| Objective Molecular typing and source-tracking analysis of strains of Bacillus anthracis isolated from Yunnan Province from 2014 to 2015.To understand genetic characteristics and genotypes,to provide scientific basis for the prevention and control of anthrax.MethodsOne strain of Bacillus anthracis isolated from Midu county in 2014(named YN1)and two strains of Bacillus anthracis isolated from Dongchuan district in 2015(named YN2 and YN3 respectively)were used as test strains.The genomic DNA of Bacillus anthracis was extracted.Two molecular typing methods namely canonical single-nucleotide polymorphism(canSNP)analysis and multi-locus variable number tandem repeat analysis(MLVA)with 15 markers.(the loci were VrrA,VrrB1,VrrB2,VrrC1,VrrC2,CG3,Pxo1-aat,Pxo2-at,VNTR12,VNTR16,VNTR17,VNTR19,VNTR23,VNTR32 and VNTR35 respectively)were used.Using the molecular typing library of Chinese Bacillus anthracis strains as a reference for typing and clustering analysis.At the same time,the whole genome sequence of three strains was sequenced by second generation sequencing and make a preliminary analysis.Results1.Can SNP results:The nucleotide structure of the B.anthracis YN1 strain genome at the 13 SNPs were characterized as T,G,A,T,A,T,T,A,T,G,G,T and A at A.Br.001,A.Br.002,A.Br.003,A.Br.004,A.Br.006,A.Br.007,A.Br.008,A.Br.009,B.Br.001,B.Br.002,B.Br003,B.Br.004 and A/B.Br.001 respectively.YN1 strain is classified as the A branch(A.Br.)001/002 canSNP subgroup;The nucleotide structure of the B.anthracis YN2 and YN3 strains genome at the 13 SNPs were characterized as T,G,A,T,A,T,T,A,T,G,G,T and A at at A.Br.001,A.Br.002,A.Br.003,A.Br.004,A.Br.006,A.Br.007,A.Br.008,A.Br.009,B.Br.001,B.Br.002,B.Br003,B.Br.004 and A/B.Br.001 respectively.YN2 and YN3 strains belonging to the A branch(A.Br.)001/002 canSNP subgroup;2.MLVA15 results: for the 15 VNTR locus,the amplification product fragment size of the B.anthracis YN1 strain was 314 bp,229bp,162 bp,80bp,532 bp,158bp,135 bp,141bp,115 bp,273bp,378 bp,96bp,197 bp,571bp,115 bp respectively.The amplification product fragment size of YN2 and YN3 are 314 bp,229bp,162 bp,580bp,532 bp,158bp,132 bp,141bp,115 bp,273bp,386 bp,96bp,197 bp,571bp,115 bp respectively.The results showed that Bacillus anthracis YN1 strain formed a separate branch and was a new genotype,it was classified as MLVA15-CHN51 genotype.While Bacillus anthracis YN2 and YN3 strains belonging to MLVA15-6 genotype.3.The whole genome sequencing results: After sequencing,the YN1 genome size was 5.74 Mb,containing 5948 genes,the average gene length was 778 nt,G+C content was 35.1(%),containing 90 t RNA,11 5SrRNA genes,1 23 SrRNA,324 tandem repeats,7 prophage genes,21 gene islands,26 Contigs;The YN2 genome size was 5.58 Mb,containing 6,202 genes,an average gene length was 778 nt,and G+C content was35.1%.containing 92 t RNA,11 5SrRNA genes,1 23 SrRNA,352 tandem repeats,8prophage genes,19 gene islands,27 Contigs;The YN3 genome size was 5.58 Mb,containing 5931 genes,with an average gene length was776 nt,G+C content was 35.1%.containing 90 tRNA,11 5SrRNA genes,1 16 SrRNA and 1 23 SrRNA,325 tandem repeats,9 prophage genes,21 gene islands,32 Contigs.ConclusionIn this study,we founded that YN1 strain was a new MLVA15 genotype,named MLVA15-CHN51.The canSNP genotype of YN2 and YN3 strains are the same,and MLVA15 genotype showed the two strains were clustered in the same branch.Combing with the epidemiological investigation of anthrax outbreak in 2015,we can infer that the patients in this outbreak were infected with the same infectious source.The three strains have some differences at the genomic level,which is worth further discussion. |
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