Animal models

All animal experiments were performed using protocol M2000027–00 approved by the Vanderbilt University Medical Center Institutional Animal Care and Use Committee and in compliance with NIH guidelines, the Animal Welfare Act, and US Federal law. Animal studies were conducted using 8- to 12-week old male C57BL/6 (Jackson Laboratories) mice housed in groups of up to five at the Vanderbilt University Medical Center Animal Facilities. Briefly, C57BL/6 mice received cefoperazone in their drinking water (0.5 mg/ml) for 5 days. Following a 2-day recovery period, mice were gavaged orally with PBS or infected with 10⁵ C. difficile spores to an endpoint of three days. CDI symptomology was monitored daily by weight loss. Mice were humanely euthanized using compressed CO₂. Intestinal segments were dissected, embedded in 2.6% carboxymethylcellulose (CMC), and frozen in liquid nitrogen. Samples were stored at −80°C prior to sectioning. Mice provided a cholestyramine or control diet (Dyets, Inc., Bethlehem, PA) were placed on these diets at the same time they were given cefoperazone, and remained on their respective modified diets for the duration of the experiment. Mice provided 3% DSS in their drinking water for 2 days were first treated with cefoperazone for 5 days, followed by a 2-day recovery period. Mice receiving rectal infusion of purified, recombinant TcdA or TcdB (or PBS) were placed on 0.5 mg/ml cefoperazone in their drinking water for 5 days, followed by a 2-day recover period, before receiving 50 μg of either toxin (Markham et al., 2021). Mice were euthanized after 8 hours.