JAC Advance Access published online on October 27, 2008
Journal of Antimicrobial Chemotherapy, doi:10.1093/jac/dkn443
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Research letter |
Potential evolution of hydrolysis spectrum for AmpC β-lactamase of Escherichia coli
1 Service de Bactériologie, CHU Amiens, Hôpital Nord, Place Victor Pauchet, 80000 Amiens, France 2 Service de Bactériologie-Virologie-Hygiène, Inserm U914, Hôpital de Bicêtre, rue du Général Leclerc, 94278 Le K.-Bicêtre, France
* Correspondence address. Service de Bactériologie, CHU Amiens, Hôpital Nord, Place Victor Pauchet, 80000 Amiens, France. Tel: +33-03-22-66-84-30; Fax: +33-03-22-66-84-98; E-mail: hedi.mammeri{at}bct.aphp.fr
Key Words: ESAC , cephalosporins , imipenem , cephalosporinases , carbapenems
Escherichia coli harbours a chromosome-encoded ampC gene that is expressed naturally at a very low level due to a weak promoter.1 Spontaneous mutations in the promoter region may induce constitutive overproduction of the AmpC β-lactamase, thus conferring resistance to narrow-spectrum cephalosporins.1
Recently, a novel mechanism of resistance to β-lactams has been identified, cephalosporinases with broadened substrate activity in several clinical isolates of Enterobacteriaceae.1,2 These extended-spectrum AmpC (ESAC) β-lactamases are derived from wild-type cephalosporinases by structural modifications in the R1 or the R2 binding sites that contribute to the binding of the C-3 and C-7 side chains of cephalosporins, respectively.2 The R1 binding site is mainly formed by the 
loop, whereas the R2 binding site is formed by the N-terminal extremity of the helix H-11 (Asn-346), the C-terminal extremity of the helix H-9 (Asn-289) and the R2 loop that contains the H-10 helix.2 The resulting ESAC β-lactamases exhibit increased catalytic efficiencies towards extended-spectrum cephalosporins (ESCs), including cefepime and cefpirome, and also slightly against imipenem.2,3 These variants significantly reduce the susceptibility to ESCs, and also the susceptibility to imipenem and ertapenem in strains lacking membrane permeability.2,4 A recent epidemiological survey revealed that it is an emerging mechanism of resistance in E. coli.1
All the ESAC β-lactamases characterized so far have presented only a single structural alteration responsible for the broadened hydrolysis spectrum.1,2 The objective of the present work was to predict the potential evolution of the hydrolysis spectrum displayed by the AmpC β-lactamase of E. coli by combining several amino acid replacements in the R1 and the R2 binding sites: the E219K replacement, which occurred in a particular region of the R1 binding site, the third motif, and the S287N, V298L and N346I replacements, which occurred in particular regions of the R2 binding site, the helix H-9, the R2 loop and the helix H-11, respectively.2 The S287N and the V298L substitutions have already been described in ESAC β-lactamases produced by E. coli isolates,5 whereas the N346I and the E219K replacements have been reported in the plasmid-borne CMY-10 β-lactamase and in the chromosome-borne cephalosporinase SRT-1 produced by the Serratia marcescens GN16694 strain, respectively.2
These four mutations were obtained alone or in combination by site-directed mutagenesis using a site-directed mutagenesis kit (Stratagene), the primers AmpC-EC2-E219K-1 (5'-CCAGGGGCGTTAGATGCTAAAGCTTATGGTGTGAAGTCG-3'), AmpC-EC2-E219K-2 (5'-CGACTTCACACCATAAGCTTTAGCATCTAACGCCCCTGG-3'), AmpC-EC2-S287N-1 (5'-CCTGACAGCATCATTAACGGCAATGGCAATAAAATTGC-3'), AmpC-EC2-S287N-2 (5'-GCAATTTTATTGCCATTGCCGTTAATGATGCTGTCAGG-3'), AmpC-EC2-V298L-1 (5'-GCACTGGCAGCACACCCTTTAAAAGCGATTACGC-3'), AmpC-EC2-V298L-2 (5'-GCGTAATCGCTTTTAAAGGGTGTGCTGCCAGTGC-3'), AmpC-EC2-N346I-1 (5'-GCTAACAAAAACTATCCCATTCCAGCGAGAGTCGCC-3') and AmpC-EC2-N346I-2 (5'-GGCGACTCTCGCTGGAATGGGATAGTTTTTGTTAGC-3') and the recombinant plasmid pEC2, which codes for a narrow-spectrum cephalosporinase of E. coli, as template.5 The mutant β-lactamase genes were sequenced on both strands using an Applied Biosystems sequencer (ABI 377).
