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Antimicrobial Agents and Chemotherapy, October 2004, p. 4050-4053, Vol. 48, No. 10
0066-4804/04/$08.00+0     DOI: 10.1128/AAC.48.10.4050-4053.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

AmpC ß-Lactamase in an Escherichia coli Clinical Isolate Confers Resistance to Expanded-Spectrum Cephalosporins

Hedi Mammeri,1 Hasan Nazic,1,2 Thierry Naas,1 Laurent Poirel,1 Sophie Léotard,1 and Patrice Nordmann1*

Service de Bactériologie-Virologie, Hôpital de Bicêtre, Assistance Publique/Hôpitaux de Paris, Faculté de Médecine Paris-Sud, Université Paris XI, Le Kremlin-Bicêtre, France,1 Department of Microbiology, Faculty of Medicine, Istanbul University, Istanbul, Turkey2

Received 5 February 2004/ Returned for modification 14 April 2004/ Accepted 26 June 2004


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ABSTRACT
 
Cloning, sequencing, and biochemical analysis identified a novel AmpC-type ß-lactamase conferring resistance to extended-spectrum cephalosporins in an Escherichia coli clinical isolate. This enzyme, exhibiting 14 amino acid substitutions compared to a reference AmpC cephalosporinase of E. coli, hydrolyzed ceftazidime and cefepime significantly.


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TEXT
 
AmpC-type ß-lactamases confer resistance to aminopenicillins and narrow-spectrum cephalosporins (14) but spare oxyimino-cephalosporins (ceftazidime and cefotaxime) unless these ß-lactamases are expressed at a high level (16). In recent years, rare AmpC-type ß-lactamases that confer resistance to cefepime and cefpirome have been recovered from Serratia marcescens and Enterobacter cloacae (2, 7, 9).

For this study, we report findings respecting an Escherichia coli isolate that produced a chromosomal cephalosporinase possessing an extended spectrum activity.

E. coli KL was recovered from the urine specimen of an 81-year-old woman who was admitted from her home into the emergency medical unit of the Bicêtre hospital (Kremlin Bicêtre, France) for pyelonephritis in January 2001 (Table 1). No antibiotic treatment was prescribed during the month preceding her hospitalization. The infection was treated with imipenem successfully. E. coli KL was selected for further study on the basis of its uncommon pattern of resistance to ß-lactam antibiotics, including reduced susceptibility to cefepime. It was also resistant to cotrimoxazole and fluoroquinolones.


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  		TABLE 1. Clinical features and ß-lactamase content of the strains

Transformation experiments using plasmid DNA from E. coli KL extracted with a plasmid Midi kit (QIAGEN, Courtaboeuf, France) were performed as previously described (3) and yielded an E. coli DH10B(pNAT) strain that had a resistance phenotype consistent with a penicillinase biosynthesis (Table 2). A PCR product obtained with primers preTEM-1 (5'-GTATCCGCTCATGAGACAATA-3') and preTEM-2 (5'-TCTAAAGTATATATGAGTAAACTTGGTCTG-3') and with E. coli DH10B(pNAT) as the template shared 100% identity with TEM-1. The resistance to cephalosporins of E. coli KL was not transferable, suggesting that resistance to extended-spectrum cephalosporins was chromosomally mediated.


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  		TABLE 2. MICs of ß-lactams for E. coli isolate and for recombinant E. coli DH10B(pBK-KL), E. coli DH10B(pNAT), E. coli 154297 clinical isolate, recombinant E. coli DH10B (pBK-S4) AmpC S4 and E. coli DH10B

Whole-cell DNA of E. coli KL was extracted as described previously (3). Primers AmpC AK (5'-GTCCGGATCCCATTACCCTGGCGCATCGT-3') and AmpC BK (5'-CGAGAATTCGGACCCGATGGAATTTTAC-3') were designed to amplify a 2,500-bp fragment including the entire blaampC gene and its promoter. The amplification product was cloned using a PCR-Script Cam cloning kit (Stratagene, Amsterdam, The Netherlands), and recombinant plasmid was electroporated, yielding an E. coli DH10B(pBK-KL) strain.

E. coli clinical isolate 154297 was used to clone a wild-type AmpC ß-lactamase gene (Table 1). Sau3AI-restricted DNA fragments from E. coli 154297 clinical isolates yielded recombinant E. coli DH10B(pBK-S4) and E. coli DH10B(pBK-OXA-30) strains.

