Biochemical properties of a carbapenem-hydrolyzing beta-lactamase from Enterobacter cloacae and cloning of the gene into Escherichia coli

Abstract

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 13-lactamases with isoelectric points (pI) of 6.9 and >9.2 were detected in strain E.cloacae NOR-1; the higher pl 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 13-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 13-lactamase. This enzyme (pl 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 ,3-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 13-lactam antibiotics.Hybridization studies performed with a 1.2-kb Hindlll fragment from pPTN2 failed to determine any homology with ampC of E. cloacae, with other known 13-lactamase genes commonly found in members of the family Enterobacteriaceae (blaTEM-l and blasHv.3 derivatives), and with previously described carbapenemase genes such as those from Xanthomonas maltophilia, Bacilus cereus, Bacteroidesfragilis (cfA), and Aeromonas hydrophila (cphA). This work describing the biochemical properties of a novel chromosome-encoded 13-lactamase from E. cloacae indicates that this enzyme differs from all the previously described carbapenemases. This is the first reported cloning of a Enterobacteriaceae.

Carbapenemases: the Versatile β-Lactamases

“…The IMI (for "imipenem-hydrolyzing ␤-lactamase") and NMC-A (for "not metalloenzyme carbapenemase") enzymes have been detected in rare clinical isolates of E. cloacae in the United States, France, and Argentina (149,175,181,183). NMC-A and IMI-1 have 97% amino acid identity and are related to SME-1, with approximately 70% amino acid identity (146,183).…”

“…These ␤-lactamases have been detected in Enterobacter cloacae, Serratia marcescens, and Klebsiella spp. as single isolates or in small outbreaks (134,149,243). Bacteria expressing these enzymes are characterized by reduced susceptibility to imipenem, but MICs can range from mildly elevated (e.g., imipenem MICs of Յ4 g/ml) to fully resistant.…”

Metallo-β-Lactamases: the Quiet before the Storm?

“…The enzymes characterized from Enterobacteriaceae include NmcA, Sme1-3, IMI-1, KPC1-3, and GES-2 (107,133,139,142,197,200). Despite the avidity of these enzymes for carbapenems, they do not always mediate high-level resistance and not all are inhibited by clavulanic acid (108).…”

Klebsiella Pneumoniaeoxa-48 in a Urology Patient: Case Report

“…Th e genes conferring resistance to ß-lactams including broad spectrum and extended-spectrum ß-lactamases (bla SHV , bla TEM , bla CTX-M and bla PER-1 ) [18][19][20][21][22] , plasmid-mediated AmpC ß-lactamases 23 , class A carbapenemases (bla KPC , bla SME , bla IMI and bla NMC ,) [24][25][26] , class B (bla VIM, bla IMP and bla NDM ) [27][28][29] , and carbapenem hydrolyzing oxacillinases (bla OXA-48 ) 30 and to fl uoroquinolones (qnrA, qnrB and qnrS) 31 were determined by PCR using protocols and conditions as described previously. Th e DNA was extracted by boiling method.…”