Spore germination is essential for the major nosocomial pathogen Clostridium difficile to initiate and transmit infection, yet clinical isolates vary widely in their responsiveness to bile salt germinants. The clinical significance of this variation is currently unknown, although recent reports suggest that strains with reduced sensitivity to the bile salt germinant taurocholate may be more likely to cause severe disease. A critical barrier to determining whether variation in germinant sensitivity impacts disease severity is the absence of known factors that control germinant sensitivity. Since identifying such factors is complicated by the extensive variation between clinical isolates, we will use targeted mutagenesis and genetic screens to identify key regulators that either increase or decrease C. difficile germinant sensitivity. The effect of these regulators on the germinant sensitivity, germination kinetics and germination heterogeneity will be determined using an innovative Raman spectroscopy and differential interference microscopy (DIC) method that can simultaneously measure key events during spore germination at the single-cell level, unlike existing methods. By identifying factors that regulate germinant sensitivity, the proposed studies will provide key insights into how C. difficile senses the gut environment in order to initiate infection, guide the development of therapeutic strategies that alter the levels of specific bile salts, and may reveal new therapeutic targets. Furthermore, the isogenic strains developed in the proposed work will allow the impact of germinant sensitivity on disease severity to be rigorously tested in animal models of infection in future studies.