Genomic study of Bacillus anthracis and Bacillus species isolated from anthrax outbreaks in South Africa

Abstract

Anthrax is a zoonotic disease that naturally occurs in wild and domestic animals. The causative agent of anthrax is Bacillus anthracis that consist of a single chromosome and the two virulence plasmids (pXO1 and pXO2) with the capsule and toxin gene regions that makes up a total genome size of 5.5 Mb. Bacillus anthracis and B. endophyticus isolated from anthrax outbreaks (mainly 2008/9 outbreaks in the northen Cape Province, NCP) in South Africa had similar microbiological and genetic features. Whole genome sequencing (WGS) was used to characterize and differentiate B. anthracis from the closely related B. endophyticus as well as other Bacillus species. The identification of morphological and homologous capsule (polyglutamate) genes present in both B. anthracis and B. endophyticus species resolved the non-conformance of the diagnosis of anthrax. Bacillus anthracis is considered to be monomorphic and single nucleotide polymorphisms (SNPs) are the ideal signatures for subtyping monomorphic species. Globally, most B. anthracis strains cluster into the major A-clade into different sub-clades using phylogenetic analysis. In South Africa, anthrax outbreaks in animals have been reported in the Kruger National Park (KNP) and NCP regions with most B. anthracis strains clustering in the A-clade. The rare Kruger B-clade reported to be restricted to the endemic Pafuri (northern) region in KNP was also investigated. In order to subtype and understand these outbreaks, the study generated a B. anthracis genetic database based on WGS to identify SNPs across sequenced genomes. The sequenced genomes were further compared with other available genomes. In conclusion, this study discovered novel SNPs and defined the phylogenetic structure of B. anthracis in South Africa. WGS-SNP analysis could trace some of the anthrax case outbreaks using similar SNP profiles of B. anthracis strains from different case outbreaks. WGS-SNPs specific to NCP were identified and developed into high-resolution melting SNP (melt-MAMA assays) discriminative assays for the purpose of molecular epidemiology. The sequenced KNP B-clade consisted of heterogeneous B. anthracis genomes that were distinctive from the previously sequenced genomes in KNP. South Africa has diverse SNP sub-clades and branches in the A-clade that are not commonly found world-wide.