Investigating Anthrax-Associated Virulence Genes among Archival and Contemporary Bacillus cereus Group Genomes

<i>Bacillus anthracis</i> causes anthrax through virulence factors encoded on two plasmids. However, non-<i>B. anthracis</i> organisms within the closely related, environmentally ubiquitous <i>Bacillus cereus</i> group (BCG) may cause an anthrax-like disease in humans through the partial adoption of anthrax-associated virulence genes, challenging the definition of anthrax disease. To elucidate these phenomena and their evolutionary past, we performed whole-genome sequencing on non-<i>anthracis</i> BCG isolates, including 93 archival (1967-2003) and 5 contemporary isolates (2019-2023). We produced annotated genomic assemblies and performed a pan-genome analysis to identify evidence of virulence gene homology and virulence gene acquisition by linear inheritance or horizontal gene transfer. At least one anthrax-associated virulence gene was annotated in ten isolates. Most homologous sequences in archival isolates showed evidence of pseudogenization and subsequent gene loss. The presence or absence of accessory genes, including anthrax-associated virulence genes, aligned with the phylogenetic structure of the BCG core genome. These findings support the hypothesis that anthrax-associated virulence genes were inherited from a common ancestor in the BCG and were retained or lost across different lineages, and contribute to a growing body of work informing public health strategies related to anthrax surveillance and identification.