Siu L.K.Lu P.-L.Chen J.-Y.Lin F.M.SHAN-CHWEN CHANG2020-12-302020-12-3020030066-4804https://www.scopus.com/inward/record.uri?eid=2-s2.0-0038338397&doi=10.1128%2fAAC.47.7.2138-2144.2003&partnerID=40&md5=e3058796120d3a6e4afa5d3c7e55bf1ahttps://scholars.lib.ntu.edu.tw/handle/123456789/535766Two Escherichia coli isolates were recovered from the blood of two cancer patients and were demonstrated to produce high levels of the AmpC β-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 β-lactamase and resistance to oxyimino-cephalosporins. The failure of the two patients to respond to treatment with oxyiminocephalosporins highlights the important role of such a resistance mechanism in the clinical setting.[SDGs]SDG3amikacin; amoxicillin plus clavulanic acid; ampicillin; aztreonam; beta lactamase; beta lactamase AmpC; cefazolin; cefotaxime; cefoxitin; ceftazidime; ceftriaxone; cephalosporin derivative; ciprofloxacin; clavulanic acid; imipenem; RNA; RNA polymerase; tetracycline; thymidine; unclassified drug; adult; article; bacterium isolate; cancer; case report; Enterobacter infection; Escherichia coli; gene insertion; gene sequence; human; isoelectric point; male; minimum inhibitory concentration; molecular cloning; nonhuman; Northern blotting; nuclease protection assay; nucleotide sequence; point mutation; priority journal; promoter region; protein expression; sequence homology; Aspergillus Nuclease S1; Bacterial Proteins; Base Sequence; beta-Lactamases; Cephalosporin Resistance; Cephalosporins; Cloning, Molecular; DNA Fingerprinting; Escherichia coli; Escherichia coli Infections; Gene Expression Regulation, Bacterial; Humans; Microbial Sensitivity Tests; Molecular Sequence Data; Plasmids; Promoter Regions (Genetics); RNA, Ribosomal, 16Sjournal article10.1128/AAC.47.7.2138-2144.200312821459