DNA sequence analysis showed that recombinant plasmid pBK-OXA-30 carried a ß-lactamase gene sharing 100% identity with blaOXA-30 whereas ß-lactamase AmpC KL presented 14 and 10 amino acid changes compared to wild-type ß-lactamases AmpC S4 and AmpC K12, respectively (4) (Fig. 1). No amino acid changes occurred in the KIA {alpha}-helix (lysine, isoleucine, and alanine residues at positions 306, 307, and 308) that corresponds to the H-10 helix in the cephalosporinase of E. cloacae (2, 8, 18), in accordance with the results reported for some cefepime-resistant enterobacterial strains (1, 2, 9, 10). Moreover, no mutation occurred at position 86 in AmpC KL ß-lactamase (residue 64 in the mature AmpC enzyme of S. marcescens), in accordance with the results reported for the expanded-spectrum AmpC ß-lactamase produced by the in vitro-selected S. marcescens 520R strain (15). Thus, the molecular basis of the extended-spectrum hydrolysis of AmpC KL remained unknown since none of the 10 amino acid changes compared to the results seen with AmpC of E. coli K12 has been identified previously as the source of hydrolysis spectrum extension. Compared to the results seen with the wild-type promoter of E. coli K12, DNA sequence analysis of the blaampC KL promoter showed seven point substitutions, including a C->T transition at position –42 that made a perfect TTGACA box upstream of a typical wild-type –35 box, modifying the transcription initiation site (5, 13). Moreover, an insertion of 7 bp (GCCAATG) was found at position +11.



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  		FIG. 1. Comparison of amino acid sequences of AmpC KL and AmpC S4 with those of other AmpC ß-lactamases. The sources of the enzymes are as follows: AmpC K12 is from E. coli K12 (5), AmpC CHE is from E. cloacae CHE (2), and AmpC HD is from S. marcescens (15). ß-Lactamases AmpC KL, AmpC CHE, and AmpC HD confer resistance to cefepime. The arrow indicates cleavage of the peptide leader site for AmpC K12. Amino acid residues of AmpC CHE and AmpC HD that were deleted compared to those of the wild-type cephalosporinase appear in black dashes on a grey background (2, 15). Dashes have been introduced to optimize the alignment, and dots indicate amino acid residues identical to those of AmpC KL. Conserved residues of class C ß-lactamases are underlined.

Isoelectric focusing analysis (3) of the culture extract of E. coli KL gave two ß-lactamase activities with pI values of 8.5 and 5.4 that comigrated with ß-lactamases extracted from recombinant E. coli DH10B(pBK-KL) and E. coli DH10B (pNAT). E. coli 154297 produced a single ß-lactamase with a pI value of 7.3 consistent with the production of OXA-30. E. coli DH10B(pBK-S4) produced a single ß-lactamase AmpC S4 with a pI value of 8.5.

Susceptibility testing by an agar dilution technique (11) showed that E. coli KL was resistant to amino- and carboxypenicillins, to narrow-spectrum cephalosporins, and to ceftazidime, had intermediate susceptibility to piperacillin, and was susceptible to cefotaxime, ceftriaxone, aztreonam, cefepime, cefpirome, and imipenem. MICs of ß-lactams for E. coli DH10B (pBK-KL) showed that the pattern of resistance of the recombinant strain mirrored that of the parental strain except with respect to the susceptibility to ticarcillin. E. coli DH10B(pBK-S4), which produced a wild-type cephalosporinase, was resistant to amoxicillin and to narrow-spectrum cephalosporins and was susceptible to the extended-spectrum cephalosporins, including ceftazidime, cefepime, and cefpirome (Table 2). It was noteworthy that the MICs of cefepime and cefpirome for E. coli DH10B(pBK-KL) were sixfold higher than for E. coli DH10B(pBK-S4), indicating that ß-lactamase AmpC KL conferred significant weaker susceptibility to these compounds.

AmpC KL and AmpC S4 were purified as previously described (9) to near (>99%) homogeneity, as deduced from sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis (data not shown). The specific activities, as determined with 100 µM cephalothin as the substrate, were 10 µmol · min–1 · mg of protein–1 and 11 µmol · min–1 · mg of protein–1 for AmpC KL and AmpC S4, respectively.

Purified ß-lactamases AmpC S4 and AmpC KL were used for kinetic measurements (Km and kcat) (9). The kinetic parameters for penicillins for both AmpC enzymes were similar, whereas the catalytic efficiency of the purified ß-lactamase AmpC KL against the cephalosporins was higher than that for the enzyme AmpC S4 (Table 3). The increased catalytic efficiency, due to a decrease of the Km values, was important for the extended-spectrum cephalosporins, such as ceftazidime, cefepime, and cefpirome. However, the results seen with respect to catalytic efficiency against cefotaxime and ceftriaxone between AmpC KL and S4 ß-lactamases were almost identical.


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  		TABLE 3. Kinetic parameters of ß-lactamases AmpC KL and AmpC S4

Conclusion. Reduced susceptibility to cefepime and cefpirome in E. coli is mostly related to clavulanic-acid-inhibited extended-spectrum ß-lactamase porin modification (17) combined with overexpression of cephalosporinase (12) and oxacillinase (see, for example, MICs for the OXA-30 producer shown in Table 2) (6). Herein, we described for the first time an E. coli isolate that produced an AmpC ß-lactamase conferring resistance to expanded-spectrum cephalosporins.

Nucleotide sequence accession numbers. The nucleotide sequences of blaampC KL and blaampC S4 genes have been submitted to the GenBank nucleotide database and have been assigned accession numbers AY533244 and AY533245, respectively.


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


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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, October 2004, p. 4050-4053, Vol. 48, No. 10
0066-4804/04/$08.00+0     DOI: 10.1128/AAC.48.10.4050-4053.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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