Molecular characterization of an enterobacterial metallo beta-lactamase found in a clinical isolate of Serratia marcescens that shows imipenem resistance

Antimicrob Agents Chemother. 1994 Jan;38(1):71-8. doi: 10.1128/AAC.38.1.71.

Abstract

A clinical isolate of Serratia marcescens (TN9106) produced a metallo beta-lactamase (IMP-1) which conferred resistance to imipenem and broad-spectrum beta-lactams. The blaIMP gene providing imipenem resistance was cloned and expressed in Escherichia coli HB101. The IMP-1 was purified from E. coli HB101 that harbors pSMBNU24 carrying blaIMP, and its apparent molecular mass was calculated to be about 30 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Kinetic studies of IMP-1 against various beta-lactams revealed that this enzyme hydrolyzes not only various broad-spectrum beta-lactams but also carbapenems. However, aztreonam was relatively stable against IMP-1. Although clavulanate or cloxacillin failed to inhibit IMP-1, Hg2+, Fe2+, or Cu2+ blocked the enzyme's activity. Moreover, the presence of EDTA in the reaction buffer resulted in a decrease in the enzyme's activity. Carbapenem resistance was not transferred from S. marcescens TN9106 to E. coli CSH2 by conjugation. A hybridization study confirmed that blaIMP was encoded on the chromosome of S. marcescens TN9106. By nucleotide sequencing analysis, blaIMP was found to encode a protein of 246 amino acid residues and was shown to have considerable homology to the metallo beta-lactamase genes of Bacillus cereus, Bacteroides fragilis, and Aeromonas hydrophila. The G+C content of blaIMP was 39.4%. Four consensus amino acid residues, His-95, His-97, Cys-176, and His-215, which form putative zinc ligands, were conserved in the deduced amino acid sequence of IMP-1. By determination of the amino acid sequence at the N terminus of purified mature IMP-1, 18 amino acid residues were found to be processed from the N terminus of the premature enzyme as a signal peptide. These results clearly show that IMP-1 is an enterobacterial metallo beta-lactamase, of which the primary structure has been completely determined, that confers resistance to carbapenems and other broad-spectrum beta-lactams.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Base Sequence
  • Culture Media
  • DNA, Bacterial / analysis
  • Drug Resistance, Microbial
  • Electrophoresis, Polyacrylamide Gel
  • Escherichia coli / metabolism
  • Humans
  • Imipenem / pharmacology*
  • Isoelectric Focusing
  • Kinetics
  • Microbial Sensitivity Tests
  • Molecular Sequence Data
  • Molecular Weight
  • Nucleic Acid Hybridization
  • Plasmids
  • Serratia Infections / microbiology
  • Serratia marcescens / drug effects
  • Serratia marcescens / enzymology*
  • Serratia marcescens / genetics
  • beta-Lactamase Inhibitors
  • beta-Lactamases / analysis*
  • beta-Lactamases / isolation & purification

Substances

  • Culture Media
  • DNA, Bacterial
  • beta-Lactamase Inhibitors
  • Imipenem
  • beta-Lactamases

Associated data

  • GENBANK/D50438
  • GENBANK/S71932