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Antimicrobial Agents and Chemotherapy, May 1999, p. 1206-1210, Vol. 43, No. 5
Department of Microbiology, UMDS, St.
Thomas' Hospital, London, United Kingdom
Received 22 July 1998/Returned for modification 12 November
1998/Accepted 10 March 1999
Three cefoxitin-resistant Escherichia coli isolates
from stool specimens of a patient with leukemia were either resistant, intermediate, or sensitive to imipenem. Conjugation experiments showed that cefoxitin resistance, but not imipenem resistance, was transferable. All isolates were shown by isoelectric focusing to
produce two In the laboratory,
imipenem-resistant Escherichia coli K-12 strains
have been constructed by the introduction of the
carbapenem-hydrolyzing enzyme from Aeromonas
hydrophila, CphA, into porin-deficient mutants (7). However, there has been only one description of a
carbapenem-resistant E. coli strain isolated from a
clinical specimen, and the mechanism of resistance was not determined
(6). Carbapenem resistance among other members of the family
Enterobacteriaceae has previously been found associated with
either expression of a carbapenem-hydrolyzing enzyme (18, 20, 22,
28), a penicillin-binding protein alteration (17), or
high-level expression of a chromosomally encoded class C
In this study, we report the mechanisms responsible for
intermediate-level and high-level resistance to carbapenems in clinical isolates of E. coli. We show that in the presence of an
outer membrane protein deficiency, the level of expression of a
plasmid-mediated class C Bacterial strains and plasmids.
Three cefoxitin-resistant
E. coli isolates from stool specimens from a patient
with leukemia at St. Thomas' Hospital were studied. The isolates,
which were collected within a few days of each other, were identified
as E. coli with the API-20E identification system
(Analytab Products, Montalier Vercier, France) and by amplification and
sequencing of the nadC-ampC region of the E. coli chromosome.
0066-4804/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Carbapenem Resistance in Escherichia
coli Associated with Plasmid-Determined CMY-4
-Lactamase
Production and Loss of an Outer Membrane Protein
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
-lactamases with isoelectric points of 5.4 (TEM-1, confirmed by sequencing of a PCR product) and >8.5
(consistent with a class C -lactamase). The gene
coding for the unknown -lactamase was cloned and
sequenced and revealed an enzyme which had 99.9% sequence identity
with the plasmid-determined class C -lactamase CMY-2.
The cloned -lactamase gene differed from
blaCMY-2 at one nucleotide position that
resulted in an amino acid change, tryptophan to arginine
at position 221. We propose that this enzyme be designated CMY-4. Both
the imipenem-resistant and -intermediate isolates lacked a
38-kDa outer membrane protein (OMP) that was present in the
imipenem-sensitive isolate. The lack of an OMP alone did not
explain the difference in carbapenem susceptibilities
observed. However, measurement of -lactamase activities
(including measurements under conditions where TEM-1
-lactamase was inhibited) indicated that the
imipenem-intermediate isolate expressed six- to eightfold less
-lactamase than did the other isolates. This study
illustrates that carbapenem resistance in E. coli can
arise from high-level expression of plasmid-mediated class C
-lactamase combined with an OMP deficiency. Furthermore,
in the presence of an OMP deficiency, the level of expression of
a plasmid-mediated class C -lactamase is an
important factor in determining whether E. coli
isolates are fully resistant to carbapenems.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
-lactamase combined with reduced outer membrane permeability (11, 15, 21). Recently, there have been
described carbapenem-resistant Klebsiella pneumoniae
isolates where resistance was attributed to a lack of an outer membrane
protein in combination with the expression of the plasmid-mediated
class C -lactamase ACT-1 (4).
-lactamase is an important
factor in determining whether E. coli isolates are
fully resistant to carbapenems.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
-lactamase synthesis (24), and E. coli TB1(pTZ18U), carrying the
blaTEM-1 gene, were used as controls in
-lactamase assays. Plasmid vector pUC18, used in DNA
cloning, was obtained from Pharmacia Biotech Ltd. (St. Albans, United Kingdom).
