Objective To study the differences of chromosomal structure among Yersinia pestis strains isolated from China,and to investigate the reasons of chromosomal rearrangement events occurred in Yersinia pestis as well as the possibility of strain identification and phylogenetic analysis based on the chromosomal rearrangement features.Methods According to the genome sequence data downloaded from web of National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/genome),alignment of all the coding sequences (CDSs) among five strains(American strain CO92 as reference and other four completely sequenced strains from Inner Mongolia,Jianchuan of Yunnan,Yulong of Yunnan,Naqu of Tibet in China named 91001,D182038,D106004 and Z176003 as comparison strains) was performed,and then the chromosome of Yersinia pestis was divided into several large DNA segments (named chromosomal plate in the text) according to the similarity of CDSs.Plate arrangement patterns in each strain' s chromosome and gene content of breakpoint regions were determined.Finally,genetic relationships among Yersinia pestis strains were analyzed on the basis of rearrangement diversity from paired-comparison.Results Yersinia pestis chromosomes of strains CO92,D182038,D106004,91001 were composed of 44 relatively independent plates,except strain Z176003.Gene order was very stable within each plate,while it was movable between the plates.Comparing with the reference strain CO92,13 rearrangement events occurred in the chromosomes of both strain D182038 and strain D106004,and 14 rearrangement events involved in Z176003,while 37 rearrangement events occurred in 91001.Paired-comparison data showed that only 8 plates order differences were existed between D106004 and Z176003.Forty-three breakpoint regions were identified on the chromosome of strain CO92,and 39 of them contained insertion sequences,and 25 of them were IS100.Conclusions Yersinia pestis genome represents a high degree of genetic flux,and chromosomal structures of strains are significantly different from each other.Chromosomal rearrangement events is closely related to the large number of insertion sequences in the Yersinia pestis chromosome.Rearrangement diversity among Yersinia pestis strains could reflect their genetic relationships.

Objective To explore the genetic relationship between the Chinese and the foreign species of Francisella tularensis.Methods Based on our own findings and from the literature,17 SNP,4 INDEL,and 12 VNTR were selected for phylogenetic analysis on 39 strains of F.tularensis,including 10 strains of Chinese F.tularensis and 29 strains of foreign F.tularensis that had been sequenced and published.SNP-INDEL and MLVA were used for the separation and combination.Results Data from the combined analysis indicated that 3 strains of Chinese F.tularensis with Japanese FSC022 were assigned to B5;3 strains,with Swedish FSC200 to B1;3 strains with American OSU18 to B2 and 1 strain with French FTNF002-00,German F92,and American OR96246 to B4,respectively.10 strains of Chinese F.tularensis were assigned to 4 clades and the result demonstrated a wide diversity of F.tularensis subsp.holarctica in China.Conclusion A set of simple and robust typing tools for F.tularensis subsp.holarctica were established in this study.Based on the results,F.tularensis subsp.holarctica might have had its origins in Asia.

OBJECTIVETo perform laboratory diagnosis and tracking source of a suspected tularemia patient in Beijing.

METHODSA suspected tularemia patient was reported in Beijing city on July 19, 2012. Genomic DNA was extracted from the blood sample of the patient, then general PCR and sequencing of amplicons were conducted using 3 specific genes (fopA, tul4 and 16S rRNA) Francisella tularensis (F.tularensis), and 2 genotyping primers (C1C4 and RD1). Two other laboratories repeated the PCR and sequencing of the fopA in parallel. At the same time, real-time PCR fluorescent ration was performed using 4 targets (fopA, ISFtul2, 23kDa, and tul4), and phylogenetic analysis was carried out using 11 canonical single nucleotide polymorphisms (SNPs) and 4 insertions or deletions.

RESULTSAll the 3 specific genes were amplified positively, and sequenced fragments were 409, 407 and 1 053 bp, respectively. The patient was infected by F. tularensis comparing with the whole genome published. Next, amplicons of 151 and 924 bp were obtained by the 2 typing primers after sequencing, respectively. The segment lengths suggested that the patient was infected by the subsp. holarctica. All of the two other laboratories obtained positive data for the PCR and sequencing of the fopA. In addition, all the 4 targets tested positive by real-time PCR for F. tularensis. The Ct value of the fopA, ISFtul2, 23kDa and tul4 were 30, 25, 28, and 30, respectively. The phylogenetic analysis indicated that the whole genome of this case was assigned to a known clade from Russia, which was subgroup B3.

CONCLUSIONThis case was confirmed to be a tularemia patient, and a new subgroup of F. tularensis type B was found in China.

Beijing ; DNA Primers ; DNA, Bacterial ; genetics ; Francisella tularensis ; classification ; Genes, Bacterial ; Genotype ; Humans ; Phylogeny ; Polymorphism, Single Nucleotide ; RNA, Ribosomal, 16S ; genetics ; Real-Time Polymerase Chain Reaction ; Russia ; Tularemia ; epidemiology ; microbiology

OBJECTIVETo study the types of subspecies of Francisella tularensis from China and to investigate the genetic relationships between F. tularensis strains from China and from other countries.

METHODSTen strains of F. tularensis isolated from China were amplified by using typing primers C1/C4 and RD1. On the basis of the lengths of the polymerase chain reaction (PCR) products, it was concluded that these strains of F. tularensis belonged to the same subspecies. At the same time, the fopA, tul4, and 16S rRNA genes of the 10 strains were amplified, and a three-gene based phylogenetic analysis was performed using the Molecular Evolutionary Genetics Analysis software version 4.0.

