Advance Access published May 29, 2003
Journal of Antimicrobial Chemotherapy
DOI: 10.1093/jac/dkg256
Effect of D240G substitution in a novel ESBL CTX-M-27
R. Bonnet1*, C. Recule2, R. Baraduc1, C. Chanal1, D. Sirot1, C. De Champs1 and J. Sirot1
1Laboratoire
de Bactériologie, Faculté de Médecine, Service de Bactériologie-Virologie, 28 Place Henri-Dunant,
63001 Clermont-Ferrand Cedex; 2Laboratoire de Bactériologie, CHU de Grenoble,
Chemin Maquis du Grésivaudan, 38 700 La Tronche, France
Escherichia coli clinical strain Gre-1 collected in 2000 from a French hospital harboured a novel CTX-Mencoding gene, designated blaCTX-M-27. CTX-M-27 differed from CTX-M-14 only by the substitution D240G and
was the third CTX-M enzyme harbouring this mutation after CTX-M-15 and CTX-M-16. The Gly-240-harbouring enzyme CTX-M-27 conferred to E. coli higher MICs of ceftazidime (MIC, 8 versus 1 mg/L) than did the Asp240-harbouring CTX-M-14 enzyme. Comparison of CTX-M-14 and CTX-M-27 showed that residue Gly-240
decreased Km for ceftazidime (205 versus 940 µM), but decreased hydrolytic activity against good substrates, such as cefotaxime (kcat, 113 versus 415 s–1), probably owing to the alteration of β3 strand positioning
during the catalytic process.
Keywords: CTX-M, β-lactamase, D240G mutation, ESBL
Introduction
The first extended-spectrum β-lactamase (ESBL) of the CTX-M type
(MEN-1/CTX-M-1) was reported at the beginning of the 1990s.1,2
Initially characterized in Europe, CTX-M-producing strains have
been observed over a wide geographic area including the Near and
Far East,3–8 South America,3,9–12 Africa13 and Europe.1,2,14–24 CTX-M
enzymes have been observed in different species of the Enterobacteriaceae family such as: Escherichia coli, Salmonella enterica serovar
Typhimurium, Klebsiella pneumoniae, Proteus mirabilis, Citrobacter freundii, Citrobacter amalonaticus, Enterobacter aerogenes
and Enterobacter cloacae; and in the species Vibrio cholerae El
Tor.12
The CTX-M enzymes form a rapidly growing family that comprises currently at least 34 enzymes of which 19 have been described
in the last 3 years. They are generally much more active against cefotaxime than against ceftazidime and aztreonam.1 The flexibility of
the β3 strand and Ω loop, and residues Asn-104, Ser-237, Asp-240
and Arg-276 are involved in the cefotaxime-hydrolyzing activity of
CTX-M enzymes.1,5,17–19,25,26
There have been recent reports of CTX-M mutants exhibiting an
increased enzymic activity against ceftazidime: the P167S mutant of
CTX-M-18 (also designated CTX-M-14), designated CTX-M-19,21
and D240G mutants of CTX-M-3 and CTX-M-9, designated CTXM-15 and CTX-M-16, respectively.8,10,14
In France, we isolated a CTX-M-producing strain that produced
a novel D240G variant designated CTX-M-27 and derived from
CTX-M-14 as well as CTX-M-19. The biochemical characterization
of the two β-lactamases CTX-M-14 and CTX-M-27 and molecular
modelling give insights into the role of the D240G substitution in
CTX-M enzymes.
Materials and methods
Clinical strain
Table 1 shows the strains and plasmids used in this study. Clinical strain
Gre-1 was isolated in 2000 from a patient hospitalized in Grenoble,
France. E. coli transformants producing CTX-M-1416 were used for
comparison.
Susceptibility to β-lactams
MICs were determined by a dilution method on Mueller–Hinton
agar (Sanofi Diagnostics Pasteur, Marnes la Coquette, France) with an
inoculum of 104 cfu per spot. Antibiotics were provided as powders by
SmithKline Beecham Pharmaceuticals, Nanterre, France (amoxicillin,
ticarcillin and clavulanate), Lederle Laboratories, Paris-La Défence,
France (piperacillin and tazobactam), Eli Lilly, Paris, France (cefalothin), Roussel-Uclaf, Paris-La Défence, France (cefotaxime, cefpirome),
Glaxo Wellcome, Marly-le-Roi, France (ceftazidime), and Sanofi
Winthrop, Gentilly, France (aztreonam).
