Anthrax is a zoonosis caused by the environmentally maintained, spore-forming bacterium Bacillus anthracis, affecting humans, livestock, and wildlife nearly worldwide. Bacterial spores are ingested, inhaled, and may be mechanically transmitted by biting insects or injection as occurs during heroin-associated human cases. Herbivorous hoofstock are very susceptible to anthrax. When these hosts die of anthrax, a localized infectious zone (LIZ) forms in the area surrounding the carcass as it is scavenged and decomposes, where viable populations of vegetative B. anthracis and spores contaminate the environment. In many settings, necrophagous flies contaminate the outer carcass, surrounding soils, and vegetation with viable pathogen while scavenging. Field observations in Texas have confirmed this process and identified primary browse species (e.g., persimmon) are contaminated. However, there are limited data available on B. anthracis survival on environmental substrates immediately following host death at a LIZ. Toward this, we simulated fly contamination by inoculating live-attenuated, fully virulent laboratory-adapted, and fully virulent wild B. anthracis strains on untreated leaves and rocks for 2, 5, and 7 days. At each time point after inoculation, the number of vegetative cells and spores were determined. Sporulation rates were extracted from these different time points to enable comparison of sporulation speeds between B. anthracis strains with different natural histories. We found all B. anthracis strains used in this study could multiply for 2 or more days post inoculation and persist on leaves and rocks for at least seven days with variation by strain. We found differences in sporulation rates between laboratory-adapted strains and wild isolates, with the live-attenuated strain sporulating fastest, followed by the wild isolates, then laboratory-adapted virulent strains. Extrapolating our wild strain lab results to potential contamination, a single blow fly may contaminate leaves with up to 8.62 x 105 spores per day and a single carcass may host thousands of flies. Replication outside of the carcass and rapid sporulation confirms the LIZ extends beyond the carcass for several days after formation and supports the necrophagous fly transmission pathway for amplifying cases during an outbreak. We note caution must be taken when extrapolating replication and sporulation rates from live-attenuated and laboratory-adapted strains of B. anthracis.

The Bacillus anthracis exosporium nap is the outermost portion of spore that interacts with the environment and host systems. Changes to this layer have the potential to impact wide-ranging physiological and immunological processes. The unique sugar, anthrose, normally coats the exosporium nap at its most distal points. We previously identified additional mechanisms rendering B. anthracis anthrose negative. In this work, several new ant−B. anthracis strains are identified and the impact of anthrose negativity on spore physiology is investigated. We demonstrate that live-attenuated Sterne vaccines as well as culture filtrate anthrax vaccines generate antibodies targeting non-protein components of the spore. The role of anthrose as a vegetative B. anthracis Sterne signaling molecule is implicated by luminescent expression strain assays, RNA-seq experiments, and toxin secretion analysis by western blot. Pure anthrose and the sporulation-inducing nucleoside analogue decoyinine had similar effects on toxin expression. Co-culture experiments demonstrated gene expression changes in B. anthracis depend on intracellular anthrose status (cis) in addition to anthrose status of extracellular interactions (trans). These findings provide a mechanism for how a unique spore-specific sugar residue affects physiology, expression and genetics of vegetative B. anthracis with impacts on the ecology, pathogenesis, and vaccinology of anthrax.