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

A clinical isolate of Serratia marcescens (TN9106) produced a metallo β- lactamase (IMP-1) which conferred resistance to imipenem and broad-spectrum β-lactams. The bla(IMP) 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 bla(IMP), 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 β-lactams revealed that this enzyme hydrolyzes not only various broad- spectrum β-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 bla(IMP) was encoded on the chromosome of S. marcescens TN9106. By nucleotide sequencing analysis, bla(IMP) was found to encode a protein of 246 amino acid residues and was shown to have considerable homology to the metallo β-lactamase genes of Bacillus cereus, Bacteroides fragilis, and Aeromonas hydrophila. The G+C content of bla(IMP) 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 β- lactamase, of which the primary structure has been completely determined, that confers resistance to carbapenems and other broad-spectrum β-lactams.