Determination of MICs. MICs were determined by agar dilution on diagnostic sensitivity test agar (CM261; Oxoid, Basingstoke, United Kingdom) with an inoculum of about 104 organisms per spot as described previously (25). E. coli NCTC 10418 was used as the control strain.
Antibiotics and reagents. The following companies kindly supplied antibiotic powders of known potency: Bristol-Myers Company (cefepime), Cyanamid (piperacillin), Eli Lilly Company Ltd. (moxalactam), E. R. Squibb & Sons (aztreonam), Glaxo Group Research Ltd. (ceftazidime), Merck Sharp & Dohme (cefoxitin and imipenem), Roussel Laboratories Ltd. (cefotaxime), SmithKline Beecham (ampicillin and clavulanic acid), and Zeneca (meropenem). Restriction endonucleases and T4 DNA ligase were purchased from New England Biolabs (Hitchin, United Kingdom).
Isoelectric focusing.
Cells were harvested from 20-h brain
heart infusion (BHI) broth cultures by centrifugation and resuspended
in 0.5 ml of phosphate buffer (0.1 M, pH 7), and the
-lactamase was released by sonication. Sonication was
performed for 20 s with a W-385 sonicator (Heat Systems-Ultrasonics, Inc.) with the following settings: 5-s cycle time,
50% duty cycle, 1.5 output control setting. Enzymes were identified by
isoelectric focusing in agarose-IEF (Pharmacia Biotech) gels containing
Pharmalyte (pH range, 3 to 10; Pharmacia Biotech) and subsequent
staining with nitrocefin (100 µg/ml). Preparations of strains known
to produce TEM-1, OXA-1, SHV-1, or SHV-5 -lactamase were
used as standards.
-Lactamase studies.
Each strain was grown overnight at
37°C in BHI broth, with shaking (200 rpm). A 1:20 dilution was
performed in fresh prewarmed BHI broth, and the culture was shaken for
a further 2 h. Cells were harvested by centrifugation and
resuspended in 0.5 ml of sterile distilled water, and the
-lactamase was released by sonication (as described
above). -Lactamase activity was measured by monitoring the rate of
hydrolysis of nitrocefin (20 µM) at 482 nm in a Biochrom 4060 spectrophotometer (Pharmacia Biotech). Assays were performed in both
the presence and the absence of clavulanate at 37°C in 0.1 M
phosphate buffer (pH 7.0). Inhibition studies were performed on samples
that had been preincubated for 10 min at 37°C in the presence of
clavulanate (1 µM) before -lactamase measurement. Specific -lactamase activities are expressed as
nanomoles of nitrocefin hydrolyzed per minute standardized against the
amount of protein in the sample.
Plasmid studies. Conjugation was performed with overnight cultures of the clinical isolate and E. coli J62.1 which were mixed in a ratio of 1:2 (donor to recipient) in BHI broth and grown for a further 16 h. Transconjugants were selected for by plating the mixture onto MacConkey agar containing rifampin (200 µg/ml) and cefotaxime (2 µg/ml). Plasmid extracts were prepared by the alkaline lysis procedure (23) unless otherwise stated. Plasmid DNA was introduced into competent XL-1 Blue by a heat shock procedure (23).
