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Antimicrobial Agents and Chemotherapy, July 2003, p. 2138-2144, Vol. 47, No. 7
0066-4804/03/$08.00+0     DOI: 10.1128/AAC.47.7.2138-2144.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

High-Level Expression of AmpC ß-Lactamase Due to Insertion of Nucleotides between -10 and -35 Promoter Sequences in Escherichia coli Clinical Isolates: Cases Not Responsive to Extended-Spectrum-Cephalosporin Treatment

L. K. Siu,1* Po-Liang Lu,2 J.-Y. Chen,1 F. M. Lin,1 and Shan-Chwen Chang3

Division of Clinical Research, National Health Research Institutes,1 Department of Internal Medicine, National Taiwan University Hospital, Taipei,3 Department of Internal Medicine, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan2

Received 25 October 2002/ Returned for modification 24 January 2003/ Accepted 31 March 2003

Two Escherichia coli isolates were recovered from the blood of two cancer patients and were demonstrated to produce high levels of the AmpC ß-lactamase with isoelectric points of >9.0. The hypertranscription of ampC RNA was observed by Northern blot hybridization in both isolates. One isolate (isolate EC44) had a point mutation (G->A at position -28) and insertion of thymidine between positions -20 and -19 of the ampC promoter gene (GenBank accession no. AE000487). The single nucleotide insertion of T between positions -19 and -20 created an optimal distance (17 bp) in the Pribnow box for ampC hyperproduction. The other isolate (isolate EC38) had two point mutations (G->A at position -28 and C->T at position +58) and a 2-base (GT) insertion between positions -14 and -15. Although the insertion of GT between positions -14 and -15 may create a new promoter next to the original promoter, cloning of the ampC region with truncated nucleotides of the original -35 region of EC38 failed to verify the hypothesis that a new promoter would be created by such a nucleotide insertion. Instead, multiple start sites for ampC transcription at -1, +1, +2, and +3 were observed in an S1 nuclease protection assay. These results suggest that the RNA polymerase is flexible in the selection of a start site in ampC hypertranscription. In conclusion, nucleotide insertions between the -35 and -10 ampC promoter sequences was the mechanism for the hyperproduction of AmpC ß-lactamase and resistance to oxyimino-cephalosporins. The failure of the two patients to respond to treatment with oxyimino-cephalosporins highlights the important role of such a resistance mechanism in the clinical setting.

* Corresponding author. Mailing address: Division of Clinical Research, National Health Research Institutes (99), 128, Yen-Chiu-Yuan Rd., Sec. 2, Taipei 11529, Taiwan. Phone: 886 2 26524094. Fax: 886 2 27890254. E-mail: lksiu{at}mail.nhri.org.tw.

Antimicrobial Agents and Chemotherapy, July 2003, p. 2138-2144, Vol. 47, No. 7
0066-4804/03/$08.00+0     DOI: 10.1128/AAC.47.7.2138-2144.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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