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.

Bacillus anthracis comparative genome analysis in support of the Amerithrax investigation

“…For example, although current genotyping systems are able to differentiate strains of B. anthracis, they are unable to distinguish rare subpopulations within individual cultures. B. anthracis is highly monomorphic, and accurate genotyping relies on a combination of canonical SNPs and multiple-locus variable-number tandem repeat analyses (1,4,(24)(25)(26). We started with the naïve approach that a single reference genome and sample would be sufficient for comparison.…”

“…T he causative agent of anthrax in mammals, Bacillus anthracis, is a recently emerged pathogen with characteristics of a young evolutionary group (1). It occurs primarily as quiescent spores that can persist for long periods of time in the environment between rapid proliferation growth phases within a host (2).…”

“…Because of this episodic and highly specialized life cycle, the species is characterized by relatively little genetic variation (3)(4)(5). The most divergent B. anthracis strains are believed to share >99.99% nucleotide sequence identity (1,6). B. anthracis has long been considered a potential biological weapon because of the stability of its spores, its potential for aerosolization, and its high rate of infectivity and lethality.…”

“…Genotyping methods, such as multiple-locus variable-number tandem repeat analysis and an additional typing system based on 12 canonical SNPs, could identified colony isolates as the Ames strain yet were insufficient to distinguish between isolates (1,3,4,(10)(11)(12). Because of the limitations of existing typing systems, it was thought that only whole-genome sequencing of Ames isolates would provide the level of discrimination required for microbial forensics and attribution.…”

Identification of an African Bacillus anthracis Lineage That Lacks Expression of the Spore Surface-Associated Anthrose-Containing Oligosaccharide

“…This is interesting in that B. anthracis strains from West Africa belong to a particular genetic lineage, the genotype cluster A␤ (12; P. Pilo [Bern], unpublished observations), as determined by variable-number tandem repeat (VNTR) analysis according the typing scheme of Keim et al (9,29). In this respect, the 8-nucleotide tandem repeat AAA AAAAG that is present in 2 copies in the anthrose biosynthesis aminotransferase gene (BAS3320) in most B. anthracis strains worldwide except those from West Africa, where the repeat is present in 3 copies, is proposed as a valuable new VNTR that allows an increase in the resolution of subtyping and is directly associated with the anthrose phenotype.…”