Cefoperazone-treated Mouse Model of Clinically-relevant Clostridium difficile Strain R20291

The overall goal of this protocol is to provide a step by step guide for implementing the cefoperazone mouse model of Clostridium difficile infection, with special emphasis on clinical disease monitoring, bacteria enumeration, toxin cytotoxicity, and histopathologic changes post challenge. This detailed protocol will help researchers accurately execute a mouse model of Clostridium difficile infection, which is essential for studying the pathogenesis of this bacterium and accessing novel therapeutics. The main advantage of using this mouse model is that exposure to one antibiotic for five days renders mice susceptible to colonization of C.difficile and for a prolonged clinical course of infection concurrent with human disease.

To begin, take the C.difficile stock strain of interest and vortex. In a laminar flow hood, dilute the stock in sterile water in a screw cap tube to a concentration of ten to the fifth spores per 25 microliters. Label the mixture as spore inoculum and place the tube into a 65 degree Celsius water bath for 20 minutes.

Next, in the laminar flow hood, pipette 50 microliters of the heated spore inoculum into a sterile microcentrifuge tube and pass it into the anaerobic chamber until further use. Load the remaining spore inoculum into a one milliliter syringe attached to a sterile bulb tipped gastric gavage needle. 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. Next, take the spore inoculum aliquot stored previously in the anaerobic chamber, and any remaining gavage spore inoculum, and make one to ten serial dilutions in 180 microliters of 1x PBS.

Perform four to five of these dilutions. Using aseptic technique, transfer 100 microliters of each serial dilution into an individual TCCFA plate. Taking a new sterile L-shaped spreader each time, evenly distribute the dilution inocula on each plate.

Incubate the plates under anaerobic conditions for 24 hours at 37 degrees Celsius. Finally, count the C.difficile colonies on each dilution plate and calculate the colony forming units to obtain the infective dose administered to the mice. To collect fecal samples from mice for assessment, first lightly but firmly restrain the subject mouse by the loose skin on the neck and back, ensuring the animal is not in distress.

Using the pinky finger, gently lift the animal's tail, exposing the anus. Hold a sterile microcentrifuge tube of known weight directly under the animal's anus, ensuring the tube does not directly touch the mouse. When the animal defecates, collect the fecal pellet in the tube.

Store the tube at room temperature until all necessary samples are collected. Weigh the tubes containing feces on an analytical scale and record the mass to four decimal places. Calculate the weight of the fecal pellet by subtracting the weight of the tube.

Next, calculate a one to ten dilution of the contents and resuspend into 1x PBS. Use the tip of the pipette to gently disrupt the fecal matter into solution. Incubate the samples at room temperature for 30 minutes, allowing the contents to settle.

Maintaining the solutions anaerobically, make serial dilutions for each sample in 1x PBS. Transfer 100 microliters of each serial dilution onto TCCFA plates. Use a sterile L-shaped spreader and evenly apply the media to the plates.

Incubate the plates in anaerobic conditions at 37 degrees celsius for 24 hours. Finally, enumerate the C.difficile colonies and calculate the colony forming unit per gram of fecal content. This figure shows mouse weight in the days following challenge with C.difficile R20291.

At day zero, the baseline weight is set to 100. Average mouse bodyweight was measured each day following, for 14 days. A significant decrease in weight was observed in challenged mice on days two to seven post challenge.

Mice appeared to recover and regain weight after this point. Here, C.difficile load in the feces in the days post challenge are shown. At 24 hours post challenge, all mice were colonized with 10 to the seventh CFUs per gram of feces.

This load had decreased by day seven onwards, though C.difficile is still present. Histopathologic examination of mice over the time course of the infection showed the most significant pathologic changes in the cecum. Total cecal histologic scores were significantly different between day zero and days two and four post challenge.

Generally, individuals new to this mouse model may struggle with the calculation and preparation of the spore inoculum used to challenge the mice and the calculation of fecal C.difficile bacterial load. While attempting this protocol it's important to remember that modifications of the dose or strain of C.difficile spores administered to mice may alter the outcome of this model.