Research letters References
Journal of Antimicrobial Chemotherapy doi:10.1093/jac/dkn317 Advance Access publication 28 July 2008
Do CTX-M b-lactamases hydrolyse ertapenem? Delphine Girlich, Laurent Poirel and Patrice Nordmann* Service de Bacte´riologie-Virologie, INSERM U914 ‘Emerging Resistance to Antibiotics’, Assistance Publique/ Hoˆpitaux de Paris, Faculte´ de Me´decine et Universite´ Paris Sud, Hoˆpital de Biceˆtre, 94275 K.-Biceˆtre, France Keywords: ESBL, Ki, hydrolysis *Corresponding author. Tel: 33-1-45-21-36-32; Fax: þ33-1-45-21-63-40; E-mail:
[email protected] Sir, The current emergence and dissemination of clavulanic acidinhibited extended-spectrum b-lactamases (ESBLs) represents a global threat, as they are difficult to trace and eradicate, with CTX-M-type ESBLs being of major concern in communityacquired clinical infections. Among b-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 extendedspectrum cephalosporin and clavulanic acid. Surprisingly, a synergy image is sometimes observed between ertapenem- and
(a)
(b) AMC
AMC
ETP
ETP
Figure 1. Double-disc synergy test on Mueller– Hinton agar plates with ertapenem (ETP) and amoxicillin/clavulanate (AMC) on CTX-M-15 (a) and on TEM-3 (b) E. coli. The distances between the discs were optimized according to the inhibition diameters.
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1. Martı´nez-Martı´nez L, Pascual A, Jacoby GA. Quinolone resistance from a transferable plasmid. Lancet 1998; 351: 797 –9. 2. Robicsek A, Jacoby GA, Hooper DC. The worldwide emergence of plasmid-mediated quinolone resistance. Lancet Infect Dis 2006; 6: 629–40. 3. Yamane K, Wachino J, Suzuki S et al. New plasmid-mediated fluoroquinolone pump, QepA, found in an Escherichia coli clinical isolate. Antimicrob Agents Chemother 2007; 51: 3354– 60. 4. Gay K, Robicsek A, Strahilevitz J et al. Plasmid-mediated quinolone resistance in non-Typhi serotypes of Salmonella enterica. Clin Infect Dis 2006; 43: 297– 304. 5. Cavaco LM, Hendriksen RS, Aarestrup FM. Plasmid-mediated quinolone resistance determinant qnrS1 detected in Salmonella enterica serovar Corvallis strains isolated in Denmark and Thailand. J Antimicrob Chemother 2007; 60: 704– 6. 6. Hopkins KL, Day M, Threlfall EJ. Plasmid-mediated quinolone resistance in Salmonella enterica, United Kingdom. Emerg Infect Dis 2008; 14: 340–2. 7. Hakanen A, Kotilainen P, Jalava J et al. Detection of decreased fluoroquinolone susceptibility in salmonellas and validation of nalidixic acid screening test. J Clin Microbiol 1999; 37: 3572– 7. 8. Aarestrup FM, Wiuff C, Molbak K et al. Is it time to change fluoroquinolone breakpoints for Salmonella spp.? Antimicrob Agents Chemother 2003; 47: 827–9.
