Abstract

The extended-spectrum β-lactamase CTX-M-15 confers resistance to ceftazidime, unlike the majority of CTX-M-type enzymes. Kinetic parameters were determined from purified CTX-M-15 and CTX-M-3, which differ by the single amino acid substitution Asp-240 to Gly, according to the Ambler numbering of class A β-lactamases. Relative molecular masses of CTX-M-15 and CTX-M-3 were ∼29 kDa and pI values were 8.9 and 8.4, respectively. CTX-M-15 had higher affinities for β-lactams (lower Km values) than those of CTX-M-3 but catalytic efficiency (kcat/Km values) was variable depending on the β-lactam substrate. Only CTX-M-15 showed a measurable catalytic efficiency for ceftazidime. Clavulanic acid and tazobactam were good inhibitors of both enzymes. MICs of β-lactams for Escherichia coli reference strains expressing cloned β-lactamase genes in the same genetic background were similar except for ceftazidime. This work underlines the fact that some CTX-M enzymes may hydrolyse ceftazidime and thus confer resistance to this expanded-spectrum cephalosporin in Enterobacteriaceae.

Received 17 July 2002; returned 3 September 2002; revised 10 September 2002; accepted 11 September 2002

Introduction

In addition to the classical TEM and SHV enzymes, sev- eral plasmid-mediated Ambler class A extended-spectrum β-lactamases (ESBLs) have been reported. Among them, the CTX-M-type β-lactamases are currently spreading worldwide in Enterobacteriaceae.1 The name ‘CTX-M’ refers to their potent hydrolytic activity for cefotaxime.1,2 The CTX-M enzymes confer high-level resistance to cefotaxime, ceftriaxone and aztreonam, but have only marginal effects on MICs of ceftazidime for both wild-type and laboratory-derived strains of enterobacteria.1 According to amino acid sequence data, they may be grouped in four clusters: CTX-M-1 (CTX-M-1, -3, -10, -11, -12, -15), CTX-M-2 (CTX-M-2, -4, -5, -6, -7, -20, Toho-1), CTX-M-8 and CTX-M-9 (CTX-M-9, -13, -14, -16, -18, -19 and Toho-2) (accession nos AJA16344 and 41346).16

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 β-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-240→Gly (Ambler numbering)]7 compared with CTX-M-3.5 It has so far also been found in Japan (β-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.

Materials and methods

Bacterial strains, cloning experiments and sequencing

CTX-M-15-producing Escherichia coli 2 was from India.5Citrobacter freundii isolate 2526/96, which was identified in Poland in 1996, was used as a blaCTX-M-3-containing strain.4E. 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 Mueller–Hinton (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 β-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). β-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 β-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 SDS–PAGE analysis were carried out as reported previously.6

Purified β-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 β-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

Results and discussion

Recombinant plasmids and susceptibility testing

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 β-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.

Biochemical analysis of CTX-M-15 and CTX-M-3

The specific activities of purified β-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 SDS–PAGE 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 β-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.

In the case of ceftazidime, higher MIC values for CTX- M-15 producer than that for CTX-M-3 producer could be explained by different kinetic parameters. CTX-M-15, but not CTX-M-3, demonstrated a detectable, although relatively low, catalytic activity against ceftazidime, along with a low affinity for this substrate (high Km value). Similar observations had previously been reported for the two other ceftazidime-hydrolysing CTX-M-type enzymes, i.e. CTX-M-16 and CTX-M-19.35

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 β-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-240→Glu 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 β-lactamases, which are spread worldwide.16,8,10

Acknowledgements

This work was funded by a grant from the Ministère de l’Education Nationale et de la Recherche (UPRES-EA), Faculté de Médecine Paris-Sud, Université Paris XI, Paris, France.

*

Corresponding author. Tel: +33-1-45-21-36-32; Fax: +33-1-45-21-63-40; E-mail: nordmann.patrice@bct.ap-hop-paris.fr

Table 1.

 MICs of β-lactams for E. coli DH10B alone or harbouring recombinant plasmids pCTX-M-15 and pCTX-M-3 expressing CTX-M-15 and CTX-M-3, respectively