Detection of ESBLs was carried out with the standard double disc
synergy tests as described previously.27 Antibiotic discs for agar tests
were obtained from Sanofi Diagnostics Pasteur.
..................................................................................................................................................................................................................................................................
*Corresponding author. Fax: +33-4-73-27-74-94; E-mail: Richard.Bonnet@u-clermont1.fr
...................................................................................................................................................................................................................................................................
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Received 27 January 2003; returned 14 February 2003; revised 13 March 2003; accepted 13 March 2003
R. Bonnet et al.
Table 1. Strains and plasmids used in the study
Relevant genotype and produced β-lactamase (pI)
Strain or plasmid
Strain
E. coli Gre-1
E. coli DH5α
Plasmid
pClGre-1
pClCF-1
pBK-CMV
Source
clinical strain producing TEM-1 (5.4) and CTX-M-27 (8.2)
SupE44 hsdS20 (rB–, mB–) recA13 ara-14 proA2 lacY1 galK2 rpsL20 xyl-5
mtl-1
this study
(28)
recombinant pBK-CMV plasmid which contains a 0.9-kb fragment encoding
CTX-M-27 (8.2)
recombinant pBK-CMV plasmid, which contains a 0.9-kb fragment encoding
CTX-M-14 (7.9)
plasmid vector
this study
Isoelectric focusing was carried out with polyacrylamide gels containing
ampholines with a pH range of 3.5–10 as previously described.10
β-Lactamases of known pIs were used as standards: TEM-1 (pI 5.4),
TEM-24 (pI 6.5), SHV-1 (pI 7.6) and SHV-5 (pI 8.2).
Amplification of CTX-M-encoding genes
The detection of CTX-M-encoding genes was carried out with the
primers CTX-MA (5′-CGCTTTG CGATGTGCAG-3′) and CTX-MB
(5′-ACCGCGATATCGTTGGT-3′) (temperature of annealing of 54°C).
They amplify a 550 bp internal fragment from positions 264–814
(blaCTX-M-1 numbering), which correspond to conserved regions of
blaCTX-M-type genes. The complete ORF of the blaCTX-M-27 gene was
amplified with the primers CTX-M-9A (5′-CTGATGTAACACGGATTGAC-3′) and CTX-M-9C (5′-AGCGCCCCATTATTGAGAG-3′)
(temperature of annealing of 54°C).
β-Lactamase gene cloning
Stratagene
were precipitated by addition of 0.2 M [7% (v/v)] spermine and centrifuged at 48 000g for 60 min at 4°C. The clarified supernatant was
dialysed overnight against MES–NaOH 20 mM (pH 5.5). The CTX-M
purification was carried out as previously described10 by ion-exchange
chromatography with an SP Sepharose column (Amersham Biosciences
Europe, Orsay, France) and gel-filtration chromatography with a
Superose 12 column (Amersham Pharmacia Biotech). The total protein
concentration was estimated by the Bio-Rad protein assay (Bio-Rad,
Richmond, CA, USA), with bovine serum albumin (Sigma Chemical
Co., St Louis, MO, USA) used as a standard.
The purity of CTX-M extracts was estimated as previously
described10 by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and staining with Coomassie Blue R-250 (Sigma
Chemical Co.). The renaturation of proteins and the detection of the
β-lactamase activity were carried out as previously described10 with
renaturation buffer Tris–HCl (100 mM)–Triton X-100 [2% (v/v); pH
7.0] and 0.5 mM nitrocefin (Oxoid, Paris, France) in 100 mM phosphate
buffer (pH 7.0), respectively.
