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JAC Advance Access originally published online on March 10, 2005
Journal of Antimicrobial Chemotherapy 2005 55(5):768-772; doi:10.1093/jac/dki074
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© The Author 2005. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions{at}oupjournals.org

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

Dobryan M. Tracz1, David A. Boyd1, Louis Bryden1, Romeo Hizon1, Sandra Giercke2, Paul Van Caeseele2 and Michael R. Mulvey1,*

1 National Microbiology Laboratory, Public Health Agency of Canada, 1015 Arlington St, Winnipeg, Manitoba, Canada, R3E 3R2; 2 Cadham Provincial Laboratory, Winnipeg, Manitoba, Canada

* Corresponding author. Tel: +1-204-789-2133; Fax: +1-204-789-5020; Email: michael_mulvey{at}phac-aspc.gc.ca

Objectives: To characterize the mechanism of cefoxitin resistance in clinical isolate Escherichia coli N99-0001.

Methods: Plasmid analysis, PCR for ß-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.

Keywords: cefoxitin resistance , E. coli , RT–PCR


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