JAC Advance Access originally published online on June 22, 2007
Journal of Antimicrobial Chemotherapy 2007 60(3):490-494; doi:10.1093/jac/dkm227
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Extension of the hydrolysis spectrum of AmpC ß-lactamase of Escherichia coli due to amino acid insertion in the H-10 helix
Service de Bactériologie-Virologie, Hôpital de Bicêtre, Assistance Publique/Hôpitaux de Paris, Faculté de Médecine Paris-Sud, Université Paris Sud, 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
Received 28 December 2006; returned 22 February 2007; revised 2 May 2007; accepted 30 May 2007
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Objectives: To characterize the naturally occurring expanded-spectrum ß-lactamase from an Escherichia coli clinical isolate and to compare it with a wild-type ß-lactamase.
Methods: The chromosome-borne ampC genes from E. coli BER and E. coli EC2 were PCR amplified, sequenced and cloned into an expression vector. Antimicrobial susceptibilities of the parental isolate and the recombinant strains were determined by agar dilution methods. Kinetic parameters were determined from purified AmpC BER and AmpC EC2.
Results: AmpC BER was overexpressed in its original clinical isolate because of mutations in the promoter region of its gene at positions –42 and –18. The analysis of the ampC coding sequence revealed a 6 bp insertion when compared with the wild-type sequence leading to the tandem duplication of two alanine residues inside the H-10 helix. AmpC BER-producing recombinants were resistant to ceftazidime, had reduced susceptibility to other oxyiminocephalosporins (cefotaxime and cefepime), but had a greater susceptibility to cefoxitin when compared with the recombinant expressing the wild-type ß-lactamase AmpC EC2. The affinity of AmpC BER for cephalosporins and imipenem was increased, whereas the hydrolysis rate was decreased for all these compounds. In addition, the IC50 values of clavulanic acid and tazobactam for AmpC BER were increased.
Conclusions: This work sheds new light on structure–function relationships of expanded-spectrum AmpC ß-lactamases.
Keywords: expanded-spectrum AmpC , cefepime , E. coli
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Introduction |
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Most class C ß-lactamases produced by Gram-negatives hydrolyse many ß-lactam antibiotics in vitro, including cephamycins (cefoxitin and cefotetan) and oxyiminocephalosporins, such as ceftazidime, cefotaxime and ceftriaxone, and monobactams such as aztreonam, but usually to a lesser extent.1 In many enterobacterial species, the expression of the chromosomal ampC gene is low and inducible.2 Escherichia coli behaves differently as its chromosomal AmpC is constitutively produced at a very low level because of a weak promoter as well as a transcriptional attenuator.3 Spontaneous mutations affecting the promoter region of the ampC gene in E. coli induce constitutive overproduction of the AmpC ß-lactamase and confer resistance to early generation cephalosporins.3,4 Zwitterionic cephalosporins, such as cefepime and cefpirome, and imipenem, which penetrate very efficiently through the outer membrane of Gram-negatives and are usually poor substrates for AmpC ß-lactamases, are active in vitro against organisms producing high levels of cephalosporinases.5
Recently, a novel mechanism of resistance due to the production of cephalosporinases with broadened substrate activity has been reported in several clinical isolates of the Enterobacter cloacae, Enterobacter aerogenes, Serratia marcescens and E. coli.6 Those expanded-spectrum AmpC (ESAC) ß-lactamases confer reduced susceptibility to all cephalosporins including cefepime and cefpirome. These enzymes are structurally related to wild-type cephalosporinases, but have structural modifications in four regions of the proteins that are in the vicinity of the active site: the 
loop,7–11 the H-10 helix,12–17 the H-2 helix18 and the C-terminal end of the protein.19
Most of the ESAC ß-lactamases are encoded by ampC genes which are chromosomally located.6 However, a recent study showed that the plasmid-encoded cephalosporinase CMY-10 exhibited kinetic parameters consistent with ESAC ß-lactamase because of a shorter R2 loop, which is located beyond the H-10 helix in the vicinity of the active site.20
The aim of this study was to characterize a novel ESAC ß-lactamase, which presented the peculiarities of a novel structural modification confering a pattern of resistance sharing biochemical features with class A extended-spectrum ß-lactamases (ESBLs).