Determination of β-lactamase kinetic constants
The recombinant DNA manipulations were carried out as described
by Sambrook et al.28 T4 DNA ligase was purchased from Boehringer
Mannheim, Germany. The CTX-M-encoding sequence was cloned
as follows: the complete ORF, which was amplified with proof-reading
Taq polymerase Tfu (Appligene Oncor, Illkirch, France) and primers
reported above, was ligated in the SmaI site of the phagemid pBK-CMV
(Stratagene, La Jolla, CA, USA). E. coli DH5α28 was transformed by
electroporation. The transformants harbouring the recombinant CTX-Mencoding plasmid were selected on Mueller–Hinton agar supplemented
with 2 mg of cefotaxime per L.
The steady-state kinetic parameters (Km and kcat) of the β-lactamases
were obtained by an improved computerized micro-acidimetric method
derived from the technique of Labia et al.30 using 702 SM Titrino pHstat
(Metrohm, Herisau, Swiss). They were determined by the analysis of the
complete hydrolysis time-courses and the kinetic progress curves were
fitted by non-linear least-squares regression. The concentrations of the
inhibitors (clavulanate and tazobactam) required to inhibit enzyme
activity by 50% (IC50s) were determined as previously described with
penicillin G.10 The specific activity and IC50s were monitored with penicillin G (200 mM) as the reporter substrate. The kinetic constants were
determined three times.
DNA sequencing
The sequence was determined by direct sequencing of PCR products,
carried out by the dideoxy chain termination procedure of Sanger
et al.29 on an ABI 1377 automatic sequencer using the ABI PRISM
Dye Terminator Cycle Sequencing Ready Reaction Kit with Ampli
Taq DNA polymerase FS (Perkin-Elmer/Applied Biosystems Division,
Foster City, CA, USA).
Sequence analysis
The nucleotide sequence and the deduced protein sequence were analysed with the software available over the Internet at the National Center
of Biotechnology (http://www.ncbi.nlm.nih.gov/). A hydrophobic blot
was obtained with the method of Nielsen et al.31 Multiple sequence alignment and pairwise comparisons of sequences were carried out with the
help of ClustalW 1.74 software.32
β-Lactamase preparation
The CTX-M-producing E. coli DH5α was grown in 6 L of brain heart
infusion broth containing cefotaxime at 2 mg/L for 18 h at 37°C. The
bacteria collected by centrifugation were suspended with MES–NaOH
20 mM (pH 5.5) and disrupted by ultrasonic treatment (4 × 30 s, each time
at 20 W). After centrifugation (10 000g for 10 min at 4°C), nucleic acids
Molecular modelling
Molecular modelling was carried out using Hyperchem v6.3 software
(Hypercube Inc., Gainesville, FL, USA) on the basis of the crystallographic structure of the Glu-166→Ala Toho-1 mutant25 by introducing
the mutations as part as an automated procedure. The residues, the
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Isoelectric focusing
(16)
D240G substitution in CTX-M-27
Table 2. MICs for clinical strain E. coli Gre-1 and the corresponding transformant E. coli DH5α
(pClGre-1) in comparison with CTX-M-14-producing transformant E. coli DH5α (pClCF-1)16
β-Lactam MICs (mg/L) for
β-Lactam
E. coli DH5α
(pClGre-1b)
CTX-M-27
E. coli DH5α
(pClCF-1c)
CTX-M-14
E. coli DH5α
(pBK-CMVd)
–
>2048
64
>2048
128
1024
4
>2048
1024
256
0.06
64
16
32
16
0.06
>2048
4
>2048
8
256
2
1024
512
16
0.06
4
1
8
8
0.06
>2048
8
>2048
16
256
2
512
512
16
0.06
2
0.50
4
1
0.06
2
2
2
2
2
2
4
<2
0.06
0.06
0.06
0.06
0.12
0.12
0.06
aClinical strain E. coli Gre-1 harbouring natural plasmid pGre-1, which encodes β-lactamases TEM-1 and CTXM-27.
bE. coli DH5α harbouring recombinant plasmid pClGre-1, which encodes β-lactamase CTX-M-27.
cE. coli DH5α harbouring recombinant plasmid pClCF-1, which encodes β-lactamase CTX-M-14.16
dE. coli DH5α harbouring plasmid vector pBK-CMV (Stratagene).
eCLA, clavulanate at fixed concentration of 2 mg/L.
fTZB, tazobactam at fixed concentration of 4 mg/L.
catalytic water molecule and the water molecules of the Toho-1 crystal
were minimized using the Amber96 parameters33 and a distancedependent dielectric constant by conjugate gradient energy minimization
until the r.m.s. gradient was <0.1 kcal/mol.
