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 re sistance to imipenem and remained susceptible to extended-spectrum cep halosporins. Clavulanic acid partially restored the susceptibility of the strain to imipenem. Two beta-lactamases with isoelectric points (p I) of 6.9 and >9.2 were detected in strain E. cloacae NOR-1; the highe r pI corresponded to AmpC cephalosporinase. Plasmid DNA was not detect ed in E. cloacae NOR-1 and imipenem resistance could not be transferre d into Escherichia coli JM109. The carbapenem-hydrolyzing beta-lactama se gene was cloned into plasmid pACYC184. One recombinant plasmid, pPT N1, harbored a 5.3-kb Sau3A fragment from E. cloacae NOR-1 expressing the carbapenem-hydrolyzing beta-lactamase. This enzyme (pI 6.9) hydrol yzed ampicillin, cephalothin, and imipenem more rapidly than it did me ropenem and aztreonam, but it hydrolyzed extended-spectrum cephalospor ins only weakly and did not hydrolyze cefoxitin. Hydrolytic activity w as partially inhibited by clavulanic acid, sulbactam, and tazobactam, was nonsusceptible to chelating agents such as EDTA and 1,10-o-phenant hroline, and was independent of the presence of ZnCl2. Its relative mo lecular mass was 30,000 Da. Induction experiments concluded that the c arbapenem-hydrolyzing beta-lactamase biosynthesis was inducible by cef oxitin and imipenem. Subcloning experiments with HindIII partial diges ts of pPTN1 resulted in a recombinant plasmid, designated pPTN2, which contained a 1.3-kb insert from pPTN1 and which conferred resistance t o beta-lactam antibiotics. Hybridization studies performed with a 1.2- kb HindIII fragment from pPTN2 failed to determine any homology with a mpC of E. cloacae, with other known beta-lactamase genes commonly foun d in members of the family Enterobacteriaceae (bla(TEM-1) and blas(SHV -3) derivatives), and with previously described carbapenemase genes su ch as those from Xanthomonas maltophilia, Bacillus cereus, Bacteroides fragilis (cfiA), and Aeromonas hydrophila (cphA). This work describin g the biochemical properties of a novel chromosome-encoded beta-lactam ase from E. cloacae indicates that this enzyme differs from all the pr eviously described carbapenemases. This is the first reported cloning of a carbapenem-hydrolyzing beta-lactamase gene from a member of the f amily Enterobacteriaceae.