Persistence in time: the hunt for Bacillus anthracis at a historic tannery site in Austria reveals genetic diversity thought extinct.

Identifying and analyzing historic anthrax loci may provide a treasure trove to fill in the gaps of persistence in time and genetic diversity of Bacillus anthracis. In countries where anthrax has become a disease of the past, detailed knowledge of the exact location and stability of spores in soil reservoirs is limited. Reviewing archival records may provide valuable clues to unearthing such forgotten sites. Knowledge of anthrax diversity in Austria is scarce, as the only available isolates-originating from the last outbreak in Austria in 1988-cluster in the B.Br.004 (CNEVA) canonical single-nucleotide polymorphism (canSNP) group. Thus, we analyzed archival records on anthrax incidents in Austria to locate historic B. anthracis soil reservoirs. In parallel, we tested the performance of different soil processing protocols for the isolation of B. anthracis spores to establish a suitable workflow for screening historical anthrax loci. Using an optimized workflow, we were able to isolate viable B. anthracis spores 80 years after the last occurrence of anthrax at an abandoned tannery identified through our archival work. Genome analysis of the isolated strains allowed to improve the phylogeographic resolution within the hitherto poorly covered A.Br.064 (V770) canSNP group by linking historical records to genetic information. Furthermore, our results re-emphasize that B. anthracis can survive for decades at historic sites and may pose a health threat when such sites are eventually reactivated by climatic factors or human intervention. IMPORTANCE: Bacillus anthracis is a continuing threat from a One Health perspective since it leads to severe infections in animals and humans. Ongoing climate change or human activities can reactivate historical B. anthracis loci, previously considered inactive or forgotten. Therefore, knowledge of historic anthrax incidents at abandoned animal processing facilities, such as tanneries or farmyards, along with robust detection protocols, is of prime interest when monitoring this important zoonosis. As shown here, archival records of possible origins of anthrax-contaminated goods received at tanneries are valuable sources and support these efforts. Investigation for viable spores at such historical sites could not only provide new insights into the past genetic diversity and population structure of B. anthracis but also provide important information for taking appropriate measures to prevent future outbreaks originating from these sites.