Archival and Newly Isolated Historical Bacillus anthracis Strains Populate the Deeper Phylogeny of the A.Br.075(Sterne) Clade

The anthrax pathogen Bacillus anthracis can remain dormant as spores in soil for many years. This applies to both natural foci and to sites of anthropogenic activity such as tanneries, abattoirs, or wool factories. The A.Br.075 (A-branch) clade (also known as A.Br.Sterne) is prominent not only because it comprises several outbreak strains but even more so because spore preparations of its namesake, the Sterne strain, are counted among the most utilized anthrax animal vaccines. In this study, we genome-sequenced and analyzed 56 additional B. anthracis isolates of the A.Br.075 clade. Four of these we recently retrieved from soil samples taken from a decades-long abandoned tannery. The other 52 strains originated from our archival collection from the 20th century. Notably, the extended phylogeny of the A.Br.075 clade indicated that many of the newly added chromosomes represent basal members, some of which are among the most basal strains from this lineage. Twelve new strains populate a very deep-branching lineage we have named A.Br.Ortho-Sterne (also known as A.Br.076). A further 11 isolates amend the clade named A.Br.Para-Sterne (A.Br.078). Finally, some of the terminal clusters of the clade named A.Br.Eu-Sterne appear to be replete with (near) identical isolates, possibly a result of widespread use of the Sterne vaccine and of its re-isolation from vaccination-related animal anthrax outbreaks. From the accrued new phylogenetic information, we designed and tested a variety of new SNP-PCR assays for rapid and facile genotyping of unassigned B. anthracis genomes. Lastly, the successful isolation of live B. anthracis from a long-abandoned tannery reemphasizes the need for continued risk awareness of such sites.