Nucleotide sequence accession number
The blaCTX-M-27 nucleotide sequence data appear in the GenBank
nucleotide sequence database under accession number AY156923.
Results and discussion
Characterization of the CTX-M-27-producing clinical isolate
E. coli clinical isolate Gre-1 exhibited resistance to broad-spectrum
cephalosporins (MIC of cefotaxime, 256 mg/L; MIC of ceftazidime,
16 mg/L; MIC of aztreonam, 32 mg/L) (Table 2) and a positive
double-disc synergy test. Isoelectric point determination with
benzylpenicillin as substrate revealed the presence of two different
β-lactamases (Table 1), but with cefotaxime as substrate, only one
enzyme, of pI value 8.2, showed strong cefotaxime-hydrolysing
activity.
Cloning and DNA sequencing of β-lactamase genes
The strain Gre-1 exhibited a positive amplification with primers
CTX-MA and CTX-MB, which were designed to conserve sequences
of blaCTX-M genes. DNA sequencing of PCR products showed that
strain Gre-1 harboured a blaCTX-M-14-type gene. The complete ORF of
the blaCTX-M gene was amplified with the primers CTX-M-9A and
CTX-M-9C and cloned downstream of the LacZ promoter of plasmid
pBK-CMV.
The sequencing of the blaCTX-M ORF revealed that strain Gre-1
harboured a new gene, designated blaCTX-M-27, which differed from
blaCTX-M-1416 by the substitution A → G at ORF position 725. On
the basis of amino acid sequence alignment (data not shown) with
CTX-M signal peptide sequences previously determined by direct
amino acid sequencing1,6 and from hydropathy plots, the deduced
amino acid sequence comprised a signal peptide of 28 amino acids.
Thus, the putative mature enzyme CTX-M-27 consisted of 263
amino acid residues with a calculated molecular weight of 27 915 Da.
This sequence differed by one or two substitutions at positions 231
and 240 (according to the numbering of Ambler et al. 34) from those of
CTX-M-9 (A231V, D240G), CTX-M-14 (D240G) and CTX-M-16
(A231V). Thus, CTX-M-27 was the third CTX-M enzyme harbouring the substitution D240G after CTX-M-15 and CTX-M-16,8,10,
which derive from CTX-M-3 and CTX-M-9, respectively.
β-Lactam susceptibility of the CTX-M-27-producing E. coli
transformant
MICs of β-lactams for the E. coli DH5α transformants producing
CTX-M-27 (pClGre-1) and CTX-M-14 (pClCF-1)16 are listed in
Table 2. The CTX-M-producing strains exhibited a high level of
resistance to amino- and carboxy-penicillins (MICs > 2048 mg/L),
piperacillin (MIC, 256 mg/L), cefalothin and cefuroxime (MICs,
512–1024 mg/L). Similar levels of resistance to cefotaxime (MICs,
16 mg/L) and aztreonam (MICs, 4–8 mg/L) were observed. However, the Gly-240-harbouring enzyme CTX-M-27, like CTX-M-15
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Amoxicillin
Amoxicillin + CLAe
Ticarcillin
Ticarcillin + CLA
Piperacillin
Piperacillin + TZBf
Cefalothin
Cefuroxime
Cefotaxime
Cefotaxime + CLA
Cefpirome
Cefepime
Aztreonam
Ceftazidime
Ceftazidime + CLA
E. coli Gre-1
(pGre-1a)
