1 Service de Bactériologie-Virologie, Hôpital de Bicêtre, Assistance Publique/Hôpitaux de Paris, Faculté de Médecine Paris-Sud, 94275 Le Kremlin-Bicêtre, France; 2 Sera & Vaccines Central Research Laboratory, 00725 Warsaw, Poland
Received 17 July 2002; returned 3 September 2002; revised 10 September 2002; accepted 11 September 2002
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Abstract |
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Keywords: ß-lactamase, CTX-M, expanded-spectrum ß-lactamases
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Introduction |
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Two novel point-mutant derivatives of CTX-M-9, CTX-M-16 and CTX-M-19, have been reported to hydrolyse ceftazidime significantly.3,6 Additionally, we have reported recently the DNA sequence of another ß-lactamase, CTX- M-15, from Indian enterobacterial isolates that were resistant to both cefotaxime and ceftazidime.5 CTX-M-15 has a single amino acid change [Asp-240Gly (Ambler numbering)]7 compared with CTX-M-3.5 It has so far also been found in Japan (ß-lactamase UOE-1; GenBank accession no. AY013478), Bulgaria8 and Poland,9 where CTX-M-3 is widespread.10
Since CTX-M-15-producing isolates had a significant degree of resistance to ceftazidime,5 we have purified CTX-M-15 and CTX-M-3 and compared their kinetic parameters (kinetics of CTX-M-3 has not been studied before). Additionally, this report provides detailed kinetic data that are available only for a very few CTX-M-type enzymes.
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Materials and methods |
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CTX-M-15-producing Escherichia coli 2 was from India.5 Citrobacter freundii isolate 2526/96, which was identified in Poland in 1996, was used as a blaCTX-M-3-containing strain.4 E. coli reference strain DH10B was used for cloning and expression experiments.6 Cloning was carried out with PCR products generated with primers PROM+ (5'-TGCTCTGTGGATAACTTGC-3') and preCTX-M-3B (5'-CCGTTTCCGCTATTACAAAC-3') annealing to the 3'-end of insertion sequence ISEcp1 located upstream of blaCTX-M-15 and downstream of blaCTX-M-15/-3, respectively (accession no. AY044436).3,4 Whole-cell DNA from E. coli 2 and C. freundii 2526/96 was used as template.5 PCR amplimers were cloned into the SrfI site of the pPCRScript-Cam (SK+) plasmid (Stratagene Inc., La Jolla, CA, USA). Recombinant plasmids were transformed into electrocompetent E. coli DH10B cells and selected on MuellerHinton (MH) agar plates containing 100 mg/L ampicillin and 30 mg/L chloramphenicol. Sequencing of inserts of recombinant plasmids was carried out as described previously.6
Susceptibility testing
MICs of selected ß-lactams were determined by the agar dilution technique on MH agar plates as described previously,6 and interpreted according to the NCCLS guidelines.11
Biochemical analysis of CTX-M-15 and CTX-M-3
Cultures of E. coli DH10B with plasmids pCTX-M-15 and pCTX-M-3 were grown overnight at 37°C in 4 L of trypticase soy broth containing ampicillin (100 mg/L) and chloramphenicol (30 mg/L). ß-Lactamase extracts were obtained using purification steps with a Q-Sepharose column, then an S-Sepharose column followed by elution at 50 mM NaCl, as described previously.6 ß-Lactamase-positive fractions were pooled and dialysed against 50 mM phosphate buffer (pH 7), and subsequently concentrated 10-fold with Centrisart-C30 microcentrifuge filters (Sartorius, Goettingen, Germany).6
Analytical isoelectric focusing (IEF) using an ampholine-containing polyacrylamide gel and purity of the enzymes and relative molecular masses estimated by SDSPAGE analysis were carried out as reported previously.6
Purified ß-lactamases were then used for kinetic measurements at 30°C in 100 mM sodium phosphate buffer (pH 7.0). The initial rates of hydrolysis were determined with an ULTROSPEC 2000 UV spectrophotometer (Amersham Pharmacia Biotech), as described previously.6 The 50% inhibitory concentrations (IC50 values) were determined as reported previously.6 Specific activities of the purified ß-lactamases were evaluated as previously reported; one unit of enzyme activity was defined as the activity that hydrolysed 100 µmol of cefalothin per minute.6
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Results and discussion |
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The DNA inserts of the two recombinant plasmids pCTX- M-15 and pCTX-M-3 were sequenced, confirming that they contained the blaCTX-M-15 and blaCTX-M-3 genes, respectively. The 3'-end of ISEcp1 was located 48 and 128 bp upstream of the start codon of blaCTX-M-15 and blaCTX-M-3, respectively (data not shown), indicating that the surrounding sequences of these two blaCTX-M genes were different.
