Mutation in Serratia marcescens AmpC beta-lactamase producing high-level resistance to ceftazidime and cefpirome

Starting from a clinical isolate of Serratia marcescens that produced a chr omosomally encoded AmpC p-lactamase inducibly, we isolated by step,vise sel ection two laboratory mutants that showed high levels of resistance to some cephalosporins. The 98R mutant apparently overproduced the unaltered P-lac tamase constitutively, but the 520R mutant produced an altered enzyme, also constitutively. Ceftazidime and cefpirome MICs for the 520R mutant were mu ch higher (512 and 64 mug/ml, respectively) than those for the 98R mutant ( 16 and 16 mug/ml, respectively). Yet the MICs of cephaloridine and piperaci llin for the 520R mutant were four- to eightfold lower than those for the 9 8R mutant. Cloning and sequencing of the ampC alleles showed that in the 52 0R mutant enzyme, the Thr64 residue, about two turns away from the active-s ite serine, was mutated to isoleucine, This resulted in a >1,000-fold incre ase in the catalytic efficiency (kcat/K-m) of the mutated AmpC enzyme towar d ceftazidime, whereas there was a > 10-fold decrease in the efficiency of the mutant enzyme toward cefazolin and cephaloridine. The outer membrane pe rmeability of the 520R strain to cephalosporins was also less than in the 9 8R 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.