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Antimicrobial Agents and Chemotherapy, August 2001, p. 2331-2339, Vol. 45, No. 8
0066-4804/01/$04.00+0   DOI: 10.1128/AAC.45.8.2331-2339.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Mutation in Serratia marcescens AmpC beta -Lactamase Producing High-Level Resistance to Ceftazidime and Cefpirome

Alessandro Raimondi,1 Francesca Sisto,1 and Hiroshi Nikaido2,*

Institute of Medical Microbiology, University of Milan, 20133 Milan, Italy,1 and Department of Molecular and Cell Biology, University of California, Berkeley, California2

Received 13 December 2000/Returned for modification 14 March 2001/Accepted 12 May 2001

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 µg/ml, respectively) than those for the 98R mutant (16 and 16 µg/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 (kcat/Km) 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.

* Corresponding author. Mailing address: Department of Molecular and Cell Biology, Room 229, Stanley Hall, University of California, Berkeley, CA 94720-3206. Phone: (510) 642-2027. Fax: (510) 643-9290. E-mail: nhiroshi{at}uclink4.berkeley.edu.

Antimicrobial Agents and Chemotherapy, August 2001, p. 2331-2339, Vol. 45, No. 8
0066-4804/01/$04.00+0   DOI: 10.1128/AAC.45.8.2331-2339.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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