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Bacterial strains
Clinical isolate E. coli BER was identified using API 20E system (bioMérieux, Marcy l’Étoile, France). E. coli TOP10 and azide-resistant E. coli J53 were used for transformation and conjugation experiments, respectively.17 E. coli strain EC2 was previously characterized as a wild-type AmpC producer.17
Plasmid DNA extraction, conjugation and transformation experiments
Plasmid DNA was extracted using the plasmid Midi kit (Qiagen, Courtaboeuf, France). Transfer by conjugation or transformation of ß-lactam resistance marker was attempted from E. coli BER, as described previously.19 In addition, as E. coli BER was resistant to gentamicin and chloramphenicol, selection of transformants was also attempted on agar plates containing these antibiotics.
Amplification of the ampC genes and sequence analysis
PCR amplifications of ampC genes were performed with primers Int-B2 (5'-TTCCTGATGATCGTTCTGCC-3') and Int-HN (5'-AAAAGCGGAGAAAAGGTCCG-3'), yielding a 1315 bp amplification product that contained the entire ampC gene of E. coli, including its own promoter sequence. Sequence analyses were performed with PAUP version 3.1.1 and software available at the internet web sites www.ncbi.nlm.nih.gov and http://www.ebi.ac.uk/clustalw/.
Amplification with primers Int-B1 (5'-TTTTGTATGGAACCAGACC-3') and Int-HN of ampC genes from E. coli BER and EC2 gave PCR products of 1120 bp containing only the coding regions without their own promoters. These PCR products were cloned into pCR-BluntII-Topo (Invitrogen), and the recombinant plasmids were subsequently transformed into E. coli strain TOP10, as described previously.17
Antimicrobial agents and MIC determination
The antibiotic agents and their sources have been described elsewhere.21 MICs were determined by an agar dilution technique on Mueller–Hinton agar (Sanofi-Diagnostics Pasteur, Paris, France) with an inoculum of 104 cfu per spot and were interpreted according to the guidelines of the Clinical and Laboratory Standards Institute.22
Recombinant E. coli TOP10 strains were grown overnight at 37°C in 4 L of trypticase soy (TS) broth containing amoxicillin (100 mg/L) and kanamycin (30 mg/L), resuspended in 40 mL of 100 mM phosphate buffer (pH 7), disrupted by sonication and centrifuged at 20 000 g for 1 h at 4°C, as described previously.14 ß-Lactamase extracts were filtered through a 0.45 µm pore-size filter (Millipore, Saint-Quentin-en-Yvelines, France), dialysed overnight at 4°C against 20 mM Tris (pH 7.5) and loaded onto a pre-equilibrated Q-Sepharose column (Amersham Pharmacia Biotech). The flow-through fractions containing the ß-lactamase were recovered and dialysed against 50 mM phosphate buffer (pH 6) before loading onto a pre-equilibrated S-Sepharose column (Amersham Pharmacia Biotech). The enzymes were eluted by a linear NaCl gradient (0–1 M) in the same buffer. Eluted fractions with highest ß-lactamase activity (nitrocefin test) were pooled and dialysed against 100 mM phosphate buffer (pH 7). To assess the purity of the extracts and to determine the molecular weight of the AmpC ß-lactamases, purified enzymes were subjected to SDS–PAGE analysis.21 The molecular weights were also determined in silico using the software available over the internet from the Expasy web site (http://expasy.org/tools/pi_tool.html).
