Groups of 5 week old C57BL/6J WT mice (male and female) were treated with ursodiol at three distinct doses (50, 150, and 450 mg/kg dissolved in corn oil; Ursodiol U.S.P., Spectrum Chemical, CAS 128-13-2) given daily via oral gavage for 21 days (Fig 1). These distinct doses were selected for a proof of concept experiment in order to achieve sufficient intestinal concentrations of ursodiol to alter the life cycle of Clostridioides difficile in vivo [32]. The total volume gavaged was consistent between the three distinct doses in order to control for the volume of corn oil administered. Ursodiol dosing was adjusted once weekly, based on current weight. Two independent experiments were performed, with a total of n = 8 mice (female/male) per treatment group. Mice were weighed daily over the course of the experiment. Fecal pellets were collected twice daily, flash-frozen and stored at -80°C until further analysis. A control group of mice were necropsied prior to initiating any treatments (pretreatment group). This pretreatment group serves as a microbiome and bile acid metabolome baseline prior to mice receiving ursodiol treatment. An additional control group of mice underwent daily handling similar to the treatment groups, but were not administered ursodiol (no treatment control). Necropsy was performed at day 21 in all ursodiol treated mice and the no treatment control mice. Gastrointestinal contents and tissue from the ileum and cecum were collected, flash frozen in liquid nitrogen, and stored at -80°C until further analysis. Serum and bile aspirated from the gallbladder was obtained flash frozen in liquid nitrogen, and stored at -80°C until further analysis.

Groups of 5-week old C57BL/6J WT mice were treated with ursodiol at three distinct doses (50, 150, and 450 mg/kg) given daily via oral gavage for 21 days. Fecal collection was performed twice daily throughout the experiment. Two independent experiments were performed, with a total of n = 8 (4 females/4males) mice per treatment group. Mice were monitored and weighed daily throughout the experiment. A control group of mice were necropsied prior to initiating any treatments (pretreatment group). Necropsy was performed at day 21 for all ursodiol treated mice (open circles).

On several occasions, mice had evidence of corn oil within the oral cavity or on their muzzles immediately after the gavage. These mice were monitored closely for signs of aspiration pneumonia for 36 hr following this event. Two mice, one from the ursodiol 50 mg/kg group and another from the ursodiol 450 mg/kg group, inadvertently aspirated gavaged ursodiol, containing corn oil, and subsequently developed respiratory distress within 12–24 hr following the aspiration event. The clinical signs were most consistent with lipid induced pneumonitis and both mice were humanely euthanized and excluded from the study.

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