clavulanic-acid-containing discs performed with CTX-M-producing Enterobacteriaceae.1 This observation suggests that ertapenem might be hydrolysed by CTX-M-type b-lactamases (Figure 1). Therefore, we have compared the b-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, SaintQuentin-en-Yvelines, France). The primers used in this study were CTX-M-2F (50 -AATGTATATTGAAGGCCGAGGG-30 ), CTX-M-2R (50 -ATACCTCGCTCCATTTATTGC-30 ), CTX-M3F (50 -TCGTCTCTTCCAGAATAAG-30 ), CTX-M-3R (50 -TAC CTATTACAAACCGTCGGTG-30 ), CTX-M-9F (50 -CTGATGTA ACACGGATTGAC-30 ), CTX-M-9R (50 -AGCGCCCCATTATT GAGAG-30 ), CTX-M-15F (50 -TCGTATCTTCCAGAATAAGG30 ) 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 (Hoˆpital Biceˆtre, K.-Biceˆtre, France). Transformants were selected overnight at 378C on nutrient agar containing 30 mg/L chloramphenicol. Cultures of E. coli harbouring the recombinant plasmids pCTX-M were grown overnight at 378C in 10 mL of Trypticase soy broth with 100 mg/L amoxicillin. After sonication and centrifugation, b-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 mmol 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 b-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 b-Lactamase activities of the CTX-Ms towards ertapenem were very low,
Research letters Table 1. b-Lactamase activity of E. coli Wi harbouring recombinant plasmid pACYC184 containing the blaCTX-M-2, blaCTX-M-3, blaCTX-M-9 or blaCTX-M-15 genes, of E. coli Top 10 harbouring pTEM-3 and of the clinical isolate Klebsiella pneumoniae YC harbouring KPC-2
b-Lactamase activity (mU/mg of protein)b
E. coli Wi pCTX-M-2
E. coli Wi pCTX-M-3
E. coli Wi pCTX-M-9
E. coli Wi pCTX-M-15
E. coli Top 10 pTEM-3a
K. pneumoniae YC pKPC-2
0.8 + 0.2
0.7 + 0.3
0.4 + 0.1
1.6 + 0.5
0.6 + 0.2
98 + 6
a
pTEM-3 is a natural plasmid containing the blaTEM-3 gene.6 E. coli Top 10 pTEM-3 and K. pneumoniae YC expressing KPC-2 were used as negative and positive controls, respectively. b One unit of b-lactamase is defined as 1 mmol of ertapenem hydrolysed/min. The b-lactamase activities are geometric mean determinations for three independent cultures. The standard deviations are indicated.
References 1. Woodford N, Dallow JW, Hill RL et al. Ertapenem resistance among Klebsiella and Enterobacter submitted in the UK to a reference laboratory. Int J Antimicrob Agents 2007; 29: 456–9. 2. Jacoby GA, Mills DM, Chow N. Role of b-lactamases and porins in resistance to ertapenem and other b-lactams in Klebsiella pneumoniae. Antimicrob Agents Chemother 2004; 48: 3203–6. 3. Lartigue MF, Poirel L, Poyart C et al. Ertapenem resistance of Escherichia coli. Emerg Infect Dis 2007; 13: 315–7. 4. Poirel L, Gniadkowski M, Nordmann P. Biochemical analysis of the ceftazidime-hydrolysing extended-spectrum b-lactamase CTXM-15 and of its structurally related b-lactamase CTX-M-3. J Antimicrob Chemother 2002; 50: 1031–4. 5. Naas T, Cuzon G, Villegas MV et al. Genetic structures at the origin of acquisition of the b-lactamase blaKPC gene. Antimicrob Agents Chemother 2008; 52: 1257–63. 6. Kitzis MD, Billot-Klein D, Goldstein FW et al. Dissemination of the novel plasmid-mediated b-lactamase CTX-1, which confers resistance to broad-spectrum cephalosporins, and its inhibition by b-lactamase inhibitors. Antimicrob Agents Chemother 1988; 32: 9–14. 7. Dixon M. The determination of enzyme inhibitor constants. Biochem J 1953; 55: 170–1. 8. Tzouvelekis LS, Tzelepi E, Tassios PT et al. CTX-M-type b-lactamases: an emerging group of extended-spectrum enzymes. Int J Antimicrob Agents 2000; 14: 137–42.
Journal of Antimicrobial Chemotherapy doi:10.1093/jac/dkn314 Advance Access publication 30 July 2008
Funding
In vitro activity of ME1036 versus other b-lactams against penicillin-resistant Streptococcus pneumoniae serotypes exhibiting higher amoxicillin than penicillin MIC
This work was funded by a grant from the Ministe`re de l’Education Nationale et de la Recherche (UPRES-EA 3539), Universite´ Paris XI, and mostly by a grant of the European Community (6th PCRD, LSHM-CT-2005-018705).
Asuncio´n Fenoll1, Lorenzo Aguilar2*, Olga Robledo1, Marı´a-Jose´ Gime´nez2, Juan-Jose´ Granizo3, Donald Biek4 and David Tarrago´1
Transparency declarations None to declare.
1
Spanish National Reference Pneumococcal Laboratory, Instituto de Salud Carlos III, ctra. Majadahonda-Pozuelo 1156
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approximately 100-fold lower than that of the KPC-2 b-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 b-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 mM) was used as the substrate and ertapenem as the inhibitor (0.001 – 0.1 mM). 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 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.