Purified ß-lactamases AmpC BER and AmpC EC2 were used for kinetic measurements (Km and kcat), which were carried out at 30°C in 100 mM sodium phosphate (pH 7.0). The rates of hydrolysis were determined with a Pharmacia ULTROSPEC 2000 spectrophotometer and were analysed using the SWIFT II software (Amersham Pharmacia Biotech). Km and kcat values were determined by analysing the ß-lactam hydrolysis under initial rate conditions by using the Eadie–Hofstee linearization of the Michaelis–Menten equation, as described previously.23 When the Km value was very low, Ki was determined instead of Km using cefaloridine as the substrate and the kcat value was determined from initial rates at saturating substrate concentrations ([S]>>Km). Values were the mean of three independent measures.
The concentrations of inhibitors giving a 50% reduction in hydrolysis of cefalotin (IC50) were measured after 10 min of pre-incubation of the enzymes with the inhibitors at 37°C and cefalotin as the substrate at 100 µM.
The PCR-based phylotyping analysis described by Clermont et al.24 was applied to E. coli BER. The method, which uses a combination of two genes (chuA and yjaA) and an anonymous DNA fragment, allows the determination of the main phylogenetic groups of E. coli, A, B1, B2 and D.
Nucleotide sequence accession number
The nucleotide sequence of the blaampC BER gene from E. coli isolate BER has been assigned to the GenBank nucleotide database under the accession number EF125541.
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The E. coli clinical isolate BER was recovered from a urinary sample of a kidney-transplanted 52-year-old woman admitted in the nephrology ward of the Bicêtre Hospital (K-Bicêtre, France). This isolate was selected for further study on the basis of its uncommon pattern of susceptibility to ß-lactam antibiotics, including reduced susceptibility to cefepime. E. coli BER was susceptible to aminoglycosides and co-trimoxazole, but was resistant to fluoroquinolones. Phylogenetic typing revealed that E. coli BER belonged to phylogenetic group A.
The analysis of the plasmid DNA content of E. coli BER identified a single plasmid of 15 kb (data not shown). Transformation and conjugation experiments failed to transfer any amoxicillin resistance marker, suggesting a very likely chromosomal location of the ß-lactamase gene, whereas the gentamicin and the chloramphenicol resistance markers were co-transferred by transformation.
Whole-cell DNA of E. coli BER was extracted and the entire ampC gene was amplified and analysed, as described previously.17 The analysis of the nucleotide sequence showed mutations in the promoter region, according to the wild-type promoter of E. coli.25 The C 
T mutation at position –42 resulted in a consensus TTGACA box upstream of the native –35 sequence and the T A transversion at position –18 resulted in a new –10 box separated by 17 bp from the new –42 box giving rise to a strong promoter.3
The analysis of the coding sequence revealed a 6 bp insertion according to the ampC EC2 gene, which coded for a narrow-spectrum cephalosporinase described previously.17 This insertion led to the tandem duplication of two alanine residues, which are located in the H-10 helix, at positions 294 and 295.
The expansion of the substrate specificity in ESAC ß-lactamases was attributable to amino acid changes in specific locations of the proteins that are located in the vicinity of the active site.6 Amino acid replacements in the H-2 helix,18 in the loop,10,11 in the vicinity of the H-10 helix12,15,17 or in the C terminal end of the protein19 accounted for a broadened substrate specificity. In addition, amino acid deletions in the vicinity of the H-10 helix13,14,16 and amino acid insertion in the loop8,9 were also shown to result in an increase of the hydrolysis spectrum. In this work, we describe a novel structural modification leading to broadened hydrolysis spectrum that consisted of amino acid insertion in the H-10 helix.
Cloning experiments using PCR products from E. coli BER and E. coli EC2 as template yielded recombinant strains E. coli TOP10 (pBER) and E. coli TOP10 (pEC2), respectively.
