Whole Genome-Sequencing and Phylogenetic Analysis of a Historical Collection of Bacillus anthracis Strains from Danish Cattle

Abstract

Bacillus anthracis, the causative agent of anthrax, is known as one of the most genetically monomorphic species. Canonical single-nucleotide polymorphism (SNP) typing and whole-genome sequencing were used to investigate the molecular diversity of eleven B. anthracis strains isolated from cattle in Denmark between 1935 and 1988. Danish strains were assigned into five canSNP groups or lineages, i.e. A.Br.001/002 (n = 4), A.Br.Ames (n = 2), A.Br.008/011 (n = 2), A.Br.005/006 (n = 2) and A.Br.Aust94 (n = 1). The match with the A.Br.Ames lineage is of particular interest as the occurrence of such lineage in Europe is demonstrated for the first time, filling an historical gap within the phylogeography of the lineage. Comparative genome analyses of these strains with 41 isolates from other parts of the world revealed that the two Danish A.Br.008/011 strains were related to the heroin-associated strains responsible for outbreaks of injection anthrax in drug users in Europe. Eight novel diagnostic SNPs that specifically discriminate the different sub-groups of Danish strains were identified and developed into PCR-based genotyping assays.

Fig 1. Position of the eleven Danish strains within the B. anthracis phylogenetic tree based on whole-genome SNP analysis.

Minimum spanning tree based on 6596 chromosomal SNPs (A), 222 pXO1 SNPs (B) and 166 pXO2 SNPs (C). The 13 different canSNP groups are color-coded: C.Br.A1055 in white, B.Br.CNEVA in yellow, B.Br.001/002 and B.Br.Kruger in orange, A.Br.011/009 in light blue, A.Br.008/011 in blue, A.Br.WNA in dark blue, A.Br.005/006 in pink, A.Br.003/004 in red, A.Br.001/002 in green, A.Br.WNA in dark green, A.Br.Aust94 in brown and A.Br.Vollum in purple. The position of the 11 Danish isolates (in bold and underligned), the African IEMVT89 and 40 available whole genome-sequenced strains is marked. The length of each branch is proportional (logarithmic scale) to the number of SNPs identified between strains. Indicated in red are the position and name of some new or published SNPs specific to various canSNP groups: A05 (A.Br.005/006 group); A.Br.008 (A.Br.008/009 group); A08/D and A08/D1 (A.Br.008/011); A.Br.011 (A.Br.011/009); A.Br.009 (A.Br.WNA); A.Br.002 and A01 (A.Br.001/002 and A.Br.Ames); A02, A02/A, A02/B, A02/B1 (A.Br.001/002 subgroup A02); A.Br.001 and A01/A-DK (A.Br.Ames); A.Br.013, A.Br.015a, A.Br.15b, A.Br.026 (A.Br.Aust94). Based on a parsimony approach, the trees sizes are, respectively, 6730 (A), 227 (B) and 168 SNPs (C), i.e. containing approximately 1.9 (A), 2.2 (B) or 1.2 (C) % of homoplasia. Available from the Dryad Digital Repository, see S1 File.