Nucleic acid techniques. Total DNA extractions were performed as described by Ausubel et al. (1). DNA cloning of the blaCMY-4 gene was achieved from a Sau3AI partial digest of total DNA ligated into the BamHI site of pUC18. E. coli XL-1 Blue was transformed with the recombinant DNA, and clones carrying the blaCMY-4 gene were selected by plating the transformants onto Luria-Bertani agar plates containing cefotaxime (2 µg/ml). DNA sequencing of both strands of the cloned insert was performed with a primer-walking strategy with fluorescein-labelled sequencing primers custom made by Pharmacia Biotech. Sequencing reactions were performed on QIAprep plasmid DNA preparations (Qiagen, Crawley, United Kingdom) with the reagents contained within a Thermo Sequenase cycle sequencing kit (Amersham International, Amersham, United Kingdom) according to the manufacturer's instructions. Template preparation and PCR amplification of the blaTEM gene were performed as described previously (26). Amplification of the blaCMY-4 gene was performed with an annealing temperature of 55°C and primers TR8A3U (5'-GATTCCTTGGACTCTTCAG-3') and TR8A3R (5'-TAAAACCAGGTTCCCAGATAGC-3') based on the sequence of blaCMY-4 deduced in this study. The blaCMY-4 PCR product was labelled with digoxigenin-11-dUTP by the incorporation of digoxigenin-11-dUTP in the PCR mixture according to the instructions of the manufacturer (Boehringer Mannheim UK, Lewes, England). Southern blotting and DNA hybridization were performed as described by Sambrook et al. (23). Amplification of the ampR gene was performed with an annealing temperature of 55°C and primers PSN006 (5'-ATGACGCGTAGCTATATCCCTCTT-3') and PSN007 (5'-TTATTTGTGCAGCACCCCGGT-3') based on the sequence of the ampR gene from C. freundii described by Lindquist et al. (13).
Outer membrane protein studies. Cell membranes of a 16-h culture were disrupted for 2 min with a W-385 sonicator (Heat Systems-Ultrasonics, Inc.) with the following settings: 5-s cycle time, 50% duty cycle, and 1.5 output control setting. Cell debris was removed by centrifugation at 6,000 × g for 15 min at 4°C, and the supernatant was subjected to ultracentrifugation at 50,000 × g for 45 min to collect the membranes. Cytoplasmic membrane proteins were differentially solubilized for 20 min at room temperature with 1.7% sodium lauryl-sarcosinate in 50 mM Tris, pH 7.6. The suspension was recentrifuged at 50,000 × g for 45 min at 4°C, and the pellet containing the outer membrane proteins was resuspended in 100 µl of sterile distilled water. The outer membrane proteins were separated on a vertical sodium dodecyl sulfate-containing polyacrylamide gel (acrylamide/bisacrylamide ratio of 3:0.27; 5% stacking gel with a 10% separating gel) and were visualized after staining with polyacrylamide gel electrophoresis Blue 83 (BDH, Lutterworth, United Kingdom). Molecular weights of the proteins were determined from a calibration curve derived from the migratory positions of protein standards of known molecular weights run on the same gel. The standard proteins used in this study had the molecular weights 94,000, 67,000, 43,000, 30,000, 20,100, and 14,400 and were provided in a calibration kit supplied by Pharmacia Biotech.
Nucleotide sequence accession number. The nucleotide sequence reported in this study can be obtained from EMBL with accession no. AJ007826.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Bacterial strains and plasmids. The three clinical isolates investigated in this study were cultured from stool specimens from a patient with leukemia at St. Thomas' Hospital. They were identified as E. coli by API-20E substrate profile analysis and shown to have identical XbaI-digested genomic DNA profiles by pulsed-field gel electrophoresis (data not shown). Plasmid profiles revealed the presence of five plasmids in the strains with the following approximate sizes: 3, 5.5, 7, 30, and 45 kb. All plasmids except the smallest one were transferred to the recipient strain E. coli J62.1 after mating with strain 69. Transconjugants of strains 68 and 79 were found to contain the 5.5-, 7-, and 45-kb plasmids.
When plasmid preparations were used to transform E. coli XL-1 Blue, all transformants were found to contain the 7-kb plasmid either alone (strains 68 and 69) or in combination with the 45-kb plasmid (strain 79).Antimicrobial susceptibilities.
