevance of the efflux mechanism in the resistance of clinical
strains has rarely been demonstrated (15), mainly because of
the lack of an experimental approach for accurate measurement of the
rates of export of antibiotics in bacterial cells. In this study, all
11 resistant isolates that emerged under therapy turned out to be
MexA-MexB-OprM efflux mutants, on the basis of differences in OprM
expression compared with that of their susceptible pretherapy
counterparts. This shows that such efflux mutants have a clinical
significance and that their possible emergence under antimicrobial
therapy must be taken into account in the management of patients
infected with P. aeruginosa.
Most of MexA-MexB-OprM mutants obtained in vitro have been selected by
fluoroquinolones, which have been used alone (28, 30) or in
combination with -lactams (14, 19, 20). Clearly, the
small number of patients in this study does not allow us to draw
general conclusions about therapeutic factors that may affect the
emergence of such efflux mutants in vivo. However, it is interesting that nearly all mutants developed under combination therapy. The role
of aminoglycosides (which are not substrates for the MexA-MexB-OprM pump system) (8, 20, 22) in preventing the emergence of MexA-MexB-OprM mutants is unclear. Four of our patients did receive an
aminoglycoside combined with a -lactam or a fluoroquinolone, but the
emergence of MexA-MexB-OprM-associated resistance occurred after the
completion of aminoglycoside therapy. Because of its important clinical
implications, the latter point warrants further assessment.
The MexR protein is the transcriptional repressor of the
mexA-mexB-oprM operon (26). Knockout mutants of
mexR show a modest twofold increase in mexA
transcription with a concomitant two- to fourfold decrease in
antibiotic susceptibility. The only mutation so far reported to lead to
overexpression of the efflux system is a single amino acid change at
position 70 of MexR (position 69 in reference 26) in
the nalB-type mutant strain OCR1. In the present study,
alterations of the mexR gene were found in 10 of the 11 posttherapy isolates. Mutants with frameshift mutations in the
mexR gene were predominant, while only two strains (strains 128 and 12) presented amino acid substitutions. Comparison of the
amounts of OprM on Western blots did not show any significant difference in amounts between mutants with frameshift mutations and the two mutants with amino acid substitutions.
Similarly, MIC ratios (Table 1), which were rather homogeneous for
-lactams but which varied quite strongly for chloramphenicol and
trimethoprim (2- to 
16-fold), did not enable us to establish a
correlation between the type of mexR mutation and the levels
of mexA-mexB-oprM expression. This suggests that the point
mutations seen in isolates 128 and 12 do not result in mutants with a
gain of function, as in strain OCR1.
Complementation with the cloned wild-type mexR gene significantly decreased the MICs of all antibiotics tested for strain 128 but reduced the MICs of only some of them for the other mexR mutants, mutants 17, 109, and 283. It was noted that for all the other posttherapy isolates, resistance levels decreased consistently only for nalidixic acid, even for strain 92, which did not show any mutation in mexR. One explanation for this puzzling observation might be that the presence of an additional mutation(s) outside of the mexR gene could interfere with the regulation of the mexA-mexB-oprM operon or affect other efflux pumps or antibiotic resistance mechanisms. In support of this, it has been reported that clinical strains of P. aeruginosa resistant to quinolones may exhibit alterations in DNA gyrase along with LPS defects (29) or decreased levels of drug accumulation (39). Alternatively, it is possible that some of the resistant mutants might require adjusted expression of the intact mexR gene to recover a wild-type phenotype. Further investigation is needed to better understand the regulation of the mexA-mexB-oprM operon and the emergence of multidrug resistance in P. aeruginosa.
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ACKNOWLEDGMENTS |
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We thank C. Godard and C. Bailly for technical assistance.
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FOOTNOTES |
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* Corresponding author. Mailing address: Laboratoire de Bactériologie, Hôpital Jean Minjoz, F-25030 Besançon, France. Phone: 33-381668286. Fax: 33-381668914. E-mail: patrick.plesiat{at}ufc-chu.univ-fcomte.fr.
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Abstract
Introduction
Materials and methods
Results
Discussion
References
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