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JAC Advance Access published online on March 10, 2005
Journal of Antimicrobial Chemotherapy, doi:10.1093/jac/dki074
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JAC © The British Society for Antimicrobial Chemotherapy 2005; all rights reserved
Received December 9, 2004
Revised January 25, 2005
Accepted January 26, 2005

Brief report

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. Tracz 1, David A. Boyd 1, Louis Bryden 1, Romeo Hizon 1, Sandra Giercke 2, Paul Van Caeseele 2, and Michael R. Mulvey 1*

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

* To whom correspondence should be addressed.
Michael R. Mulvey, E-mail: Michael_mulvey{at}phac-aspc.gc.ca


  Abstract

Objectives: 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.

Keywords: cefoxitin resistance; E. coli; RT-PCR.
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