TEM-1, CTX-M-27
R. Bonnet et al.
Table 3. Substrate profile of β-lactamases CTX-M-14,16 and CTX-M-27
CTX-M-27
Substrate
Penicillin G
Amoxicillin
Ticarcillin
Piperacillin
Cefalothin
Cefuroxime
Cefotaxime
Cefpirome
Ceftazidime
Aztreonam
CTX-M-14
kcat (s–1)
Km (µM)
kcat/Km (µM–1 s–1)
kcat (s–1)
Km (µM)
kcat/Km (µM–1 s–1)
11 ± 0.5
5 ± 1.0
1 ± 0.3
9 ± 0.5
232 ± 30.0
79 ± 4.0
113 ±15.0
63 ± 2.5
3 ± 0.3
0.4 ± 0.2
6 ± 0.2
10 ± 0.4
13 ± 0.3
8 ± 0.3
83 ± 1.0
45 ± 2.0
150 ± 8.0
520 ± 50.0
330 ± 22.0
17 ± 1.0
1.8
0.5
0.07
1.1
2.8
1.75
0.75
0.12
0.009
0.02
290 ± 4.0
100 ± 2.0
45 ± 1.5
200 ± 2.8
2700 ± 72.0
320 ± 12.0
415 ± 15.0
940 ± 53.0
3 ± 0.5
10 ± 4.0
20 ± 1.0
20 ± 1.5
24 ± 2.2
48 ± 1.8
175 ± 5.0
40 ± 2.0
130 ± 10.0
1000 ± 98.0
630 ± 40.0
200 ± 10.0
14.5
5
2
4
15.4
14.4
3.2
0.94
0.004
0.05
and CTX-M-16,8,10 exhibited higher MICs of ceftazidime than its
parental enzyme CTX-M-14 (8 versus 1 mg/L), which contains the
residue Asp-240.
Clavulanate and tazobactam restored partially or totally the activities of the β-lactams (MICs, 0.06–16 mg/L).
Kinetic constants
The purified protein appeared on SDS–polyacrylamide gels as a
band of ∼28 kDa for CTX-M-27 (Figure 1). The specific activity of
purified (≥97% pure) CTX-M-27 was 22 µmol min–1 mg–1 of protein
with 200 mM benzylpenicillin as the substrate.
The kinetic constants of CTX-M-14 and CTX-M-27 are shown in
Table 3. Common enzymic features were observed. There were better
affinities for penicillins (Km, 6–48 µM) than for cephalosporins,
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Figure 1. Electrophoresis analysis of CTX-M-27 purified extracts. (a) SDS–
PAGE stained with Coomassie Brilliant Blue R-250. (b) Zymogram detection of
β-lactamase activity with nitrocefin after renaturation treatment of SDS–PAGE.
Lanes: 1, protein molecular mass reference; 2, purified extract of β-lactamase
CTX-M-27; 3, clarified extract of β-lactamase CTX-M-27.
cefalothin was the best substrate (kcat for cefalothin 10- to 20-fold
higher than that for penicillin), and kcat for cefotaxime (113–415 s–1)
was higher than for ceftazidime (3 s–1) and aztreonam (0.4–10 s–1).
The hydrolytic properties of enzyme CTX-M-27 were therefore
similar to those of previously reported CTX-M enzymes with regard
to the higher catalytic activity against cefotaxime than against ceftazidime and aztreonam.
However, CTX-M-27 had lower kcat values than CTX-M-14 with
all substrates except ceftazidime for which the hydrolytic activity
was closely related to that observed with previously reported CTXM.1,6,7,10,15,16,18 Shimamura et al.26 reported an interaction between
the Asp-240 side chain and the amino-thiazole ring of cefotaxime in
the crystallographic acyl-intermediate of Toho-1, which accounts for
its activity against cefotaxime. Residue Gly-240, which is devoid of
side chains, is unable to establish this interaction and may therefore
decrease the hydrolytic activity of CTX-M-27 against cefotaxime
and against substrates devoid of the amino-thiazole ring such as penicillins, cefuroxime and cefalothin.
The Gly-240-harbouring enzyme CTX-M-27, as previously
observed with CTX-M-16,10 had a lower Km value than the Asp-240harbouring enzyme CTX-M-14, with regard to ceftazidime. This
decrease in Km could explain the higher MIC value of ceftazidime
observed for the CTX-M-27-producing E. coli than for the CTX-M14 producer.