Based on the MIC
interpretative standards of the National Committee for Clinical
Laboratory Standards (16), the three clinical isolates and
the transconjugants of strains 69 and 79 were resistant to ampicillin,
piperacillin, aztreonam, cefoxitin, ceftazidime, cefotaxime, and the
-lactam--lactamase inhibitor combinations (Table
1). Strain 68 exhibited a lower
level of resistance to these compounds than did the other strains
but was less susceptible to imipenem, meropenem, and moxalactam
than was strain 69 (Table 1). Strain 79 was significantly more
resistant to imipenem, meropenem, cefepime, and moxalactam than
were the other strains, but like strain 68, neither the transconjugant
nor the transformant of the strain was resistant to imipenem,
indicating that the carbapenem resistance was nontransferable in both
cases. Revertants of strain 79 (79-rev), which were susceptible to
imipenem, had a similar resistance phenotype to strain 69 and
displayed increased susceptibility to cefepime and moxalactam compared
to the parental strain.
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-Lactamases.
Isoelectric focusing revealed the presence of
two -lactamases in all three clinical isolates, one with
a pI of 5.4, consistent with TEM-1, and one with a pI of >8.5. PCR
amplification with primers specific for the
blaTEM gene confirmed the presence of this gene
in the isolates, and DNA sequencing of the PCR product established that
the encoded enzyme was TEM-1. Measurement of the total
-lactamase activities of the isolates revealed that strain 68 produced approximately six- to eightfold less
-lactamase than did the other strains (Table
2). The rate of nitrocefin hydrolysis by
the -lactamases from the strains was reduced by 25 to
40% in the presence of 1 µM clavulanate, indicating that one of the
enzymes was not inhibited by clavulanate, an observation consistent with the presence of a class C
-lactamase. Since 1 µM clavulanate was effective in
reducing the -lactamase activity of an E. coli strain hyperproducing TEM-1 -lactamase to 4%
of its activity in the absence of the inhibitor (Table 2), activities measured in the presence of clavulanate should give an indication of
the level of class C -lactamase expressed. When the
-lactamase activities of the isolates, measured in the
presence of clavulanate, were compared with those of a C. freundii strain (31A12) that constitutively expressed high
levels of class C -lactamase, we found that strain
68 produced 2.6-fold less -lactamase than did the
C. freundii strain. In contrast, the
-lactamase activities of strains 79 and 69 were 2.7- and
3.5-fold higher, respectively, than that of the C. freundii strain. None of the strains exhibited inducible
-lactamase expression in the presence of
imipenem (data not shown).
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Sequencing of the cloned -lactamase gene.
In
order to determine the identity of the unknown
-lactamase, a Sau3AI DNA fragment of
the genome of imipenem-resistant strain E. coli
79 was cloned into the BamHI site of pUC18. Sequence
determination of the insert by a primer-walking strategy revealed
the presence of an open reading frame of 1,146 bp in length. A
nucleotide sequence search showed that the open reading frame had
99.9% sequence identity with a plasmid-mediated class C
-lactamase, CMY-2, from K. pneumoniae (2). The -lactamase gene cloned in this study
differed from CMY-2 by one amino acid residue, tryptophan instead of
arginine at position 221 in the deduced amino acid sequence, the result of one nucleotide difference, T instead of A, in the gene coding sequence. We proposed that this enzyme be designated CMY-4. The ampR coding gene from C. freundii was not
found upstream from the blaCMY-4 gene, nor could
the ampR gene be amplified from the isolates. The lack of
the ampR gene thus accounts for the inability to induce
-lactamase expression in the isolates. Comparison of the
upstream region of blaCMY-4 with the
corresponding region of the chromosomal -lactamase of
C. freundii OS60 (13) revealed that the
homology ended 14 bases downstream from the ampR initiation codon. Downstream from blaCMY-4 was part of the
sequence (119 nucleotides) coding for a C. freundii
outer membrane lipoprotein (3).
Location of the blaCMY-4 gene. PCR amplification of the blaCMY-4 gene followed by direct DNA sequencing of the PCR product revealed that all the isolates had the blaCMY-4 gene. In order to determine the location of the blaCMY-4 gene, a digoxigenin-labelled blaCMY-4 DNA probe was prepared and hybridized to total DNA preparations from each of the strains and their transconjugants. The probe hybridized to give a strong signal with the 7-kb plasmid and a weak signal with the larger plasmid (data not shown). No cross-reaction of the blaCMY-4 probe with the ampC gene in a chromosomal DNA preparation of E. coli J62.1 or with the chromosomal DNA of the clinical isolates was observed.
