title: Establishment and comparison of pulsed-field gel electrophoresis, multiple-locus variable number tandem repeat analysis and automated ribotyping methods for subtyping of Citrobacter strains.
abstract: OBJECTIVE
  To establish and compare the pulsed-field gel electrophoresis (PFGE), multiple-locus variable number tandem repeat analysis (MLVA) and automated ribotyping for subtyping of Citrobacter strains.
  
  
  METHODS
  PFGE protocol was optimized in terms of plug preparation procedure, restriction enzymes and configuration of electrophoretic parameters. MLVA method was evaluated by finding variable number tandem repeats in two genomes of Citrobacter strains. The ribotyping was performed by using the automated RiboPrinter system.
  
  
  RESULTS
  We optimized the plug preparation procedure, focused on the cell suspension concentration (turbidity of 2.5 to 3.5), SDS addition (no SDS needed) and lysis time (1 h), and selected the appropriate restriction enzyme (XbaI) and the electrophoretic parameters (1.0 s-20.0 s for 19 h) of PFGE. There was nearly no discriminatory power of MLVA between Citrobacter strains. For 51 Citrobacter strains, automated ribotyping gave a D-value of 0.9945, while PFGE gave a D-value of 0.9969. Both PFGE and automated ribotyping clustered strains from the same sources (with the same species from the same place at the same time identified as the same source) and divided strains from different sources (from different years, places and hosts) into different subtypes.
  
  
  CONCLUSIONS
  PFGE protocol established in this paper and automated ribotyping are suitable for application in Citrobacter subtyping.
authors.affiliation: State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China.
authors.forename: Xiao Ai
authors.initials: XA
authors.lastname: Zhang
authors.forename: Xue Mei
authors.initials: XM
authors.lastname: Bai
authors.forename: Chang Yun
authors.initials: CY
authors.lastname: Ye
authors.forename: Zhi Hong
authors.initials: ZH
authors.lastname: Ren
authors.forename: Jian Guo
authors.initials: JG
authors.lastname: Xu
mesh_terms: D001331:Automation; D002954:Citrobacter / Q000145:classification / Q000235:genetics*; D016521:Electrophoresis, Gel, Pulsed-Field / Q000379:methods*; D018598:Minisatellite Repeats / Q000235:genetics*; D058885:Multilocus Sequence Typing / Q000379:methods*; D010802:Phylogeny; D021521:Ribotyping / Q000379:methods*