The MICs of several ß-lactams for E. coli isolates BER and EC2 and their corresponding E. coli TOP10 clones are reported in Table 1. Susceptibility data showed that E. coli BER was resistant to amoxicillin, cefalotin and ceftazidime and presented a reduced susceptibility to cefotaxime, aztreonam and cefepime, whereas it remained susceptible to ticarcillin and cefoxitin (Table 1). The isolate was found to be fully susceptible to ß-lactams when cloxacillin (200 mg/L)-containing agar plates were used, thus indicating that the resistance was due to AmpC production (data not shown).26 Moreover, E. coli TOP10 (pEC2) recombinant strain, which produced AmpC EC2, presented a reduced susceptibility to narrow-spectrum cephalosporins, such as cefalotin, cefuroxime and cefoxitin, whereas recombinants producing this enzyme remained susceptible to extended-spectrum cephalosporins (Table 1). The recombinant that expressed the ESAC BER displayed a higher level of resistance to oxyiminocephalosporins, including cefepime and cefpirome, whereas susceptibility to cefuroxime and cefoxitin was increased. E. coli TOP10 (pBER) remained susceptible to imipenem.
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The pattern of resistance conferred by AmpC BER shared similarities with those conferred by class A ESBLs.1 AmpC BER conferred reduced susceptibility to oxyiminocephalosporins, including cefepime, which is usually spared by AmpC ß-lactamases, whereas the susceptibility to 7-methoxycephalosporins, such as cefoxitin, was not significantly modified. These common phenotypic features could lead to misidentification between these two types of ESAC ß-lactamases. The identification of the ESAC enzymes relies on the negativity of the synergy test between extended-spectrum cephalosporins and clavulanic acid27 and the full inhibition of these variant AmpC enzymes when cloxacillin-containing agar plates are used (P. Nordmann, personal communication).
AmpC enzymes were purified to near homogeneity (>99%) as deduced from the SDS–PAGE analysis. The specific activities, determined with 100 µM of cefalotin as substrate, were 48 and 490 µmol/min mg of protein for AmpC BER and AmpC EC2, respectively. Comparison of specific activities before and after purification showed purification factors of 27 and 35 for AmpC BER and AmpC EC2, respectively. Mature proteins had similar relative molecular masses, determined theoretically and experimentally to be 
40 kDa (data not shown).
The Km and kcat values of AmpC BER were decreased, with respect to those of AmpC EC2, for all cephalosporins and imipenem, thus indicating that the variant enzyme presented a higher affinity for these compounds but displayed lower rates of hydrolysis (Table 2). The resulting catalytic efficiencies (kcat/Km) of AmpC BER were increased against extended-spectrum cephalosporins and imipenem, whereas they were decreased against narrow-spectrum cephalosporins, which was in accordance with the susceptibility profiles. Moreover, the IC50 values of tazobactam or clavulanate for AmpC BER were slightly increased when compared with the wild-type ß-lactamase AmpC EC2 (Table 3).
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Previous studies showed that carbapenem resistance may result from the production of a large amount of chromosomal or plasmid-mediated cephalosporinases combined with an important decreased drug permeability through the outer membrane because of the loss of several porins.28 Production of ESAC ß-lactamases, which displayed an increased hydrolytic activity against imipenem, may increase the risk for the selection of carbapenem-resistant isolates among strains that have a reduced membrane permeability.
In conclusion, although inducible chromosomal AmpC ß-lactamases confer resistance to extended-spectrum cephalosporins (except cefepime and cefpirome) due to massive overexpression because of mutations in their ß-lactamase regulatory genes,2 AmpC ß-lactamases with structural alterations and broadened hydrolysis spectra also represent a possible mechanism of increased resistance to oxyiminocephalosporins. They may be particularly important in E. coli where there is no AmpC induction mechanism, meaning that a large increase in enzyme expression is more difficult to achieve.13,17
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None to declare.
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Present address. Service de Bactériologie-Hygiène, Centre Hospitalier Universitaire d'Amiens, Hôpital, Nord, 2 Place Victor Pauchet, 80080 Amiens, France. 
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This work was funded by a grant from the Ministère de l'Education Nationale et de la Recherche (UPRES-EA3539), Université Paris XI, France and mostly by a grant from the European Community (LSHM-CT-2005-018705). L. P. is a researcher from the INSERM (Paris, France).
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References |
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19
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