Outer membrane profiles. The transconjugant and transformant from the imipenem-resistant isolate were not resistant to imipenem, suggesting that an additional mechanism was acting in concert with CMY-4 production. Examination of the outer membrane profiles of the original isolates revealed that the imipenem-resistant (strain 79) and imipenem-intermediate-resistant (strain 68) isolates lacked a 38-kDa protein (Fig. 1).
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The mechanism of carbapenem resistance in clinical isolates of
E. coli has not previously been reported. From studies
of derepressed mutants of C. freundii and
Enterobacter cloacae, it is known that high-level expression
of a class C -lactamase alone is not sufficient to
confer carbapenem resistance (14, 25). But resistance can arise if the high level of -lactamase expression is
combined with a decrease in outer membrane permeability (11, 15,
21). Consistent with these studies, the
imipenem-resistant E. coli isolate
(strain 79) in this investigation expressed high levels of a class C
-lactamase, CMY-4, and lacked an outer membrane protein that was found in an imipenem-susceptible isolate.
However, unlike with derepressed C. freundii and
E. cloacae strains the high level of
-lactamase expression in the E. coli
isolates was plasmid determined and not due to overexpression of the
chromosomal -lactamase. The mechanism was thus similar
to that recently reported to explain imipenem resistance in
K. pneumoniae isolates where resistance was associated with
high-level expression of a plasmid-determined class C
-lactamase, ACT-1, in combination with the loss of a 42-kDa outer membrane protein (4).
The importance of the level of class C -lactamase
expression in conferring carbapenem resistance was illustrated by a
comparison of strain 68 with strain 79. Both strains lacked a 38-kDa
outer membrane protein and expressed the CMY-4
-lactamase, so that one might have expected both strains
to be resistant to carbapenems. However, strain 68 was not resistant to
imipenem and was more susceptible to cefoxitin,
extended-spectrum cephalosporins, and the penicillins tested than were
the other strains. Mainardi et al. (15) have proposed
residual expression of an outer membrane protein to explain
intermediate-level carbapenem resistance in a C. freundii strain, but no trace of the missing 38-kDa protein could
be detected in this case. Instead, measurement of the total -lactamase activities of the strains revealed that
strain 68 expressed six- to eightfold less -lactamase
than did the other strains. Since the MIC of cefoxitin for the
transconjugant of strain 68 was lower than that for the other
transconjugants, it is likely that this strain expressed a smaller
amount of the CMY-4 -lactamase. This would explain both
the lower level of resistance to extended-spectrum cephalosporins and
moxalactam observed and the increased susceptibility to carbapenems
exhibited by strain 68 compared to the imipenem-resistant isolate.
The reason why strain 68 produced less -lactamase than
did the other isolates is not known. From isoelectric
focusing, PCR, and DNA sequencing, it is known that the three
isolates in this study produced two -lactamases, TEM-1
and CMY-4, in addition to the E. coli chromosomal
-lactamase. DNA hybridization studies revealed that the
gene coding for the CMY-4 -lactamase was located on two different plasmids that had approximate sizes of 7 and 45 kb.
Since MICs of cephalosporin for the transconjugant of strain 68 were
lower than those for the other transconjugants, we infer that host
factors were not responsible for the variation in
-lactamase expression observed. The MICs of -lactams
for E. coli XL-1 Blue carrying the 7-kb plasmid from
either the imipenem-sensitive strain or strain 68 were
identical, suggesting that this plasmid was not responsible for the
observed difference in the levels of -lactamase expressed. Therefore, by process of elimination, the presence of the
45-kb plasmid was the most likely cause of the phenotype observed.
Whether this was due to lower CMY-4 -lactamase
production from the 45-kb plasmid or to the expression of a
regulatory factor that led to differential expression of the
blaCMY-4 gene on the 7-kb plasmid has yet to be determined.
