Bacillus anthracis spores germinate extracellularly at air-liquid interface in an in vitro lung model under serum-free conditions

Abstract

Aims: To better understand the parameters that govern spore dissemination after lung exposure using in vitro cell systems. Methods and Results: We evaluated the kinetics of uptake, germination and proliferation of Bacillus anthracis Sterne spores in association with human primary lung epithelial cells, Calu-3 and A549 cell lines. We also analysed the influence of various cell culture medium formulations related to spore germination. Conclusions: We found negligible spore uptake by epithelial cells, but germination and proliferation of spores in the serum-free extracellular environment was evident. Spore germination was appreciably higher in immortalized cell cultures than in primary epithelial cells. Additionally, spores still germinated apically at a mucus-secreting air-liquid interface lung barrier that was devoid of cell culture medium much earlier than medium-only controls. Significance and Impact of the Study: The role of lung epithelial cells in B. anthracis spore dissemination after inhalation remains poorly defined and rather controversial. These results are novel as they show spore germination is appreciably enhanced in the presence of lung cells in vitro, however, the cell line and cell state (air-liquid interface vs submerged in medium) dictates the extent of germination and in some cases proliferation.