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

Ethical Statement:
The Institutional Animal Care and Use Committee (IACUC) at North Carolina State University College of Veterinary Medicine (NCSU) approved this study. The NCSU Animal Care and Use policy applies standards and guidelines set forth in the Animal Welfare Act and Health Research Extension Act of 1985. Laboratory animal facilities at NCSU adhere to guidelines set forth in the Guide for the Care and Use of Laboratory Animals. The animals' health statuses were assessed daily, and moribund animals were humanely euthanized by CO₂ asphyxiation followed by secondary measures. Trained animal technicians or a veterinarian performed animal husbandry in an AAALAC-accredited facility during this study.

1. Administration of the Antibiotic Cefoperazone in Drinking Water to Achieve Susceptibility to C. difficile Colonization and Disease

NOTES: 5- to 8-week-old C57BL/6 WT mice (females and males) were purchased and quarantined for 1 week prior to starting the antibiotic water administration. Following quarantine, the mice were housed with autoclaved food, bedding, and water. Cage changes were performed weekly by laboratory staff in a laminar flow hood.

1. Prepare cefoperazone (0.5 mg/ml) in sterile distilled water 7 days prior to the targeted day of challenge (Figure 1).
2. Fill autoclaved water bottles with cefoperazone water (0.5 mg/ml) and place them in each mouse cage.
   NOTE: Freshly-prepared cefoperazone water should be made and changed out every 2 days during a 5-day period, as denoted in steps 1.1 and 1.2. Proper disposal of cefoperazone water following institutional environmental health and safety guidelines is recommended.
3. After 5 days on cefoperazone water, replace the bottles with autoclaved water bottles filled with sterile distilled water. Give mice this drinking water for the duration of the experiment.

2. Preparation of the C. difficile Spore Inoculum and the Oral Gavage of the Mice

NOTE: Before beginning, ensure that the following items are placed in the anaerobic chamber for at least 24 hr: 1x phosphate-buffered saline (PBS; see Materials), taurocholate cycloserine cefoxitin fructose agar (TCCFA) plates (see Materials and the supplemental file), and a sterile L-shaped spreader.

NOTE: Mice challenged with C. difficile spores should be housed in a Biosafety Level 2 animal facility.

1. Obtain a C. difficile spore stock of the desired strain (in this case, R20291) that was prepared earlier and stored at 4 °C in sterile water^21,22. Determine the enumeration of this spore stock prior to use.
2. Based on the known concentration (spores/ml) of the spore stock, calculate the desired dilution to obtain 10⁵ spores per 25 µl. Ensure the inoculum volume is adequate to inoculate all mice in the experiment (25 µl per mouse), to perform the enumeration of the inoculum (approximately 30 µl), and to account for pipetting error.
3. Vortex the original C. difficile spore stock. Then, resuspend the calculated volume (from step 2.2) of the original C. difficile spore stock in sterile water within a screw-capped microcentrifuge tube. Perform this aerobically in a laminar flow hood. Identify this diluted mixture as the "spore inoculum."
4. Place the spore inoculum into a 65 °C water bath and heat it for 20 min.
   NOTE: This step will ensure that only active C. difficile spores remain after the heat treatment.
5. Within the laminar flow hood, take 50 µl of heated spore inoculum, place it in a sterile microcentrifuge tube, and pass it into the anaerobic chamber²³. Use this sample for spore enumeration.
6. Within the laminar flow hood, load the remaining heated spore inoculum into a 1-ml syringe attached to a sterile bulb-tipped gastric gavage needle. Utilize a manual syringe stepper to ensure accurate and rapid repeat dosing between mice. Ensure that the gavage needle is filled with the heated spore inoculum prior to use.
   NOTE: A new sterile gavage needle for each mouse is not required. However, if various doses of C. difficile spores are administered, a new gavage needle is recommended for each dose.
   NOTE: C. difficile spores are highly resistant to traditional disinfectants. Therefore, the use of a sporicidal disinfectant, such as #62 Perisept Sporicidal Disinfectant, is necessary. The manufacturer recommends a 2-min contact time to destroy C. difficile spores.
7. Gavage each mouse with 25 µl of the spore inoculum. Restrain each mouse gently by grasping the animal by the loose skin of the neck and back in order to immobilize the head. Using the index finger further immobilize the animal's head and to elongate the esophagus. Ensure that the animal does not vocalize or show other signs of distress during restraint.
   NOTE: The total volume given to mice by gavage should not exceed 10 ml/kg bodyweight (0.1 ml/10 g).
8. 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 the gavage needle into the esophagus.
   NOTE: If any resistance is encountered, the gavage needle may be entering into the trachea and thus should NOT be advanced, but rather removed and repositioned immediately.
9. After the gavage needle is approximately halfway into the esophagus, inject 25 µl of the heated spore inoculum and gently withdraw the gavage needle from the esophagus.
   NOTE: Forceful advancement of the gavage needle may lead to the rupture of the esophagus or stomach. Therefore, if resistance is encountered when advancing the gavage needle, it is best to immediately remove and reposition the gavage needle.
10. Return the animal to its cage and observe it for any coughing or signs of respiratory distress, as this may indicate inadvertent tracheal administration or aspiration of the heated spore inoculum.
    NOTE: Mice are extremely susceptible to C. difficile following antibiotic treatment; therefore, precautions to prevent cross-contamination between cages is essential. This is especially important when managing antibiotic-treated but unchallenged mice as controls.
11. After inoculating all mice, place the remaining heated spore inoculum into a sterile microcentrifuge tube and pass it into the anaerobic chamber for spore enumeration.
12. For enumeration of the heated spore inoculum (from step 2.4) and the remaining gavaged heated spore inoculum, make 1:10 serial dilutions of each inoculum in 1x PBS. It is recommended to make and plate 4 – 5 dilutions (i.e., 10^-1 through 10^-5).
13. Using aseptic technique, transfer 100 µl of each serial dilution onto an individual TCCFA plate.
14. Using a sterile L-shaped spreader, evenly spread the dilution applied to the medium across the plate. Even spreading of the diluted inoculum is essential for the accurate enumeration of spores.
15. Incubate the plates anaerobically for 24 hr at 37 °C. After 24 hr, C. difficile colony-forming units (CFUs) can be enumerated to obtain the infective dose administered to mice in the 25 µl (0.025 ml) volume (see the supplemental file "Example Calculations"). C. difficile colonies are flat and pale yellow and have irregular edges and a ground glass appearance²⁴.
    NOTE: The limit of detection for bacterial enumeration is 10² CFU.