A detailed biochemical and phenotypic characterization was performed for the single variants, as described previously,3 to determine the individual effect induced by each amino acid replacement. The amounts of purified enzymes were 90, 80, 55, 70 and 60 mg/L for the β-lactamases AmpC-EC2, AmpC-EC2-E219K, AmpC-EC2-S287N, AmpC-EC2-V298L and AmpC-EC2-N346I, respectively.
This characterization showed that each single substitution was able to increase the level of resistance against ESCs (Table 1) due to different modifications of the enzymatic activity (Table 2). The S287N and the V298L substitutions led to increased affinity for ESCs (lower Km values), whereas the hydrolysis rates were slightly decreased (lower kcat values) (Table 2). In contrast, the N346I substitution decreased the affinity for these compounds but induced a weak increase in the hydrolysis rate, whereas the E219K replacement increased both the hydrolysis rate and the affinity for ESCs.
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The MIC values conferred by the double variants were then determined to assess the combined effect of two amino acid substitutions on the resistance pattern. The six double variants had identical or decreased MICs of ESCs when compared with those displayed by the corresponding single variants (Table 1).
Interestingly, the combination of two amino acid replacements at key positions in the cephalosporinase of E. coli failed to increase further the level of resistance to ESCs and imipenem, which contrasted with the general evolutionary potential of class A β-lactamases. Indeed, the hydrolysis spectrum of several class A penicillinases, such as TEM enzyme, could be expanded step-by-step by the successive addition of several substitutions, thus leading to cefotaximase activity, such as TEM-3, and thereafter to ceftazidimase activity, such as TEM-24.6 This difference underlines the structural discrepancies between the catalytic sites of class A and class C β-lactamases, especially in the loop, which is much larger in cephalosporinases.
Nucleotide sequence accession number
The nucleotide sequences of blaAmpC genes of E. coli EC2 have been deposited in the EMBL nucleotide sequence database under accession number EU497239.
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Funding |
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No external funding was received for this study.
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None to declare.
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Acknowledgements |
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Part of this work was presented at the Eighteenth European Congress of Clinical Microbiology and Infectious Diseases, Barcelona, Spain, 2008 (Poster P1224).
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1 . Mammeri H, Eb F, Berkani A, et al. Molecular characterization of AmpC-producing Escherichia coli clinical isolates recovered in a French hospital. J Antimicrob Chemother (2008) 61:498–503.
2 . Nordmann P, Mammeri H. Extended-spectrum cephalosporinases: structure, detection, and epidemiology. Future Microbiol (2007) 2:297–307.[CrossRef][Medline]
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Mammeri H, Poirel L, Nordmann P. Extension of the hydrolysis spectrum of AmpC β-lactamase of Escherichia coli due to amino acid insertion in the H-10 helix. J Antimicrob Chemother (2007) 60:490–4.
4 . Mammeri H, Nordmann P, Berkani A, et al. Contribution of extended-spectrum AmpC (ESAC) β-lactamases to carbapenem resistance in Escherichia coli. FEMS Microbiol Lett (2008) 282:238–40.[CrossRef][Web of Science][Medline]
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Mammeri H, Poirel L, Fortineau N, et al. Naturally occurring extended-spectrum cephalosporinases in Escherichia coli. Antimicrob Agents Chemother (2006) 50:2573–6.
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Chanal C, Poupart MC, Sirot D, et al. Nucleotide sequences of CAZ-2, CAZ-6, and CAZ-7 β-lactamase genes. Antimicrob Agents Chemother (1992) 36:1817–20.
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