List of strains used in this study.

Bacillus anthracis and the emerging pathogen Bacillus cereus biovar anthracis (Bcbva) are causative agents of the lethal disease anthrax. Their ability to form highly resistant endospores enables them to persist in the environment, posing significant threats to wild herbivores, livestock, and also humans globally, especially considering their potential use as bioweapons. Despite the importance of these pathogens, the exact mechanisms underlying host infection remain poorly understood. Notably, both require relatively high infectious doses to cause disease. Proposed transmission routes include soil-based transmission and dissemination by carrion flies, which contaminate the area surrounding infected carcasses. However, considering the substantial dilution of bacteria and spores in these processes alongside the high infectious dose required, sustained transmission under natural conditions appears improbable. An alternative hypothesis is that B. anthracis and Bcbva can survive and proliferate in the environment by forming biofilms, structured bacterial communities attached to surfaces, which may serve as reservoirs for infection. While biofilm formation has been demonstrated for B. anthracis, data on Bcbva are lacking. To address this, we examined the biofilm-forming abilities of multiple B. anthracis and Bcbva strains using a three-dimensional, soil-inspired porous glass bead model (PGB) system. We applied confocal laser scanning microscopy, scanning electron microscopy, and quantitative cell counts to characterize biofilm development in detail. Our results confirm that both B. anthracis and Bcbva form biofilms under somewhat soil-like conditions. Furthermore, differences observed in biofilm structure and density between the above-mentioned species suggest that Bcbva may prefer additional environmental niches beyond soil, such as plant surfaces or small water bodies, potentially expanding our understanding of its environmental persistence and infection routes.