Molecular characterization of an enterobacterial metallo beta-lactamase found in a clinical isolate of Serratia marcescens that shows imipenem resistance.
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.
Osano
E
E
Department of Bacteriology, Nagoya University School of Medicine, Japan.
Arakawa
Y
Y
Wacharotayankun
R
R
Ohta
M
M
Horii
T
T
Ito
H
H
Yoshimura
F
F
Kato
N
N
D000595:Amino Acid Sequence; D001483:Base Sequence; D003470:Culture Media; D004269:DNA, Bacterial / Q000032:analysis; D004352:Drug Resistance, Microbial; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D015378:Imipenem / Q000494:pharmacology*; D007525:Isoelectric Focusing; D007700:Kinetics; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009693:Nucleic Acid Hybridization; D010957:Plasmids; D016868:Serratia Infections / Q000382:microbiology; D012706:Serratia marcescens / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D065093:beta-Lactamase Inhibitors; D001618:beta-Lactamases / Q000032:analysis* / Q000302:isolation & purification
D003470:Culture Media; D004269:DNA, Bacterial; D065093:beta-Lactamase Inhibitors; D015378:Imipenem; D001618:beta-Lactamases