RESULTSThe 10 strains of F. tularensis from China were all identified as belonging to subspecies holarctica (type B). We found no direct relationship between the genotypes of F. tularensis subsp. holarctica and the geographical area from where they were isolated.

CONCLUSIONThe F. tularensis strains isolated from North China mainly belong to subspecies holarctica (type B). The strains of F. tularensis subsp. holarctica from China may have evolved earlier than those from Europe and North America.

China ; Francisella tularensis ; classification ; genetics ; Molecular Sequence Data ; Phylogeny

Objective To perform laboratory diagnosis and tracking source of a suspected tularemia patient in Beijing.Methods A suspected tularemia patient was reported in Beijing city on July 19, 2012.Genomic DNA was extracted from the blood sample of the patient, then general PCR and sequencing of amplicons were conducted using 3 specific genes (fopA, tul4 and 16S rRNA)Francisella tutarensis(F.tularensis), and 2 genotyping primers (C1C4 and RD1).Two other laboratories repeated the PCR and sequencing of the fopA in parallel.At the same time, real-time PCR fluorescent ration was performed using 4 targets (fopA, ISFtul2, 23kDa, and tul4), and phylogenetic analysis was carried out using 11 canonical single nucleotide polymorphisms (SNPs) and 4 insertions or deletions.Results All the 3 specific genes were amplified positively, and sequenced fragments were 409, 407 and 1 053 bp, respectively.The patient was infected by F.tularensis comparing with the whole genome published.Next, amplicons of 151 and 924 bp were obtained by the 2 typing primers after sequencing, respectively.The segment lengths suggested that the patient was infected by the subsp.holarctica.All of the two other laboratories obtained positive data for the PCR and sequencing of the fopA.In addition, all the 4 targets tested positive by real-time PCR for F.tularensis.The Ct value of thefopA, ISFtul2, 23kDa and tul4 were 30, 25, 28, and 30, respectively.The phylogenetic analysis indicated that the whole genome of this case was assigned to a known clade from Russia, which was subgroup B3.Conclusion This case was confirmed to be a tularemia patient, and a new subgroup of F.tularensis type B was found in China.

Objective To perform laboratory diagnosis and tracking source of a suspected tularemia patient in Beijing.Methods A suspected tularemia patient was reported in Beijing city on July 19, 2012.Genomic DNA was extracted from the blood sample of the patient, then general PCR and sequencing of amplicons were conducted using 3 specific genes (fopA, tul4 and 16S rRNA)Francisella tutarensis(F.tularensis), and 2 genotyping primers (C1C4 and RD1).Two other laboratories repeated the PCR and sequencing of the fopA in parallel.At the same time, real-time PCR fluorescent ration was performed using 4 targets (fopA, ISFtul2, 23kDa, and tul4), and phylogenetic analysis was carried out using 11 canonical single nucleotide polymorphisms (SNPs) and 4 insertions or deletions.Results All the 3 specific genes were amplified positively, and sequenced fragments were 409, 407 and 1 053 bp, respectively.The patient was infected by F.tularensis comparing with the whole genome published.Next, amplicons of 151 and 924 bp were obtained by the 2 typing primers after sequencing, respectively.The segment lengths suggested that the patient was infected by the subsp.holarctica.All of the two other laboratories obtained positive data for the PCR and sequencing of the fopA.In addition, all the 4 targets tested positive by real-time PCR for F.tularensis.The Ct value of thefopA, ISFtul2, 23kDa and tul4 were 30, 25, 28, and 30, respectively.The phylogenetic analysis indicated that the whole genome of this case was assigned to a known clade from Russia, which was subgroup B3.Conclusion This case was confirmed to be a tularemia patient, and a new subgroup of F.tularensis type B was found in China.

Objective To understand the homology of sequence type 562 (ST562) strains of Burkholderia pseudomallei which circulated in two separate continents (Asia and Australia) at different times.Methods Spe Ⅰ restriction fragments and 4-locus multiple locus variable number tandem repeat analysis (MLVA-4) profiles were extracted from MSHR5858 (ST562 Australia strain) and 350105 (ST562 historical strain of Hainan) genomes respectively by in silico analysis and then compared with the PFGE and MLVA-4 results of five ST562 clinical isolates from Hainan to test their homology.Synteny and homology between MSHR5858 and 350105 genomes were evaluated with bioinformatics methods.Results Five ST562 clinical strains from Hainan shared same PFGE pattern (similarity ＞97％) and this pattern coincided to the map of Spe Ⅰ restriction fragments of Australian strain MSHR5858.The amounts of genomic restriction fragments (Spe Ⅰ) for MSHR5858 and 350105 were 31 and 34 respectively,with 31 of them matched by each other.Five ST562 clinical strains of Hainan were distinct by MLVA-4 profiles,among which HPPH43 (MLVA-4 profile:10,8,10,8) was close to Australia strain MSHR5858 (10,8,8,6),containing identical repeat numbers at VNTR loci 2341k and 1788k;while HK003 (11,8,15,7) and HK061 (11,8,17,7) similar to Hainan historical strain 350105 (11,8,11,8),with same repeat numbers at loci 2341k and 1788k also.High-degree synteny and consistency on genomic contents were observed between 350105 and MSHR5858,indicating a similar origin for the 2 strains.Conclusion All inter-continental and historical ST562 strains ofB.pseudomallei had similar genomic characteristics,supporting the assumption that they had a common origin.Also,it is possible that Hainan historical strain 350105 is the ancestor of all circulating ST562 strains.