Global genetic population structure of Bacillus anthracis

Abstract

Anthrax, caused by the bacterium Bacillus anthracis, is a disease of historical and current importance that is found throughout the world. The basis of its historical transmission is anecdotal and its true global population structure has remained largely cryptic. Seven diverse B. anthracis strains were whole-genome sequenced to identify rare single nucleotide polymorphisms (SNPs), followed by phylogenetic reconstruction of these characters onto an evolutionary model. This analysis identified SNPs that define the major clonal lineages within the species. These SNPs, in concert with 15 variable number tandem repeat (VNTR) markers, were used to subtype a collection of 1,033 B. anthracis isolates from 42 countries to create an extensive genotype data set. These analyses subdivided the isolates into three previously recognized major lineages (A, B, and C), with further subdivision into 12 clonal sub-lineages or sub-groups and, finally, 221 unique MLVA15 genotypes. This rare genomic variation was used to document the evolutionary progression of B. anthracis and to establish global patterns of diversity. Isolates in the A lineage are widely dispersed globally, whereas the B and C lineages occur on more restricted spatial scales. Molecular clock models based upon genome-wide synonymous substitutions indicate there was a massive radiation of the A lineage that occurred in the mid-Holocene (3,064-6,127 ybp). On more recent temporal scales, the global population structure of B. anthracis reflects colonial-era importation of specific genotypes from the Old World into the New World, as well as the repeated industrial importation of diverse genotypes into developed countries via spore-contaminated animal products. These findings indicate humans have played an important role in the evolution of anthrax by increasing the proliferation and dispersal of this now global disease. Finally, the value of global genotypic analysis for investigating bioterrorist-mediated outbreaks of anthrax is demonstrated.

Figure 1. The relationship between canSNPs, sub-lineages and/or sub-groups: The stars in this dendrogram represent specific lineages that are defined by one of the seven sequenced genomes of B. anthracis.

The circles represent branch points along the lineages that contain specific subgroups of isolates. These sub-groups are named after the canSNPs that flank these positions. Indicated in red are the positions and names for each of the canSNPs (also see Table 1).

Figure 2. UPGMA dendrogram of VNTR data from worldwide B. anthracis isolates: Fifteen VNTR loci and UPGMA cluster analysis were used to establish genetic relationships among the 1,033 B. anthracis isolates.

In this UPGMA dendrogram, which was created using MEGA software , groups of genetically similar isolates are collapsed into black triangles that are sized in proportion to the number of isolates in that particular lineage. VNTR loci mutate at faster rates than SNPs and, hence, provide greater resolution for terminal branches. Longer branches, such as the B and C lineages, have length underestimation in this analysis due to mutational saturation. The scale bar indicates genetic distance. Also illustrated on this figure is the distribution of the canonical SNP groups relative to the MLVA phylogeny (right columns). The number of isolates (N) associated with each canSNP group is shown in the second column. The correlation between the phylogenetic clusters identified by the canSNP and MLVA analysis with regards to the world wide geographic distribution of these clusters can be seen in Figure 3.

Figure 3. Worldwide distribution of B. anthracis clonal lineages:Phylogenetic and geographic relationships among 1,033 B. anthracis isolates.

(A) Population structure based upon analysis of data from 12 canSNP (Protocol S1). The numbers of isolates (N) and associated MLVA genotypes (G) within each sub-lineage are indicated as well as the average Hamming distance (D) as estimated from VNTR data. The major lineages (A, B, C) are labelled, as are the derived sub-lineages (1–12), which are also color-coded. (B) Frequency and geographic distribution of the B. anthracis sub-lineages. The colors represented in the pie charts correspond to the sub-lineage color designations in panel A.