Uncovering a new subclade of Bacillus anthracis in India: Genomic and phylogenetic insights from recent animal isolates.

Anthrax, a zoonotic disease caused by B. anthracis, has a significant impact on both animal and human health. India reports recurrent anthrax cases, yet genomic data on circulating strains remains limited. In this study, we report whole genome sequence and comparative genome analysis of 17 B. anthracis strains collected between 2018 and 2023 from animal anthrax cases across three states in India. These strains were obtained from sheep (Ovis aries, n = 8), cattle (Bos taurus, n = 3), pigs (Sus scrofa, n = 2), deer (Axis axis, n = 2), goats (Capra hircus, n = 1), and contaminated soil (n = 1). Genomic analysis revealed the presence of 101 virulence factors, 14 antimicrobial resistance genes, and 12 prophages. Phenotypic antimicrobial susceptibility testing revealed selective resistance to fosfomycin, imipenem, and cefotaxime in a few strains. Phylogenetic analysis based on core genome single-nucleotide polymorphism (SNP) analysis categorized them into four distinct subclades, all of which fell within Clade A. Two isolates clustered with A.Br.034 (Vollum) subclade; two with A.Br.001/002 (Ames/Sterne), and five with A.Br.014 (Aust94). Interestingly, seven strains formed a new subclade that diverged from A.Br.005. The global phylogenetic tree with 986 B. anthracis shows that these seven strains, along with the strain identified in the USA, have formed a new subclade and are designated as IND2023. The core genome SNP analysis identified 22 unique SNPs specific to this subclade, underscoring its distinct evolutionary path. A Delayed Mismatch Amplification Assay (DMAA) was developed and validated to detect this new subclade, IND2023. This study elucidates the phylogenetic diversity of anthrax in the Indian subcontinent and contributes to bridging existing gaps in the global phylogenetic diversity of anthrax pathogen.