In TEM and SHV ESBLs, residues Lys and Arg at position 240 are
known to increase the enzymic activity against ceftazidime. Lys and
Arg are positively-charged residues that can form an electrostatic
bond with the carboxylic acid group on oxyimino substituents of
ceftazidime.35,36 In CTX-M-27, neutral residue Gly-240 is not able to
form electrostatic interactions with β-lactams but could favour the
accommodation of ceftazidime. In Asp-240-harbouring enzyme
CTX-M-14, the acyl-amide group of ceftazidime is probably sterically encumbered by the side chain of Asp-240, which, as a result,
could alter the interactions between the ceftazidime C-7β-amid
group and residues Ser-237 and Asn-132. The residue Gly-240,
which is devoid of side chains, could decrease this steric hindrance.
Substitution D240G did not modify the susceptibility to
β-lactamase inhibitors. The two enzymes CTX-M-14 and CTX-M-27
were susceptible to tazobactam (IC50, 0.008 and 0.007 µM, respectively), clavulanate (IC50, 0.030 and 0.020 µM, respectively) and, to a
lesser extent, sulbactam (IC50, 3.5 and 3.4 µM, respectively).
D240G substitution in CTX-M-27
Molecular modelling
Ibuka et al. reported that the β3 strand of CTX-M enzymes has
numerous Gly residues and is therefore probably more flexible than
that of TEM penicillinases.25 Residue Gly-240 in CTX-M-27 may
further increase the flexibility of the β3 strand and alter its positioning
during the catalytic process. To investigate the diminution of the
hydrolytic activity associated with residue Gly-240 in CTX-M-27,
the enzymes CTX-M-14, CTX-M-27 and CTX-M-1 were modelled
by geometric optimization after the introduction of amino acid substitutions and of the catalytic water molecule in the crystallographic
structure of Toho-1. The major differences observed between the
models obtained and the TEM-1 crystallographic structure (Figure
2c)37 are shown in Figure 2 (a and b). In the CTX-M enzymes, the
loop connecting the β5 strand and the H11 helix exhibited two additional residues and was oriented towards the C-terminal extremity of
the β3 strand unlike that in the TEM-type enzyme. In the CTX-M-14
model, the residue Asn-270 of this loop established a hydrogen bond
with the Asp-240 side chain (Figure 2a). This interaction could
favour the correct positioning of the β3 strand residues during the
catalytic process. In the crystal structure of Toho-1 acyl-enzyme
intermediates,26 the interaction between residues 270 and 240 is
mediated by a water molecule. However, the calculated energy from
the structure exhibiting indirect binding was higher than that
obtained from the structure exhibiting direct interaction of residues
270 and 240. No interaction between residues 240 and 270 was seen
in CTX-M enzymes belonging to the CTX-M-1 group, because they
are devoid of residue Asn-270. However, these enzymes harbour
residue Lys-271, which could interact with residue Asp-240, and
could replace the Asn-270 (data not shown).
Following the substitution D240G, interaction between the
residues 240 and 270 or 271 was absent in CTX-M-27 (Figure 2b).
The absence of this interaction in CTX-M-27 could work towards
modifying the flexibility of the C-terminal of the β3 strand. During
the catalytic process, this flexibility could favour the accommodation
of substrates, but could alter the geometry of the oxyanionic hole and/
or the positioning of the catalytic water molecule as the result of
modifications of the interactions between residues 240 and Asn-170.
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Figure 2. Structure of enzymes CTX-M-14 (a), and CTX-M-27 (b) obtained by molecular modelling and the crystallographic structure of the TEM-1 enzyme (c).35
Two black dots indicate the positions of the water molecules: W1, the catalytic water molecule maintained by the lateral chains of residues 166, 170 and 70; and W2, the
water molecule of the oxyanionic hole formed by the main chains of residues 70 and 237. The hydrogen bonds between residues 170, 240 and 270 are indicated by
dashed lines.
R. Bonnet et al.
In conclusion, residue Gly-240 in CTX-M-27 decreases the Km
for ceftazidime but could decrease hydrolytic activity against good
substrates, probably by modifying β3 strand-residue positioning
during the hydrolytic process.
Acknowledgements
We thank Rolande Perroux, Marlène Jan and Dominique Rubio for
technical assistance. This work was supported in part by a grant from
the Ministère de l’Education Nationale, de la Recherche et de la
Technologie.
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