 MIC (mg/L) 
β-Lactam(s)a E. coli DH10B (pCTX-M-15) E. coli DH10B (pCTX-M-3) E. coli DH10B 
Amoxicillin >512 >512 
Co-amoxiclav  32  128 
Ticarcillin >512 >512 
Ticarcillin + CLA  32  64 
Piperacillin >512  512 
Piperacillin + TZB   4   2 
Cefalothin >512 >512 
Cefuroxime >512 >512 
Cefotaxime  512  512 <0.06 
Cefotaxime + CLA   2   2 <0.06 
Cefotaxime + TZB   0.5   1 <0.06 
Ceftazidime  256  32 <0.06 
Ceftazidime + CLA   2   2 <0.06 
Ceftazidime + TZB   2   2 <0.06 
Ceftriaxone >512 >512 <0.06 
Cefepime  64  128 <0.06 
Cefpirome  512  512 <0.06 
Cefoxitin   4   2 
Moxalactam   1   0.5 0.12 
Aztreonam  64  128 0.06 
Imipenem   0.25   0.25 0.12 
 MIC (mg/L) 
β-Lactam(s)a E. coli DH10B (pCTX-M-15) E. coli DH10B (pCTX-M-3) E. coli DH10B 
Amoxicillin >512 >512 
Co-amoxiclav  32  128 
Ticarcillin >512 >512 
Ticarcillin + CLA  32  64 
Piperacillin >512  512 
Piperacillin + TZB   4   2 
Cefalothin >512 >512 
Cefuroxime >512 >512 
Cefotaxime  512  512 <0.06 
Cefotaxime + CLA   2   2 <0.06 
Cefotaxime + TZB   0.5   1 <0.06 
Ceftazidime  256  32 <0.06 
Ceftazidime + CLA   2   2 <0.06 
Ceftazidime + TZB   2   2 <0.06 
Ceftriaxone >512 >512 <0.06 
Cefepime  64  128 <0.06 
Cefpirome  512  512 <0.06 
Cefoxitin   4   2 
Moxalactam   1   0.5 0.12 
Aztreonam  64  128 0.06 
Imipenem   0.25   0.25 0.12 

aCLA, clavulanic acid at a fixed concentration of 2 mg/L; TZB, tazobactam at a fixed concentration of 4 mg/L.

Table 2.

 Steady-state kinetic parameters of purified CTX-M-15 and CTX-M-3 β-lactamases

 CTX-M-15  CTX-M-3 
Substrate kcat (s–1Km (µM) kcat/Km (µM–1 s–1 kcat (s–1Km (µM) kcat/Km (µM–1 s–1
Benzylpenicillin  40  10   270     2.5 110 
Amoxicillin  20  38 0.5   160  185  1 
Ticarcillin  2   5 0.5   40   29  1 
Piperacillin  35  13   180   66  3 
Cefalothin  35  43 0.5  2800   96  30 
Cephaloridine 130  83 1.5   130  300  0.5 
Cefuroxime  70  13    3   49  0.07 
Cefoxitin  <0.01  ND ND   <0.01  ND  ND 
Ceftazidime  2 1760 0.001   <0.01 >3000  ND 
Ceftriaxone 135  37 3.5   30   58  0.5 
Cefotaxime 150  54   380  113  3.5 
Cefepime  10 1075 0.01    0.2  170  0.001 
Cefpirome 120  195 0.6   30  316  0.1 
Imipenem  <0.01  ND ND   <0.01  ND  ND 
Aztreonam  1.5  11 0.1   190  188  1 
 CTX-M-15  CTX-M-3 
Substrate kcat (s–1Km (µM) kcat/Km (µM–1 s–1 kcat (s–1Km (µM) kcat/Km (µM–1 s–1
Benzylpenicillin  40  10   270     2.5 110 
Amoxicillin  20  38 0.5   160  185  1 
Ticarcillin  2   5 0.5   40   29  1 
Piperacillin  35  13   180   66  3 
Cefalothin  35  43 0.5  2800   96  30 
Cephaloridine 130  83 1.5   130  300  0.5 
Cefuroxime  70  13    3   49  0.07 
Cefoxitin  <0.01  ND ND   <0.01  ND  ND 
Ceftazidime  2 1760 0.001   <0.01 >3000  ND 
Ceftriaxone 135  37 3.5   30   58  0.5 
Cefotaxime 150  54   380  113  3.5 
Cefepime  10 1075 0.01    0.2  170  0.001 
Cefpirome 120  195 0.6   30  316  0.1 
Imipenem  <0.01  ND ND   <0.01  ND  ND 
Aztreonam  1.5  11 0.1   190  188  1 

Values are means of three independent measurements (standard deviations of the values were within 15%); ND, not determinable (the initial rate of hydrolysis was lower than 0.01 µM–1 s–1).

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Author notes

1Service de Bactériologie-Virologie, Hôpital de Bicêtre, Assistance Publique/Hôpitaux de Paris, Faculté de Médecine Paris-Sud, 94275 Le Kremlin-Bicêtre, France; 2Sera & Vaccines Central Research Laboratory, 00725 Warsaw, Poland