3. Monitoring Mouse Weight Loss and Clinical Signs of Disease throughout C. difficile Infection

1. Weigh animals before and after the 5-day cefoperazone antibiotic treatment, after the 2-day recovery off of antibiotics (day 0), and then for the duration of the experiment.
   NOTE: Obtain weights at a consistent time each day. Weights obtained the day of challenge (day 0) should be used as the initial baseline weight for comparison throughout C. difficile infection.
2. Place a digital scale into a biosafety cabinet. Place a sterile plastic beaker onto the scale and tare the weight of the plastic beaker. Then, gently pick up the mouse by the base of the tail and place it into the plastic beaker.
   1. Place the beaker back on the scale. Hover a hand over the top of the beaker to ensure that the mouse does not escape. It may take 2 – 3 sec to obtain a stable weight. Then, gently place the mouse back into its cage. Record this weight and repeat the process for each mouse in that cage.
      NOTE: It is imperative that precautions are taken to prevent cross-contamination between cages when obtaining weights. Each cage of mice should have its own plastic beaker for weighing. The plastic beakers should be disinfected with a sporicidal agent between each use and covered with sterile aluminum foil.
3. Monitor the challenged animals twice daily (at a minimum) for signs of clinically-severe C. difficile infection, including lethargy, loss of appetite, diarrhea, and hunched posture.
4. Humanely euthanize animals that lose 20% of their initial baseline bodyweight and/or develop severe clinical signs of C. difficile infection (listed in step 3.3).
   NOTE: Mice that display clinical signs of C. difficile infection should be weighed as needed during the experiment to ensure that they have not lost 20% of their initial baseline bodyweight. During early time points in the experiment, this might mean twice-daily measurements.

4. Bacterial Enumeration of C. difficile from Mouse Feces and Cecal Content

NOTE: Before beginning, ensure that the following items are placed into the anaerobic chamber for at least 24 hr: 1x PBS (see Materials), TCCFA plates (see Materials), a sterile L-shaped spreader, and sterile microcentrifuge tubes and/or PCR plates for dilutions.

