Biochemical properties of a carbapenem-hydrolyzing beta-lactamase from Enterobacter cloacae and cloning of the gene into Escherichia coli.
A clinical isolate of Enterobacter cloacae, strain NOR-1, exhibited resistance to imipenem and remained susceptible to extended-spectrum cephalosporins. Clavulanic acid partially restored the susceptibility of the strain to imipenem. Two beta-lactamases with isoelectric points (pI) of 6.9 and > 9.2 were detected in strain E. cloacae NOR-1; the higher pI corresponded to AmpC cephalosporinase. Plasmid DNA was not detected in E. cloacae NOR-1 and imipenem resistance could not be transferred into Escherichia coli JM109. The carbapenem-hydrolyzing beta-lactamase gene was cloned into plasmid pACYC184. One recombinant plasmid, pPTN1, harbored a 5.3-kb Sau3A fragment from E. cloacae NOR-1 expressing the carbapenem-hydrolyzing beta-lactamase. This enzyme (pI 6.9) hydrolyzed ampicillin, cephalothin, and imipenem more rapidly than it did meropenem and aztreonam, but it hydrolyzed extended-spectrum cephalosporins only weakly and did not hydrolyze cefoxitin. Hydrolytic activity was partially inhibited by clavulanic acid, sulbactam, and tazobactam, was nonsusceptible to chelating agents such as EDTA and 1,10-o-phenanthroline, and was independent of the presence of ZnCl2. Its relative molecular mass was 30,000 Da. Induction experiments concluded that the carbapenem-hydrolyzing beta-lactamase biosynthesis was inducible by cefoxitin and imipenem. Subcloning experiments with HindIII partial digests of pPTN1 resulted in a recombinant plasmid, designated pPTN2, which contained a 1.3-kb insert from pPTN1 and which conferred resistance to beta-lactam antibiotics. Hybridization studies performed with a 1.2-kb HindIII fragment from pPtN2 failed to determine any homology with ampC of E. cloacae, with other known beta-lactamase genes commonly found in members of the family Enterobacteriaceae (bla(TEM-1)) and bla(SHV-3) derivatives), and with previously described carbapenemase genes such as those from Xanthomonas maltophilia, Bacillus cereus, Bacteroides fragilis (cfiA), and Aeromonas hydrophila (cphA). This work describing the biochemical properties of a novel chromosome-encoded beta-lactamase from E. cloacae indicates that this enzyme differs from all the previously described carbapenemases. This is the first reported cloning of a carbapenem-hydrolyzing gene from a member of the family Enterobacteriaceae.
Laboratoire de Microbiologie, Hôpital Raymond Poincaré, Faculté de Médecine Paris-Ouest, Garches, France.
P
P
Nordmann
S
S
Mariotte
T
T
Naas
R
R
Labia
M H
MH
Nicolas
D015780:Carbapenems / Q000378:metabolism*; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry; D004352:Drug Resistance, Microbial; D016972:Enterobacter cloacae / Q000201:enzymology* / Q000235:genetics*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D005798:Genes, Bacterial; D006868:Hydrolysis; D007525:Isoelectric Focusing; D008826:Microbial Sensitivity Tests; D008970:Molecular Weight; D009693:Nucleic Acid Hybridization; D012689:Sequence Homology, Nucleic Acid; D001618:beta-Lactamases / Q000096:biosynthesis / Q000235:genetics*
D015780:Carbapenems; D004269:DNA, Bacterial; D001618:beta-Lactamases