High-level expression of ampC beta-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

Abstract

Two Escherichia coli isolates were recovered from the blood of two cancer patients and were demonstrated to produce high levels of the AmpC beta-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 beta-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.

FIG. 1.

Flowcharts of antimicrobial therapies for the patients harboring isolates EC38 and EC44.

FIG. 2.

RNA expression assessed by hybridization with 16S rRNA- and ampC-specific probes. Lane 1, ATCC 25922; lane 2, JP995; lane 3, EC38; lane 4, EC44.

FIG. 3.

Sequence alignment of ampC promoter for E. coli K-12, ATCC 259922, EC38, and EC40. The documented and suspected new −35 and −10 promoter sequences are shown in boldface, and the attenuator is indicated by the arrows. There is a dot above the sequence every 20 nucleotides. The initiation codon is underlined.

FIG. 4.

Primer extension assay for ATCC 25922 (lane 1) and EC38 (lane 2).

FIG. 5.

Hairpin structure identified and generated with a DNA sequence alignment program (DNASTAR Inc.).

FIG. 6.

Nuclease protection assay. The lanes marked A, G, C, and T contain the ampC sequence. The position of the RNA start site was determined according to the molecular weight of the nuclease-protected fragment. Lane 1, EC38; lane 2, ATCC 25922; lane 3, JP995.

FIG. 7.

Cloning of three different lengths of ampC insertion sequences into PCR-ScriptCamSK+ vector: EC-IS1, full-length ampC sequence with the original −10 and −35 promoter sequences; EC-IS2, full-length ampC sequence with insertion of nucleotides G and T between positions −19 and −20 inside the promoter sequence; and EC-IS3, full-length ampC sequence with insertion of nucleotides G and T between positions −19 and −20 but with truncation at position −26 of the −35 promoter sequence.