Establishing a Mouse Model of Clostridium difficile Infection

Begin with a mouse pretreated with antibiotics to deplete the gut microbiota.

Gently restrain the mouse in an upright position to elongate the esophagus.

Next, take an oral gavage needle connected to a syringe containing an inoculum of spores derived from Clostridium difficile, an anaerobic bacterial pathogen.

Insert the gavage needle through the side of the mouth and slowly advance it into the esophagus.

Once the needle reaches the mid-esophagus, slowly deliver the inoculum and gently withdraw the needle.

The administered spores pass through the stomach into the small intestine, where bile salts trigger germination into C. difficile vegetative bacteria.

These bacteria migrate to the anaerobic colon, where the depletion of competing microbiota enables adhesion to epithelial surfaces, proliferation, and colony formation.

The colonized bacteria release toxins that disrupt epithelial junctions and damage the intestinal barrier. This barrier disruption promotes bacterial translocation into deeper tissues, establishing Clostridium difficile infection.

In a laminar flow hood, take a subject mouse and immobilize the head by restraining the animal gently by the loose skin of the neck and back.

Using the index finger and ensuring the animal does not vocalize or show signs of distress, further immobilize the animal's head and elongate the esophagus. Maintain the mouse in an upright, vertical position and gently pass the gavage needle into the side of the mouth. Direct the gavage needle along the roof of the mouth and slowly advance it into the esophagus.

Once the needle is approximately halfway into the esophagus, inject 25 microliters of spore inoculum and then gently withdraw the needle. Return the animal to its cage, and observe it for any signs of coughing or respiratory distress that may indicate inadvertent tracheal administration.