Resistance to ceftazidime is associated with a S220Y substitution in the omega loop of the AmpC beta-lactamase of a Serratia marcescens clinical isolate.
OBJECTIVE
The aim of this study was to characterize the ampC beta-lactamase gene of a clinical isolate of Serratia marcescens resistant to ceftazidime.


METHODS
S. marcescens SMSA was isolated from an intra-abdominal wound of a patient previously treated with ceftazidime. A susceptible strain, SLS73, was used as a control. Susceptibility testing, PCR, DNA sequencing, molecular cloning, site-directed mutagenesis and determination of kinetic parameters were carried out to investigate the mechanism of resistance to ceftazidime.


RESULTS
MICs of ceftazidime were 64 and 0.2 mg/L for SMSA and SLS73, respectively. Sequencing of the ampC gene of SMSA was carried out. When compared with the closest AmpC enzyme, the S. marcescens S3 beta-lactamase, the novel protein showed E57Q, Q129K and S220Y substitutions. The S220Y substitution is located in the omega loop. Introduced by mutagenesis in the ampC gene of SLS73, this substitution conferred the same level of resistance to ceftazidime. The catalytic efficiency (k(cat)/K(m)) of the mutated enzyme toward ceftazidime was increased by about 100-fold.


CONCLUSIONS
We present another example of in vivo selection of broad-spectrum resistance by amino acid substitution in the omega loop of chromosomal AmpC beta-lactamase in S. marcescens.
Hidri
Nadia
N
Service de Microbiologie, Hôpital Saint Antoine, Assistance Publique-Hôpitaux de Paris, Paris, France.
Barnaud
Guilène
G
Decré
Dominique
D
Cerceau
Claude
C
Lalande
Valérie
V
Petit
Jean Claude
JC
Labia
Roger
R
Arlet
Guillaume
G
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics*; D002442:Ceftazidime / Q000494:pharmacology*; D018550:Cephalosporin Resistance / Q000235:genetics*; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011487:Protein Conformation; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012706:Serratia marcescens / Q000187:drug effects* / Q000235:genetics*; D001618:beta-Lactamases / Q000737:chemistry / Q000235:genetics*
D001426:Bacterial Proteins; D002442:Ceftazidime; C048795:AmpC beta-lactamases; D001618:beta-Lactamases