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

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Abstract
Objectives: To characterize the mechanism of cefoxitin resistance in clinical isolate Escherichia coli N99-0001. Methods: Plasmid analysis, PCR for β-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.