1 Laboratoire de Bactériologie, Faculté de Médecine, Service de Bactériologie-Virologie, 28 Place Henri-Dunant, 63001 Clermont-Ferrand Cedex; 2 Laboratoire de Bactériologie, CHU de Grenoble, Chemin Maquis du Grésivaudan, 38 700 La Tronche, France
Received 27 January 2003; returned 14 February 2003; revised 13 March 2003; accepted 13 March 2003
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Abstract |
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Keywords: CTX-M, ß-lactamase, D240G mutation, ESBL
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Introduction |
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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 ß3 strand and loop, and residues Asn-104, Ser-237, Asp-240 and Arg-276 are involved in the cefotaxime-hydrolyzing activity of CTX-M enzymes.1,5,1719,25,26
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 CTX-M-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 ß-lactamases CTX-M-14 and CTX-M-27 and molecular modelling give insights into the role of the D240G substitution in CTX-M enzymes.
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Materials and methods |
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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.
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MICs were determined by a dilution method on MuellerHinton 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.
Isoelectric focusing
Isoelectric focusing was carried out with polyacrylamide gels containing ampholines with a pH range of 3.510 as previously described.10 ß-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 264814 (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).
ß-Lactamase gene cloning
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 DH528 was transformed by electroporation. The transformants harbouring the recombinant CTX-M-encoding plasmid were selected on MuellerHinton agar supplemented with 2 mg of cefotaxime per L.
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).
ß-Lactamase preparation
The CTX-M-producing E. coli DH5 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 MESNaOH 20 mM (pH 5.5) and disrupted by ultrasonic treatment (4 x 30 s, each time at 20 W). After centrifugation (10 000g for 10 min at 4°C), nucleic acids 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 MESNaOH 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 sulphatepolyacrylamide gel electrophoresis (SDSPAGE) and staining with Coomassie Blue R-250 (Sigma Chemical Co.). The renaturation of proteins and the detection of the ß-lactamase activity were carried out as previously described10 with renaturation buffer TrisHCl (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 ß-lactamase kinetic constants
The steady-state kinetic parameters (Km and kcat) of the ß-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.
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
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-166Ala Toho-1 mutant25 by introducing the mutations as part as an automated procedure. The residues, the catalytic water molecule and the water molecules of the Toho-1 crystal were minimized using the Amber96 parameters33 and a distance-dependent 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.
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Results and discussion |
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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 ß-lactamases (Table 1), but with cefotaxime as substrate, only one enzyme, of pI value 8.2, showed strong cefotaxime-hydrolysing activity.
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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.
ß-Lactam susceptibility of the CTX-M-27-producing E. coli transformant
MICs of ß-lactams for the E. coli DH5 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, 5121024 mg/L). Similar levels of resistance to cefotaxime (MICs, 16 mg/L) and aztreonam (MICs, 48 mg/L) were observed. However, the Gly-240-harbouring enzyme CTX-M-27, like CTX-M-15 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 ß-lactams (MICs, 0.0616 mg/L).
Kinetic constants
The purified protein appeared on SDSpolyacrylamide 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 min1 mg1 of protein with 200 mM benzylpenicillin as the substrate.
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The Gly-240-harbouring enzyme CTX-M-27, as previously observed with CTX-M-16,10 had a lower Km value than the Asp-240-harbouring 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-M-14 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 ß-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-7ß-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 ß-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).
Molecular modelling
Ibuka et al. reported that the ß3 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 ß3 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 ß5 strand and the H11 helix exhibited two additional residues and was oriented towards the C-terminal extremity of the ß3 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 ß3 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).
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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 ß3 strand-residue positioning during the hydrolytic process.
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Acknowledgements |
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Footnotes |
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References |
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