Germination and Amplification of Anthrax Spores by Soil-Dwelling Amoebas

While anthrax is typically associated with bioterrorism, in many parts of the world the anthrax bacillus (Bacillus anthracis ) is endemic in soils, where it causes sporadic disease in livestock. These soils are typically rich in organic matter and calcium that promote survival of resilientB. anthracis spores. Outbreaks of anthrax tend to occur in warm weather following rains that are believed to concentrate spores in low-lying areas where runoff collects. It has been concluded that elevated spore concentrations are not the result of vegetative growth asB. anthracis competes poorly against indigenous bacteria. Here, we test an alternative hypothesis in which amoebas, common in moist soils and pools of standing water, serve as amplifiers ofB. anthracis spores by enabling germination and intracellular multiplication. Under simulated environmental conditions, we show thatB. anthracis germinates and multiplies withinAcanthamoeba castellanii . The growth kinetics of a fully virulentB. anthracis Ames strain (containing both the pX01 and pX02 virulence plasmids) and vaccine strain Sterne (containing only pX01) inoculated as spores in coculture withA. castellanii showed a nearly 50-fold increase in spore numbers after 72 h. In contrast, the plasmidless strain 9131 showed little growth, demonstrating that plasmid pX01 is essential for growth withinA. castellanii . Electron and time-lapse fluorescence microscopy revealed that spores germinate within amoebal phagosomes, vegetative bacilli undergo multiplication, and, following demise of the amoebas, bacilli sporulate in the extracellular milieu. This analysis supports our hypothesis that amoebas contribute to the persistence and amplification ofB. anthracis in natural environments.