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 P...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 （Xbal） 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. Conclusion PFGE protocol established in this paper and automated ribotyping are suitable for application in Citrobacter subtyping.展开更多
Sequence analysis of the RNA polymerase B subunit encoding gene (rpoB) has been proposed as a useful tool for bacterial identification. This method has been implemented to differentiate five well-defined Proteus speci...Sequence analysis of the RNA polymerase B subunit encoding gene (rpoB) has been proposed as a useful tool for bacterial identification. This method has been implemented to differentiate five well-defined Proteus species: P. mirabilis, P. hauseri, P. penneri, P. vulgaris, and P. myxofaciens. In this study, we evaluated the usefulness of rpoB sequencing for intraspecies discrimination of P. mirabilis. The sequence of rpoB 909 bp region was analyzed in 15 newly isolated strains and 5 of 8 years old isolates from different clinical sources. Three respective groups were obtained. The first group included 13 of the new strains showing similarity with Proteus mirabilis (ATCC 29906) strain. The second group including 3 of the old strains differs from the first group with a divergence of 0.22%. Group 3 contains only a single new strain 33. The sequence of this strain shows differences in the rpoB 909 bp region analyzed from the members of group 1 and the second group by 1.65% and 1.87% divergence respectively. According to our results, genetic differences could be detected within the P. mirabilis species. Therefore much more effort should be made to re-evaluate rpoB method and validate its usefulness as a molecular diagnostic method.展开更多
基金supported by the project (grant 2005CB522904 and 2005CB522905) from the Ministry of Scientific Technologythe project (grant 2008ZX10004-001, 2008ZX10004-008, and 2009ZX10004-101) from the Ministry of Scientific Technology and the Ministry of Health of the People’s Republic of China
文摘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 （Xbal） 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. Conclusion PFGE protocol established in this paper and automated ribotyping are suitable for application in Citrobacter subtyping.
文摘Sequence analysis of the RNA polymerase B subunit encoding gene (rpoB) has been proposed as a useful tool for bacterial identification. This method has been implemented to differentiate five well-defined Proteus species: P. mirabilis, P. hauseri, P. penneri, P. vulgaris, and P. myxofaciens. In this study, we evaluated the usefulness of rpoB sequencing for intraspecies discrimination of P. mirabilis. The sequence of rpoB 909 bp region was analyzed in 15 newly isolated strains and 5 of 8 years old isolates from different clinical sources. Three respective groups were obtained. The first group included 13 of the new strains showing similarity with Proteus mirabilis (ATCC 29906) strain. The second group including 3 of the old strains differs from the first group with a divergence of 0.22%. Group 3 contains only a single new strain 33. The sequence of this strain shows differences in the rpoB 909 bp region analyzed from the members of group 1 and the second group by 1.65% and 1.87% divergence respectively. According to our results, genetic differences could be detected within the P. mirabilis species. Therefore much more effort should be made to re-evaluate rpoB method and validate its usefulness as a molecular diagnostic method.
