Canonical single nucleotide polymorphism analysis and multiple-locus variable number tandem repeat analysis on genetic characteristics of Bacillus anthracis in China

Objective To understand the genetic characteristics of Bacillus. anthracis in China by canonical single nucleotide polymorphism（canSNP）analysis and multiple-locus variable number tandem repeat analysis (MLVA) on genotype polymorphism of B. anthracis.

Methods A total of 533 isolates of B. anthracis were collected in China. Genotype identification was carried out by using the canSNP based on 13 SNP loci and MLVA15 genotyping protocol based on 15 variable number tandem repeat (VNTR) loci. Software Bionumerics 5.1was used for clustering analysis and minimum spanning trees were constructed to analyze the genetic relationships and Simpson’s index of each loci was calculated.

Results All the 533 strains of B. anthracis belonged to the linage A of the global anthrax lineages, and had 6 sublineages, which were A.Br.001/002, A. Br.Ames, A.Br.008/009, A. Br.Vollum, A. Br.Aust94 and A. Br.005/006, respectively. Of these sublineages, A.Br.001/002 (60.98%) was predominant and widely distributed in China. A. Br.Aust94 (14.26%), A.Br.008/009 (11.82%) and A.Br.Vollum (4.69%) were mainly isolated from Xinjiang in 1980s and 1990s, and A.Br.Ames was mainly isolated from Inner Mongolia. A.Br.005/006 was a newly discovered sublineage in 2019. The geographical distribution of sublineages of B. anthracis varied with province, with 4 - 5 sublineages in Xinjiang, Guangxi, Qinghai, Inner Mongolia and Hebei, and only 1 in Sichuan and other 10 provinces. The predominant sublineages of B. anthracis were A.B.Aust94 and A.B.008/009 in Xinjiang, and A.B.001/002 in other regions. The distribution of sublineages were different among years. A. Br.001/002 was the predominant sublineage, and A. Br.008/009, A. Br.vollum and A. Br.Aust94 emerged and spread successively in the 1980s and 1990s, A .Br. Ames gradually spread after 2000, and a new sublineage A.Br.005/006 was detected in 2019. The study result revealed the genetic characteristics and the spread trend of B. anthracis in China. There were 91 genotypes indicated by MLVA15, and the results of MLVA15 genotyping were basically consistent with canSNP typing. The high resolution ability of MLVA15 was found in all sublineages.

Conclusion In this study, the canSNP and MLVA genotype databases of B. anthracis were established in China, and the distribution characteristics and spread trend of B. anthracis were revealed, which provided scientific basis for the control and molecular source tracing of the anthrax outbreak.