E. coli DH10B that harboured pCTX-M-15 and pCTX-M-3 demonstrated a typical inhibitor-susceptible ESBL-mediated resistance profile (Table 1). MICs of ß-lactams for E. coli DH10B (pCTX-M-15) mirrored those for E. coli DH10B (pCTX-M-3) except for ceftazidime; the MIC of ceftazidime for the CTX-M-15 producer was significantly higher than that for the CTX-M-3 producer.
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The specific activities of purified ß-lactamases CTX-M-15 and CTX-M-3 were 185 and 138 mU/mg of protein, respectively, with a 50-fold purification factor in both cases. Their purification level was 90% (data not shown). IEF analysis identified pI values for CTX-M-15 and CTX-M-3 of 8.9 and 8.4, respectively. The relative molecular masses of CTX- M-15 and CTX-M-3, determined by SDSPAGE analysis, were
29 kDa (data not shown).
The glycine residue in position 240 in CTX-M-15 provided lower hydrolytic activity (lower kcat values) for penicillins compared with CTX-M-3, as found for CTX-M-16 and CTX-M-9, which differ by the same amino acid substitution in position 240.6 The overall hydrolytic activity of CTX-M-15 against cephalosporins was not higher than that of CTX-M-3, depending on the cephalosporin molecule.
CTX-M-15 had higher affinities (low Km) than CTX-M-3 for all the ß-lactams studied except for cefepime. This was particularly true for aztreonam, as found for CTX-M-16 when compared with CTX-M-9.3
In general, CTX-M-15 and CTX-M-3 had strong catalytic efficiency (high kcat/Km) against benzylpenicillin, piperacillin, cefotaxime and ceftriaxone (Table 2), as reported for other CTX-M-type enzymes such as CTX-M-16 and CTX-M-18.3,6 The comparison of catalytic efficiencies of CTX- M-15 with those of CTX-M-3 revealed that cefuroxime and benzylpenicillin, respectively, were the best substrates for the two enzymes. The catalytic efficiencies of CTX-M-15 and CTX-3 did not correlate perfectly with the MIC values for E. coli producing CTX-M-15 and CTX-M-3, possibly caused by high copy number (100 copies) of the cloning vector, which may substantially increase the amount of enzymes present in the periplasmic space.
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The kinetic parameters of CTX-M-15 against ceftazidime may be explained by the glycine residue at position 240. This amino acid residue at position 240 is not conserved among class A ß-lactamases.7 Some amino acid residues in this position have been found to play a key role in the extended hydrolytic profile of several ESBLs. Amino acid residue Gly-240 is found in other ESBLs such as VEB-1, BES-1 and PER-1.3,12 Conversely, in a previous study,12 we have reported that the substitution Gly-240Glu in PER-1 caused a reduction in affinity of the enzyme for aztreonam and decreased its catalytic efficiency against cefotaxime and ceftazidime.
CTX-M-15 and CTX-M-3 were similarly prone to inhibition by clavulanic acid (IC50 values 9 and 12 nM, respectively) and by tazobactam (IC50 values 2 and 6 nM, respectively). The relatively higher susceptibility to inhibition by tazobactam compared with clavulanic acid is a feature of CTX-M-type enzymes.1
Data presented in this work indicate further that detection of CTX-M-type ESBLs can no longer be based only on a resistance pattern that includes resistance to cefotaxime and susceptibility to ceftazidime. The role of clinical usage of ceftazidime should be evaluated for selection of novel ceftazidime-hydrolysing CTX-M-type enzymes that may occur through a single amino acid substitution. This is true especially for the CTX-M-1- and CTX-M-9-type ß-lactamases, which are spread worldwide.16,8,10
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Acknowledgements |
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Footnotes |
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References |
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