title: [Expression of high-level cephalosporinase due to mutation in the AmpC attenuator of a clinical Escherichia coli strain].
abstract: OBJECTIVE
  To study the resistant phenotype of a clinical strain of Escherichia coli and to explore the effect of its attenuator mutation on AmpC expression.
  
  
  METHODS
  A clinical strain of Escherichia coli 20022 (ECO20022) resistant to cefoxitin was isolated clinically. The phenotype was examined by three-dimensional methods, isoelectric focusing (IEF), and microdilution method. The regulator genes of ECO20022 were amplified and sequenced, and the difference between them was analyzed by BLAST method. Then the regulator genes were cloned into pCAT3-basic vector (a promoterless reporter gene vector). Microdilution method was used to detect the minimal inhibitory concentration (MIC) of chloramphenicol and ampicillin to this strain with E. coli ATCC25922 as quality control bacterium. ELISA was used to detect the content of chloramphenicol acetyl transferase (CAT).
  
  
  RESULTS
  Compared to the standard E. coli K-12, there were four base substitutions, i.e., 22C-T, 26, 27TA-GT, and 32G-A in the attenuator region of ECO20022. Three-dimensional method showed that this strain was high AmpC-producing. IEF found that it produced three beta-lactamases with the values of PI of 5.4, 8.2, and 9.0 respectively. The beta-lactamase with the PI of 9.0 could be inhibited by cloxacillin but not by clavulanate. The strain was resistant to not only most of third generation cephalosporins, but also to cefepime; however it was still susceptible to carbapenem. The secondary structure of the attenuator RNA of ECO20022 was different from the traditional structure of E. coli K-12. The regulator gene was successfully cloned into pCAT3-basic vector and direct and indirect tests indicated that this regulator gene enhanced the CAT expressing level as much as 10 times that of Escherichia coli K-12.
  
  
  CONCLUSIONS
  AmpC attenuator mutation leads to high AmpC expression in Escherichia coli, resulting in a significant rise of resistance level to beta-lactamase and a great menace to clinical antibiotic therapy.
authors: Department of Respiratory Medicine, The General Hospital of People's Liberation Army, Beijing 100853, China.
authors: Xi-zhou
authors: XZ
authors: Guan
authors: You-ning
authors: YN
authors: Liu
authors: Yan-ping
authors: YP
authors: Luo
authors: Dan-yang
authors: DY
authors: She
authors: Si-jing
authors: SJ
authors: Lu
authors: Guang
authors: G
authors: Zhou
authors: Liang-an
authors: LA
authors: Chen