title: [Experimental study on phenotypic conversion of clinical chloromycetin-resistant strains of E. coli to drug-sensitive strains by using EGS technique in vitro].
abstract: OBJECTIVE
  To explore the possibility of phenotypic conversion of clinical chloromycetin (Cm)-resistant isolates of E.coli to drug-sensitive ones with external guide sequences (EGS) in vitro.
  
  
  METHODS
  Recombinant EGS plasmids directed against Cm acetyl transferase (cat) and containing kanamycin (Km) drug-resistance gene and control plasmids only containing kanamycin-resistance gene without EGS were constructed. By using CaCl(2) method, the recombinant plasmids were introduced into the clinically isolated Cm-resistant E.coli strains. Extraction of plasmids and PCR were applied to identify the EGS positive clones; The growth rate in liquid broth culture of Cm-resistant bacteria after EGS containing plasmid transformation was determined by spectrophotometer A(600). Drug sensitivity was tested in solid culture by using KB method.
  
  
  RESULTS
  Transformation studies were carried out on 16 clinically isolated Cm-resistant E.coli strains with pEGFP-C1-EGS + cat1 + cat2 recombinant plasmid. Transformants were screened on LB-agar plates containing Km after transformation using EGS. In 4 tested strains of them, transformants with specific EGS plasmid showed growth inhibition when grown in liquid broth culture containing 100 approximately 200 micro g/ml of Cm. They were sensitive to Cm on LB-agar plates containing 100 approximately 200 micro g/ml of Cm in drug-sensitivity test. Extraction of plasmids showed the existence of EGS bands. PCR amplified products of EGS. The above facts indicated that the 4 strains out of the 16 clinical isolates had been converted to drug-sensitive phenotype, and Cm-resistant clinically isolated E. coli resumed sensitivity to Cm.
  
  
  CONCLUSIONS
  EGS has the capability of converting the phenotype of clinical drug-resistant isolates to drug sensitivity.
authors.affiliation: Department of Infectious Diseases, Tongji Hospital and Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
authors.forename: Mei-ying
authors.initials: MY
authors.lastname: Gao
authors.forename: Ru
authors.initials: R
authors.lastname: Chen
authors.forename: Shou-gui
authors.initials: SG
authors.lastname: Liu
authors.forename: Jiang-nan
authors.initials: JN
authors.lastname: Feng
mesh_terms: D002701:Chloramphenicol / Q000494:pharmacology; D024881:Drug Resistance, Bacterial / Q000235:genetics*; D004722:Endoribonucleases / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology*; D015964:Gene Expression Regulation, Bacterial; D018014:Gene Transfer Techniques; D006801:Humans; D008826:Microbial Sensitivity Tests; D010641:Phenotype; D010957:Plasmids / Q000235:genetics; D016337:RNA, Catalytic / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D043262:Ribonuclease P / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology*; D014162:Transfection
chemical_list: D029968:Escherichia coli Proteins; D016337:RNA, Catalytic; D012333:RNA, Messenger; D002701:Chloramphenicol; D004722:Endoribonucleases; D043262:Ribonuclease P; C470865:ribonuclease P, E coli