Increase in ampC promoter strength due to mutations and deletion of the attenuator in a clinical isolate of cefoxitin-resistant Escherichia coli as determined by RT-PCR.
OBJECTIVE
To characterize the mechanism of cefoxitin resistance in clinical isolate Escherichia coli N99-0001.


METHODS
Plasmid analysis, PCR for beta-lactamases, and sequencing of the ampC genes was carried out. An RT-PCR method was developed to determine relative ampC expression.


RESULTS
Analysis of the ampC promoter region of E. coli N99-0001 revealed a T-->A mutation at -32, a C-->A mutation at -11, an insertion of a T between -20 and -21, and a 28 bp deletion including the entire attenuator. RT-PCR showed that ampC was expressed 140-fold higher in E. coli N99-0001 than in E. coli ATCC 25922.


CONCLUSIONS
Cefoxitin resistance in E. coli N99-0001 was due to overexpression of ampC caused by an increase in promoter strength.
Tracz
Dobryan M
DM
National Microbiology Laboratory, Public Health Agency of Canada, 1015 Arlington St, Winnipeg, Manitoba, Canada, R3E 3R2.
Boyd
David A
DA
Bryden
Louis
L
Hizon
Romeo
R
Giercke
Sandra
S
Van Caeseele
Paul
P
Mulvey
Michael R
MR
D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D002440:Cefoxitin / Q000494:pharmacology*; D024881:Drug Resistance, Bacterial; D004926:Escherichia coli / Q000187:drug effects; D005260:Female; D017353:Gene Deletion; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D011401:Promoter Regions, Genetic; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D001618:beta-Lactamases / Q000737:chemistry / Q000235:genetics
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D002440:Cefoxitin; C048795:AmpC beta-lactamases; D001618:beta-Lactamases