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Antimicrobial Agents and Chemotherapy, July 2006, p. 2573-2576, Vol. 50, No. 7
0066-4804/06/$08.00+0     doi:10.1128/AAC.01633-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Naturally Occurring Extended-Spectrum Cephalosporinases in Escherichia coli

Hedi Mammeri, Laurent Poirel, Nicolas Fortineau, and Patrice Nordmann*

Service de Bactériologie-Virologie, Hôpital de Bicêtre, Assistance Publique/Hôpitaux de Paris, Faculté de Médecine Paris-Sud, Université Paris-Sud, 94275 Le Kremlin-Bicêtre, France

Received 22 December 2005/ Returned for modification 15 February 2006/ Accepted 27 April 2006


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ABSTRACT
 
Genetic and functional characterization of the cephalosporinases produced by 65 clonally unrelated clinical Escherichia coli isolates revealed genetic diversity of the ampC genes and showed that Gln287, Cys287, Pro296, Leu298, and Phe350 substitutions were involved in extension of the hydrolysis spectrum to include ceftazidime and cefepime.


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TEXT
 
AmpC ß-lactamases (cephalosporinases) are naturally produced by a variety of enterobacterial species (2, 17). Their hydrolytic properties are similar regardless of their origin (17). AmpC overproduction confers resistance to expanded-spectrum cephalosporins (2), except to cefepime and cefpirome, which are weakly hydrolyzed by these ß-lactamases (1, 12). Since 1995, new variants deriving from cephalosporinases have been described in several enterobacterial isolates (1, 6, 7, 9, 12, 13, 14, 19). These enzymes, termed extended-spectrum AmpC ß-lactamases (ESAC), are characterized by increased catalytic efficiency against oxyiminocephalosporins, including cefepime and cefpirome (8).

Repeated isolation of AmpC-producing Escherichia coli isolates with decreased susceptibility to extended-spectrum cephalosporins prompted us to investigate the prevalence of ESAC ß-lactamases in a collection of E. coli strains recovered at the Bicêtre hospital from January 2002 to February 2005. Each isolate that was resistant to amoxicillin and to amoxicillin plus clavulanic acid and that had reduced susceptibility to ceftazidime and cefepime (MICs greater than or equal to 16 µg/ml and 0.5 µg/ml, respectively) without a positive synergy test (10) was retained for this study. Seven cephalosporinase-producing isolates, designated E. coli EC13 to E. coli EC19, were selected together with 56 E. coli isolates that did not produce AmpC at a significant level and two reference strains, E. coli KL (producing an ESAC) and E. coli 154792 (producing a typical cephalosporinase) (12).

The 65 isolates were compared by enterobacterial repetitive intergenic consensus PCR (22), whereas ESAC-producing isolates were also compared by pulsed-field gel electrophoresis analysis (16, 21). All of the isolates were genotypically unrelated (data not shown).

PCR amplifications of ampC genes were performed (13) with primers Int-B2 (5'-TTCCTGATGATCGTTCTGCC-3') and Int-HN (5'-AAAAGCGGAGAAAAGGTCCG-3'), yielding a 1,315-bp amplification product that contained the entire ampC gene, including its own promoter sequence. Sequence analyses were performed with PAUP version 3.1.1 and software available at the internet websites www.ncbi.nlm.nih.gov and http://www.ebi.ac.uk/clustalw/. It revealed that ampC genes of E. coli may be divided into several clusters (Fig. 1). Since the species E. coli is divided into four main phylogroups (A, B1, B2, and D) (4), a PCR-based phylotyping analysis was applied to the 65 strains as previously described (4). It revealed that the ampC clusters described above are related to phylogroups A and B1, B2, and D (Fig. 1). ESAC-producing strains E. coli EC13 to E. coli EC19 and E. coli KL belonged to phylogroup A or B1. This common origin can be attributed to the high prevalence of E. coli strains of these phylogroups in the digestive flora (4).


Figure 1
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  		FIG. 1. Phylogeny of the chromosomal ampC gene of the 65 E. coli isolates studied. The tree was obtained by the parsimony method. Three major groups are shown. ampC genes coding for ESAC ß-lactamases are boxed. The phylogroups of the E. coli strains are indicated by brackets.

Sequence analysis of the ampC genes of E. coli isolates EC13 to EC19 revealed mutations at position –42 or –32 or insertion of 1 bp between positions –15 and –16 in their own promoter region, which has been shown to account for AmpC expression at different levels (3, 15). Plasmids of these AmpC-producing strains were extracted (11) and transferred onto a nylon membrane (20). Hybridization of the membrane with a fluorescein-labeled probe that was made of the PCR product of the ampC-KL gene (12) failed to detect the ß-lactamase gene in the plasmid DNA contents (data not shown). In addition, transformation experiments performed as previously described (12), with plasmid DNA of AmpC-producing isolates, failed to obtain AmpC-producing transformants. All of these results argued for a chromosomal location of those ampC genes.

