JAC Advance Access published online on July 28, 2008
Journal of Antimicrobial Chemotherapy, doi:10.1093/jac/dkn317
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Research letter |
Do CTX-M β-lactamases hydrolyse ertapenem?
Service de Bactériologie-Virologie, INSERM U914 ‘Emerging Resistance to Antibiotics’, Assistance Publique/Hôpitaux de Paris, Faculté de Médecine et Université Paris Sud, Hôpital de Bicêtre, 94275 K.-Bicêtre, France
* Corresponding author. Tel: 33-1-45-21-36-32; Fax: +33-1-45-21-63-40; E-mail: nordmann.patrice{at}bct.aphp.fr
Key Words: ESBL , Ki , hydrolysis
The current emergence and dissemination of clavulanic acid-inhibited extended-spectrum β-lactamases (ESBLs) represents a global threat, as they are difficult to trace and eradicate, with CTX-M-type ESBLs being of major concern in community-acquired clinical infections. Among β-lactam molecules, carbapenems (imipenem, ertapenem and meropenem) are the drugs of choice for treating infections caused by ESBL-producing Enterobacteriaceae. Ertapenem is a broad-spectrum carbapenem that is active against Enterobacteriaceae producing ESBLs.1 However, combinations of ESBL and porin deficiency have been reported to confer resistance to ertapenem.2,3 In a clinical microbiology laboratory, identification of an ESBL may be confirmed by the observation of a synergy image between an extended-spectrum cephalosporin and clavulanic acid. Surprisingly, a synergy image is sometimes observed between ertapenem- and clavulanic-acid-containing discs performed with CTX-M-producing Enterobacteriaceae.1 This observation suggests that ertapenem might be hydrolysed by CTX-M-type β-lactamases (Figure 1).
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Therefore, we have compared the β-lactamase activity towards ertapenem from culture extracts of Escherichia coli producing the four most widespread CTX-Ms: CTX-M-2, CTX-M-3, CTX-M-9 and CTX-M-15.
In order to express the blaCTX-M genes under the same promoter and in the same bacterial genetic context, the four blaCTX-M genes (blaCTX-M-2, blaCTX-M-3, blaCTX-M-9 and blaCTX-M-15) were amplified from our collection of reference E. coli strains without their promoter sequence and cloned into the low-copy-number pACYC184 plasmid (New England Biolabs, Ozyme, Saint-Quentin-en-Yvelines, France). The primers used in this study were CTX-M-2F (5'-AATGTATATTGAAGGCCGAGGG-3'), CTX-M-2R (5'-ATACCTCGCTCCATTTATTGC-3'), CTX-M-3F (5'-TCGTCTCTTCCAGAATAAG-3'), CTX-M-3R (5'-TACCTATTACAAACCGTCGGTG-3'), CTX-M-9F (5'-CTGATGTAACACGGATTGAC-3'), CTX-M-9R (5'-AGCGCCCCATTATTGAGAG-3'), CTX-M-15F (5'-TCGTATCTTCCAGAATAAGG-3') and CTX-M-3R (also used for CTX-M-15 amplification). The PCR products were ligated into the pPCRBluntII-TOPO plasmid (Invitrogen, Life Technologies, Cergy-Pontoise, France), excised by EcoRV and BamHI digestion and subcloned into the pACYC184 plasmid, successively restricted by SphI digestion, blunt-ended with Pfu polymerase and restricted with BamHI digestion. The recombinant vectors were introduced into electrocompetent cells of E. coli Wi, a wild-type clinical isolate recovered from urinary tract infection in our hospital (Hôpital Bicêtre, K.-Bicêtre, France). Transformants were selected overnight at 37°C on nutrient agar containing 30 mg/L chloramphenicol. Cultures of E. coli harbouring the recombinant plasmids pCTX-M were grown overnight at 37°C in 10 mL of Trypticase soy broth with 100 mg/L amoxicillin. After sonication and centrifugation, β-lactamase activity of the supernatants was assayed by UV spectrophotometry, as described previously.4 One unit of enzyme activity was defined as the activity that hydrolysed 1 µmol of ertapenem/min. The total protein content was measured with bovine albumin as the standard (Bio-Rad DC Protein Assay Kit). Two other Ambler class A β-lactamases were used as controls in these enzymatic studies: KPC-2, a carbapenemase known to efficiently hydrolyse ertapenem,5 and TEM-3, an ESBL without any carbapenemase activity.6 β-Lactamase activities of the CTX-Ms towards ertapenem were very low, approximately 100-fold lower than that of the KPC-2 β-lactamase, and similar to that of TEM-3 (Table 1). The slightly higher activity of CTX-M-15 was not relevant considering the standard deviation values (Table 1). The synergistic effect of ertapenem and clavulanic acid observed on Mueller–Hinton plates with CTX-M-producing Enterobacteriaceae is not explained by a hydrolytic activity of the CTX-Ms towards ertapenem. Inhibition studies were thus conducted for β-lactamases CTX-M-15 and TEM-3 to assess the presence and magnitude of drug–drug interactions. The inhibition constants (Ki) were determined as described by Dixon,7 i.e. plots were prepared of the reciprocal of the rate of metabolite formation (1/v) as a function of inhibitor concentration at each substrate concentration. The Ki value was recovered at the intersection of the obtained lines. Cefalotin (10, 20, 50 and 100 µM) was used as the substrate and ertapenem as the inhibitor (0.001–0.1 µM). This inhibition study showed that the ertapenem Ki value was 10-fold lower for CTX-M-15 (7 nM) than for TEM-3 (65 nM). The better efficiency of CTX-M-15 towards ertapenem over TEM-3, resulting from a 10-fold lower Ki and a slightly higher Vmax (approximately 2-fold higher), may explain the synergy image observed with CTX-M producers and not with TEM-3. Similarly, it is known that tazobactam has a much higher inhibitory activity against CTX-Ms than against TEM-type ESBLs.8
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Thus, ertapenem is effective against CTX-M-producing Enterobacteriaceae, taking into account the very low level of its hydrolysis by CTX-Ms. The synergy image that may be observed between ertapenem and clavulanic acid for CTX-M producers may mostly result from the stronger inhibitory effect of clavulanic acid on CTX-Ms associated with a weak hydrolysis of ertapenem. Indeed, the IC50 value of clavulanate is 9 nM for CTX-M-15,4 whereas it is 26 nM for TEM-3.6 Finally, the synergy image observed should not lead to a false conclusion of ertapenem inefficacy, whereas a similar synergy image between cephalosporins and clavulanic acid for those CTX-M producers is related to a high hydrolysis of cephalosporins.
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This work was funded by a grant from the Ministère de l'Education Nationale et de la Recherche (UPRES-EA 3539), Université Paris XI, and mostly by a grant of the European Community (6th PCRD, LSHM-CT-2005-018705).
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Transparency declarations |
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None to declare.
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