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
Background:The prevalence of human brucellosis in Qinghai Province of China has been increasing rapidly,with confirmed cases distributed across 31 counties.However,the epidemiology of brucellosis transmission has not ...Background:The prevalence of human brucellosis in Qinghai Province of China has been increasing rapidly,with confirmed cases distributed across 31 counties.However,the epidemiology of brucellosis transmission has not been fully elucidated.To characterize the infecting strains isolated from humans,multiple-locus variable-number tandem repeats analysis(MLVA)and whole-genome single-nucleotide polymorphism(SNP)-based approaches were employed.Methods:Strains were isolated from two males blood cultures that were confirmed Brucella m elitensis positive following biotyping and MLVA.Genomic DNA was extracted from these two strains,and whole-genome sequencing was performed.Next,SNP-based phylogenetic analysis was performed to compare the two strains to 94 B.melitensis strains(complete genome and draft genome)retrieved from online databases.Results:The two Brucella isolates were identified as B.melitensis biovar 3(QH2019001 and QH2019005)following conventional biotyping and were found to have differences in their variable number tandem repeats(VNTRs)using MLVA-16.Phylogenetic examination assigned the 96 strains to five genotype groups,with QH2019001 and QH2019005 assigned to the same group,but different subgroups.Moreover,the QH2019005 strain was assigned to a new subgenotype,llj,within genotype II.These findings were then combined to determine the geographic origin of the two Brucella strains.Conclusions:Utilizing a whole-genome SNP-based approach enabled differences between the two B.m elitensis strains to be more clearly resolved,and facilitated the elucidation of their different evolutionary histories.This approach also revealed that QH2019005 is a member of a new subgenotype(Hj)with an ancient origin in the eastern Mediterranean Sea.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.
基金supported by National Key R&D Program of China(Grant No.2020YFA0907101)Major Infectious Diseases such as AIDS and Viral Hepatitis Prevention and Control Technology Major Projects(2018ZX10201002)the National Natural Scientific Foundation of China(81860588).
文摘Background:The prevalence of human brucellosis in Qinghai Province of China has been increasing rapidly,with confirmed cases distributed across 31 counties.However,the epidemiology of brucellosis transmission has not been fully elucidated.To characterize the infecting strains isolated from humans,multiple-locus variable-number tandem repeats analysis(MLVA)and whole-genome single-nucleotide polymorphism(SNP)-based approaches were employed.Methods:Strains were isolated from two males blood cultures that were confirmed Brucella m elitensis positive following biotyping and MLVA.Genomic DNA was extracted from these two strains,and whole-genome sequencing was performed.Next,SNP-based phylogenetic analysis was performed to compare the two strains to 94 B.melitensis strains(complete genome and draft genome)retrieved from online databases.Results:The two Brucella isolates were identified as B.melitensis biovar 3(QH2019001 and QH2019005)following conventional biotyping and were found to have differences in their variable number tandem repeats(VNTRs)using MLVA-16.Phylogenetic examination assigned the 96 strains to five genotype groups,with QH2019001 and QH2019005 assigned to the same group,but different subgroups.Moreover,the QH2019005 strain was assigned to a new subgenotype,llj,within genotype II.These findings were then combined to determine the geographic origin of the two Brucella strains.Conclusions:Utilizing a whole-genome SNP-based approach enabled differences between the two B.m elitensis strains to be more clearly resolved,and facilitated the elucidation of their different evolutionary histories.This approach also revealed that QH2019005 is a member of a new subgenotype(Hj)with an ancient origin in the eastern Mediterranean Sea.
