Comparative genomics of Bacillus anthracis strains from anthrax outbreaks in Kruger National Park, South Africa

Abstract

Anthrax is a zoonotic disease caused by a Gram-positive, rod-shaped, soil bacterium known as Bacillus anthracis. The global phylogenomic structure of B. anthracis consists of three major lineages namely clades A, B, and C. Anthrax is endemic in the northern part of Kruger National Park (KNP) in South Africa, which show cases of distinctive genetic diversity of B. anthracis A and B-clade strains. Until the 1990s, animals in the KNP were mainly infected by B-clade strains, but since the 1990s, only A-clade strains have been isolated from animals. In this study, whole genome sequencing (WGS) approach was employed to screen B. anthracis isolates (n=80) collected from animal carcasses and environmental samples in the KNP between 2012 and 2015. Whole genome single nucleotide polymorphism (wgSNP) and pan-genome analysis were employed to infer the population structure of B. anthracis A- and B-clades, as well as to understand the genetic differences amongst these clades, respectively. Furthermore, genetic characterization of the Bacillus collagen-like protein A (bclA), tryptophan operon and antibiotic resistance (AR) genes were investigated on A- and B-clades of the KNP B. anthracis genomes. The population structure of the KNP B. anthracis consisted of diverse strains grouping in the prominent A.Br.005/006 (Ancient A) SNP lineage. The B. anthracis isolates from 2012-2015 are dispersed amongst minor sub-clades that present a non-stabilized genetic population within this SNP lineage. Pan-genomics showed a clear distinction between A- and B-clade B. anthracis genomes with 11,374 predicted clusters of protein coding sequences. B-clade genomes contain unique accessory genes that dissociates them phylogenetically from the A-clade genomes. These consisted of multiple copies of Heme-based aerotactic transducer (hemAT), long-chain-fatty-acid--CoA ligase (fadD13) and murein hydrolases (mepH) which form part of the cell wall biosynthesis pathway of B. anthracis. The mepH gene, located on pXO2 in B-clade genomes is truncated and structurally differs from the A-clade genomes. AR profiles showed that the genomes of A and B lineages consist of fosB1 and fosB2 genes that encode for fosfomycin resistance. However, a gene copy of a multidrug gene (mdtG_2) was a unique feature of the B-clade genomes. Furthermore, one of the KNP A.Br.005/006 strain 1298 had a unique antimicrobial resistance profile (blaTEM-116_1) which conferred resistance to amoxicillin, ampicillin, cephalothin, piperacillin and ticarcillin. Genes linked to prophage regions were also identified as a unique feature in A-clade genomes. Genetic differences in the tryptophan operon showed a truncated tryptophan synthase subunit alpha (trpA) gene in the B-clade strains. Sequence length variation of the BclA protein on the exosporium of the B. anthracis endospores and the copy number of the GXX amino acid repeats identified in the collagen like region (CLR) could be used for subtyping and differentiation of B. anthracis strains. The A.Br.005/006 genomes had a higher copy number (4:3) of variable number of tandem repeats (VNTR) in comparison to B-clade genomes. Comparative genomics was successfully used to infer genetic diversity of B. anthracis strains. This study highlights the ubiquity of the A-clade, and its persistence in both enzootic and non-enzootic (areas south of the endemic region) regions of the KNP. This is the first study to report genetic variability in A- and B-clade B. anthracis strains based on the tryptophan operon and the Bacillus collagen-like protein A (bclA) in the KNP. The observed mutation in the trpA gene results into a pseudogene, inhibiting the termination step of the tryptophan biosynthesis pathway. Additionally, we were able to identify genes that contribute to the phylogenomic clustering of A and B KNP B. anthracis strains. Further studies on cell wall structure (genes and associated pathways) will provide insight on the persistence of A-clade B. anthracis genotypes over B-clade strains. This will inform adaptability and survival in the endemic regions. This study has revealed that WGS is a powerful tool that can be used to understand the evolution of B. anthracis for epidemiological surveillance and to trace anthrax outbreaks.