Amplification with primers Int-B1 (5'-TTTTGTATGGAACCAGACC-3') and Int-HN of ampC genes from E. coli isolates EC1, EC2, EC13 to EC20, EC23, EC24, EC27, EC30, EC31, EC34, EC37, EC41, EC43, EC55, EC58, E. coli KL, and E. coli 154297 gave PCR products of 1,120 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 (12), giving rise to clones harboring recombinant plasmids pEC1, pEC2, pEC13 to pEC20, pEC23, pEC24, pEC27, pEC30, pEC31, pEC34, pEC37, pEC41, pEC43, pEC55, pEC58, pKL, and pS4, respectively. In all of the recombinant plasmids, the orientation of the cloned insert was the same, with the ampC gene under the transcriptional control of the lacZ promoter flanking the cloning site.

The ß-lactamase activity against cephalothin and cefepime and the MICs of several ß-lactams were determined for recombinant strains as described previously (18). Results are shown in Tables 1 and 2. Recombinant strains E. coli TOP10(pEC1) and E. coli TOP10(pEC2) had ß-lactamase activities and MICs similar to those of strains E. coli TOP10(pEC20) to E. coli TOP10(pEC58).


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  		TABLE 1. ß-Lactamase activities of E. coli TOP10 strains harboring recombinant plasmids


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  		TABLE 2. MICs of ß-lactams for E. coli clinical isolates EC13 to EC19 and for recombinant E. coli clones TOP10(pEC1), TOP10(pEC13) to TOP10(pEC19), TOP10(pKL), and TOP10(pCR-BluntII-Topo) containing the empty vector

Comparison of amino acid sequences (Table 3), MICs (Table 2), and ß-lactamase activities (Table 1) showed that the enhancement of the hydrolysis activity against ceftazidime and cefepime was related to Ser->Gln, Ser->Cys, His->Pro, Val->Leu, and Val->Phe substitutions at positions 287, 287, 296, 298, and 350, respectively (Table 1). The effects of the S287N and V298L substitutions on the resistance levels and ß-lactamase activities are greater than those related to the S287C, H296P, and V350F substitutions (Tables 1 to 3). AmpC-EC13, AmpC-EC18, and AmpC-EC14, which have an S287N or V298L substitution, had reduced susceptibility to cefepime and cefpirome (MICs equal to 8 or 16 µg/ml), whereas AmpC-EC15, AmpC-EC16, AmpC-EC17, AmpC-EC19, and AmpC-KL, which presented an S287C, H296P, or V350F substitution, did not confer resistance to cefepime, although the MICs for the strains producing these proteins were 30- to 60-fold higher than those for wild-type E. coli.


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  		TABLE 3. Comparison of amino acid sequences of ESAC ß-lactamases and those of representative narrow-spectrum cephalosporinases

The region containing residues 287, 296, and 298 is located inside or in close proximity to helix H-10 (7, 12). This region is probably a hot spot where amino acid deletions leading to extension of the hydrolysis spectrum were already described in AmpCD from E. coli HKY28 (7), AmpC-HD from Serratia marcescens HD (13), and AmpC-CHE from Enterobacter cloacae MHN1 (1).

Interestingly, a V350F substitution is responsible for extended-spectrum hydrolysis in AmpC from E. coli belonging to phylogroup B1 but not in AmpC from E. coli belonging to phylogroup B2 (Table 2 and Fig. 1), suggesting that other residues may contribute to modify the hydrolysis spectrum in combination with a Phe350 substitution.

This study indicates that isolation of ESAC-producing E. coli strains, which are resistant to ceftazidime according to the CLSI criteria (5), occurred in clinical isolates and could be underestimated because of the slight reduction of susceptibility to cefepime and cefpirome.

Nucleotide sequence accession numbers. The GenBank accession numbers for the ampC sequences reported here are DQ092424 (EC5), DQ092425 (EC6), DQ92426 (EC7), DQ092427 (EC8), DQ092428 (EC9), DQ092429 (EC10), DQ092430 (EC11), DQ092431 (EC12), DQ092432 (EC13), DQ092433 (EC14), DQ091198 (EC15), DQ092434 (EC16), DQ091197 (EC17), AY533244 (EC18), AY533245 (EC19) SQ092420 (EC20), DQ092421 (EC26), DQ092422 (EC30), and DQ092423 (EC31).


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ACKNOWLEDGMENTS
 
This work was funded by the European Community (6th PCRD, LSHM-CT-2003-503-335) and by a grant from the Ministère de l'Education Nationale et de la Recherche (UPRES-EA3539), Université Paris XI, Paris, France. L.P. is a researcher from INSERM, Paris, France.


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FOOTNOTES
 
* Corresponding author. Mailing address: Service de Bactériologie-Virologie, Hôpital de Bicêtre, 78 rue du Général Leclerc, 94275 Le Kremlin-Bicêtre Cedex, France. Phone: 33-1-45-21-36-32. Fax: 33-1-45-21-63-40. E-mail: nordmann.patrice{at}bct.ap-hop-paris.fr. Back


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Antimicrobial Agents and Chemotherapy, July 2006, p. 2573-2576, Vol. 50, No. 7
0066-4804/06/$08.00+0     doi:10.1128/AAC.01633-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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