Extension of the hydrolysis spectrum of AmpC beta-lactamase of Escherichia coli due to amino acid insertion in the H-10 helix.
OBJECTIVE
To characterize the naturally occurring expanded-spectrum beta-lactamase from an Escherichia coli clinical isolate and to compare it with a wild-type beta-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 beta-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 beta-lactamases.
Mammeri
Hedi
H
Service de Bactériologie-Virologie, Hôpital de Bicêtre, Assistance Publique/Hôpitaux de Paris, Faculté de Médecine Paris-Sud, Université Paris Sud, 94275 K.-Bicêtre, France.
Poirel
Laurent
L
Nordmann
Patrice
P
D000596:Amino Acids / Q000737:chemistry / Q000378:metabolism*; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D002511:Cephalosporins / Q000494:pharmacology; D003001:Cloning, Molecular; D003227:Conjugation, Genetic / Q000187:drug effects; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D006868:Hydrolysis; D015378:Imipenem / Q000494:pharmacology; D007700:Kinetics; D008826:Microbial Sensitivity Tests; D009154:Mutation / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013329:Structure-Activity Relationship; D014169:Transformation, Bacterial / Q000235:genetics; D001618:beta-Lactamases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*
D000596:Amino Acids; D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D002511:Cephalosporins; D004269:DNA, Bacterial; D015378:Imipenem; C048795:AmpC beta-lactamases; D001618:beta-Lactamases