Adaptation of the Geometric Viability assay as a high throughput assay to measure Clostridioides difficile spore germination
Clostridioides difficile is the leading cause of hospital acquired infections and gastroenteritis associated death in the United States. As an obligate anaerobe, C. difficile relies on the metabolically dormant, aerotolerant spore form to transmit infection. C. difficile infection begins when spores germinate in the small intestine in response to specific bile acids and a co-germinant signal (either amino acids or divalent cations). As germination is required to initiate infection, understanding how spores sense germinants and outgrow into toxin-producing cells is an active area of research. One of the fundamental assays used to study C. difficile germination is a resource and time intensive assay, similar to a bacterial CFU assay, that relies on serial dilutions of a spore sample plated on agar media in triplicate to determine the number of viable spores in a sample. As this assay is similar to assays used to enumerate viable bacteria in a sample, we hypothesized that a recently developed high-throughput technique to measure bacterial CFUs published by Meyers et al., the Geometric viability assay (GVA), could be modified to measure the number of viable spores in a sample. We compared measurements of viable spores per mL from the conventional spore viability assay and the modified GVA protocol and found that the two measurements were highly consistent with each other (Pearson r=0.991, R2=0.9821). The modified GVA protocol was also able to measure differences in sensitivity to germination signals in strains of C. difficile hypersensitive to co-germinants and measure low levels of spontaneous germination. The GVA protocol is less resource and time intensive than the conventional assay, was easy adapt, and likely would be feasible to study germination in a wide range of spore-formers.