Molecular basis of AmpC hyperproduction in clinical isolates of Escherichia coli

Abstract

DNA sequencing data showed that five clinical isolates of Escherichia coli with reduced susceptibility to ceftazidime, ceftriaxone, and cefotaxime contain an ampC gene that is preceded by a strong promoter. Transcription from the strong promoter was 8- to 18-fold higher than that from the promoter from a susceptible isolate. RNA studies showed that mRNA stability does not play a role in the control of AmpC synthesis.

FIG. 1

Comparison of DNA sequences (135 bp) of the ampC control region and signal peptide from susceptible and AmpC-hyperproducing E. coli and Shigella strains. The sequences from E. coli K-12, hyperproducing strains C14 to C17, susceptible S. sonnei OS10, and hyperproducing S. sonnei OS112 are from reference . There is a dot above the sequence every 20 nucleotides. Identical nucleotides are indicated by dashes. Nucleotides that differ from the sequences from the susceptible E. coli strains (K-12 and E6) are in lowercase. The prototype −35 and −10 regions are in boldface lettering, and the attenuator is indicated by the arrows. The initiation codon is underlined, and the three-letter amino acid code is directly below the partial sequence of the signal peptide.

FIG. 2

Hybridization of the Northern blots prepared from RNAs extracted from E. coli E1 to E6 and probed with either the ampC or the 16S rRNA probe. The strains were cultured until exponential phase. At this point (lane −), a 10-ml aliquot was removed and RNA was extracted. Rifampin (0.2 mg/ml) was added, a 10-ml aliquot was withdrawn immediately (lane 0), and RNA was extracted. Additional aliquots were removed after 2, 4, 6, and 8 min, and RNA was extracted. Panels: A, RNA from E1 probed with the ampC probe; B, RNA from E2 probed with the ampC probe; C, RNA from E3 probed with the ampC probe; D, RNA from E4 probed with the ampC probe; E, RNA from E5 probed with the ampC probe; F, RNA from E6 probed with 16S rRNA probe.