Based on the deduced amino acid sequence, the class C
-lactamase reported in this study differed from the
plasmid-determined -lactamase, CMY-2, by one residue, an
arginine instead of tryptophan at position 221. This position is
located far from the active site in the crystal structure of the
C. freundii AmpC -lactamase (19), so that we would expect little difference in catalytic activities between the two enzymes. Both enzymes belong to a small subset of -lactamases (BIL-1, CMY-2, CMY-3, LAT-1,
and LAT-2) thought to originate from C. freundii
(2, 5, 8-10, 27). Consistent with these findings was part
of the sequence coding for an outer membrane lipoprotein
(3), found next to the blaCMY-4 gene.
This outer membrane lipoprotein-encoding gene usually lies downstream
from ampC in C. freundii, so that this is
consistent with the blaCMY-4 gene being of
C. freundii chromosomal origin.
Transposable elements responsible for the transmission of
ampC-type genes have not been described to date, but
the occurrence of -lactamase genes on both plasmids
and the chromosomes of foreign bacterial hosts has led previous workers
to imply that they exist. Bradford et al. (4) have suggested
that the gene coding for ACT-1 may occur on a transposable element
together with two other genes coding for -lactamases
with pIs of 5.4 and 7.0. The blaCMY-4 gene
characterized in this study was found on two different plasmids, which
suggests that this gene may also form part of a transposable element.
However, since the blaTEM-1 gene was not found
on the 7-kb plasmid which harbored blaCMY-4 it
is less likely that blaCMY-4 is associated with
the blaTEM-1 gene on the same transposable element.
Upstream from the ampC gene in C. freundii
usually lies the ampR gene, which codes for a
transcriptional regulatory protein involved in inducible
-lactamase expression (13). The
ampR gene was not present upstream from the
blaCMY-4 gene, and this therefore accounts for
the inability to induce CMY-4 -lactamase expression in
the E. coli isolates. Studies involving E. coli, which naturally lacks ampR, have shown that
introduction of ampC from C. freundii on a
plasmid into E. coli results in only a two- to
threefold increase in -lactamase expression over that
for E. coli carrying both ampC and
ampR (12). Despite this, the imipenem-sensitive and imipenem-resistant isolates in
this study expressed high levels of CMY-4 -lactamase and
were resistant to extended-spectrum cephalosporins, cefoxitin, and
combinations of clavulanate with either ampicillin or ceftazidime.
While one would normally associate this phenotype with derepressed
-lactamase expression, the lack of the ampR
gene makes this unlikely. Consequently, it is likely that gene and
plasmid copy number play an important role in giving rise to the high
level of -lactamase expression observed, but other
factors such as influence of insertion sequences or other regulatory
factors cannot be ruled out.
In summary, carbapenem resistance among members of the family
Enterobacteriacae is uncommon, and consequently there have
been few descriptions of carbapenem-resistant E. coli
strains in the literature. This study has investigated the mechanism
responsible for carbapenem resistance in clinical isolates of
E. coli and shown the resistance to be conferred by
high-level expression of a plasmid-mediated class C
-lactamase, in combination with the loss of an outer
membrane protein. We have also shown that in the presence of an outer
membrane protein deficiency, the level of expression of a
plasmid-mediated class C -lactamase, CMY-4, is an
important factor in determining the degree of resistance of
E. coli to carbapenems.
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ADDENDUM |
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The CMY-4 -lactamase has recently been identified
in a clinical Proteus mirabilis isolate (27a).
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ACKNOWLEDGMENTS |
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We thank Richard Anthony (St. Thomas' Hospital) for typing the isolates by pulsed-field gel electrophoresis.
This work was funded by a Project 804 grant from the Special Trustees of St. Thomas' Hospital.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Biology, Darwin Bldg., University College London, Gower St., London WC1E 6BT, United Kingdom. Phone: 44 171 504 2934. Fax: 44 171 380 7098. E-mail: p.stapleton{at}ucl.ac.uk.
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Antimicrobial Agents and Chemotherapy, May 1999, p. 1206-1210, Vol. 43, No. 5
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