Clostridioides difficile spore stocks were generated as described previously [19 (link)]. Briefly, C difficile strains were grown in 2 mL Columbia broth overnight at 37°C anaerobically. The 2-mL inoculum was then added to 40 mL Clospore media. The culture was incubated anaerobically at 37°C for 5–7 days. After the incubation, spores were harvested by centrifuging the culture at 3200 rpm for 20 minutes at 4°C, then resuspending in cold sterile water. After washing the spores at least 3 times, the spore stocks were stored at 4°C in sterile water. The stocks were heat treated at 65°C for 20 minutes to eliminate any remaining vegetative cells. The concentration of spores in each stock was determined by serially diluting the stocks in anaerobic phosphate-buffered saline (PBS) and plating on brain-heart infusion (BHI) agar supplemented with 1% sodium taurocholate. Once the colony-forming units (CFU)/mL of each stock was determined, the infection inoculum was prepared by diluting the appropriate C difficile strain spore stock to the appropriate concentration. Animals received 100 µL of inoculum each via oral gavage.
To determine C difficile colonization in infected animals, cecal contents were resuspended and serially diluted in reduced PBS. Serial dilutions were plated on BHI agar supplemented with 1% sodium taurocholate, 1 mg/mL cycloserine, and 0.032 mg/mL cefoxitin (Sigma, St. Louis, MO), then incubated at 37°C overnight in an anaerobic chamber. Bacterial burden was normalized to cecal content sample weight.
