title: Mutation in Serratia marcescens AmpC beta-lactamase producing high-level resistance to ceftazidime and cefpirome.
abstract: Starting from a clinical isolate of Serratia marcescens that produced a chromosomally encoded AmpC beta-lactamase inducibly, we isolated by stepwise selection two laboratory mutants that showed high levels of resistance to some cephalosporins. The 98R mutant apparently overproduced the unaltered beta-lactamase constitutively, but the 520R mutant produced an altered enzyme, also constitutively. Ceftazidime and cefpirome MICs for the 520R mutant were much higher (512 and 64 microg/ml, respectively) than those for the 98R mutant (16 and 16 microg/ml, respectively). Yet the MICs of cephaloridine and piperacillin for the 520R mutant were four- to eightfold lower than those for the 98R mutant. Cloning and sequencing of the ampC alleles showed that in the 520R mutant enzyme, the Thr64 residue, about two turns away from the active-site serine, was mutated to isoleucine. This resulted in a >1,000-fold increase in the catalytic efficiency (k(cat)/K(m)) of the mutated AmpC enzyme toward ceftazidime, whereas there was a >10-fold decrease in the efficiency of the mutant enzyme toward cefazolin and cephaloridine. The outer membrane permeability of the 520R strain to cephalosporins was also less than in the 98R strain, and the alteration of the kinetic properties of the AmpC enzyme together with this difference in permeability explained quantitatively the resistance levels of both mutant strains to most agents studied.
authors.affiliation: Institute of Medical Microbiology, University of Milan, 20133 Milan, Italy.
authors.forename: A
authors.initials: A
authors.lastname: Raimondi
authors.forename: F
authors.initials: F
authors.lastname: Sisto
authors.forename: H
authors.initials: H
authors.lastname: Nikaido
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D002442:Ceftazidime / Q000494:pharmacology*; D002463:Cell Membrane Permeability; D002511:Cephalosporins / Q000494:pharmacology*; D017931:DNA Primers / Q000737:chemistry; D004352:Drug Resistance, Microbial / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D007525:Isoelectric Focusing; D008969:Molecular Sequence Data; D009154:Mutation; D010641:Phenotype; D016133:Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D012706:Serratia marcescens / Q000187:drug effects / Q000201:enzymology*; D014162:Transfection; D001618:beta-Lactamases / Q000235:genetics*; D000097572:Cefpirome
chemical_list: D001426:Bacterial Proteins; D002442:Ceftazidime; D002511:Cephalosporins; D017931:DNA Primers; D001618:beta-Lactamases; D000097572:Cefpirome; C048795:AmpC beta-lactamases