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During an anthrax outbreak at the Pollino National Park (Basilicata, Italy) in 2004, diseased cattle were buried and from these anthrax-foci  Bacillus anthracis  endospores still diffuse to the surface resulting in local accumulations. Recent data suggest that  B .  anthracis  multiplies in soil outside the animal-host body. This notion is supported by the frequent isolation of  B .  anthracis  from soil lacking one or both virulence plasmids. Such strains represent an evolutionary dead end, as they are likely no longer able to successfully infect new hosts. This loss of virulence plasmids is explained most simply by postulating a soil-borne life cycle of the pathogen. To test this hypothesis we investigated possible microevolution at two natural anthrax foci from the 2004 outbreak. If valid, then genotypes of strains isolated from near the surface at these foci should be on a different evolutionary trajectory from those below residing in deeper-laying horizons close to the carcass. Thus, the genetic diversity of  B .  anthracis  isolates was compared conducting Progressive Hierarchical Resolving Assays using Nucleic Acids (PHRANA) and next generation Whole Genome Sequencing (WGS). PHRANA was not discriminatory enough to resolve the fine genetic relationships between the isolates. Conversely, WGS of nine isolates from near-surface and nine from near-carcass revealed five isolate specific SNPs, four of which were found only in different near-surface isolates. In support of our hypothesis, one surface-isolate lacked plasmid pXO1 and also harbored one of the unique SNPs. Taken together, our results suggest a limited soil-borne life cycle of  B .  anthracis .

Microevolution of Anthrax from a Young Ancestor (M.A.Y.A.) Suggests a Soil-Borne Life Cycle of Bacillus anthracis