1. Obtaining Samples for C. difficile Enumeration
   NOTE: Prior to obtaining feces or cecal content, weigh sterile microcentrifuge tubes on an analytical scale. Record the tube weight on the side of the tube, rounding to four decimal places (e.g., 0.9864 g). Denote this weight as the "tube weight." Each sample collected should be placed in a sterile, weighed microcentrifuge tube.
   1. Sterile collection of mouse feces
      1. Gently restrain each mouse by grasping the animal by the loose skin of the neck and back in order to immobilize the head. Use the pinky finger to gently restrain the animal's tail, thus exposing the anus. Ensure that the animal does not vocalize or show other signs of distress during restraint.
      2. Hold a sterile, weighed microcentrifuge tube directly under the animal's anus. It is imperative that the tube does not come into direct contact with the mouse.
         1. As the animal defecates, collect the fecal pellet into the tube. Keep the tube at room temperature until all fecal samples are collected. It can take several minutes for a mouse to defecate, thus necessitating repeat attempts to collect feces.
      3. Weigh the tubes containing feces on the analytical scale. Record the tube weight, rounding to four decimal places (e.g., 1.0021 g). Denote the obtained weight as the "final tube weight."
      4. Pass the fecal samples into the anaerobic chamber for bacterial enumeration directly after stool collection.
   2. Sterile collection of mouse cecal content at the time of necropsy
      1. After humane euthanasia by CO₂ asphyxiation followed by secondary measures, perform a necropsy to obtain the cecum²⁵. The cecum is the large, comma-shaped section of the gastrointestinal tract.
      2. Place the cecum onto a sterile plastic petri dish. Use a disposable, sterile no. 10 scalpel blade to cut the cecum open from the blind end of the pouch to expose the cecal contents.
         1. Gently extract the cecal contents by applying light pressure with the scalpel blade and sweeping the contents toward the incised portion of the cecum.
            NOTE: Care should be taken to not disrupt the cecal tissue, which will be submitted for histopathological examination.
      3. Place the cecal contents into a sterile, weighed microcentrifuge tube immediately following collection. Only about 10 mg of cecal content is required for bacterial enumeration.
      4. Weigh the tubes containing cecal content on the analytical scale. Record the tube weight, rounding to four decimal places (e.g., 1.0021 g). Denote the obtained weight as the "final tube weight."
      5. Pass the cecal content samples into the anaerobic chamber for bacterial enumeration.
2. Bacterial Enumeration of C. difficile
   1. Calculate the final weight of the contents (feces or cecal) that will be enumerated for C. difficile. Performed this by subtracting the "final tube weight" from the "tube weight."
   2. Calculate a 1:10 dilution of the contents into 1X PBS and resuspend accordingly. For fecal pellets, use the tip of the pipette to gently disrupt the pellet into solution (see the supplemental file "Example Calculations").
   3. Anaerobically incubate these resuspended samples at room temperature for 30 min, allowing the contents to settle.
   4. Make serial dilutions for each sample in 1x PBS for enumeration of the CFU per g of content (feces or cecal). Perform this anaerobically.
   5. Using aseptic technique, transfer 100 µl of each serial dilution to TCCFA plates. Using a sterile L-shaped spreader, spread the dilution applied to the media to evenly spread it across the plate. Even spreading of the dilution is essential for accurate enumeration of colonies.
   6. Incubate the plates anaerobically for 24 hr at 37 °C. At this time, enumerate C. difficile colonies to obtain the CFU per g of content (feces or cecal). C. difficile colonies are flat and pale yellow and have irregular edges and a ground glass appearance²⁴.
      NOTE: This protocol can be used to enumerate C. difficile from other murine GI content, including ileal and colonic content. PCR plates can be used for making serial dilutions.

5. Vero Cell Cytotoxicity Assay to Quantify C. difficile Toxin Cytotoxicity

NOTE: It is recommended that this assay be performed after completion of the mouse model on samples collected at necropsy and stored at -80 °C. Aseptic cell culture techniques are essential for preventing contamination of Vero cells during this assay. This protocol takes 2 days to perform. All feces and intestinal content utilized in this assay must be stored in a weighed, sterile microcentrifuge tube (denoted with the "tube weight," see section above). The final tube weight (including the contents) is measured via an analytical scale to the nearest four decimal places (see section above). Use of a multi-channel pipette is recommended for this assay.

NOTE: Before beginning, ensure that the following items are available: Vero Cells, Dulbecco's modification of Eagle medium (DMEM) 1x with 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin/streptomycin media (denoted as "DMEM 1x media;" see Materials and the supplemental file), 0.25% Trypsin-EDTA, 1x PBS, 0.4% Trypan Blue, a 96-well cell culture flat-bottom plate, a 96-well filter plate, Clostridium difficile Toxin A (aliquot in 3 µl at 1 µg/µl in Ultra-Pure water and store at -80 °C), Clostridium difficile Antitoxin, a worksheet (for calculations and plate maps; see supplemental files).

NOTE: Caution should be taken during this assay for personnel exposure to C. difficile and its toxin.

1. Splitting the Vero Cells (Day 1)
   1. Preheat the DMEM 1x media, 0.25% Trypsin-EDTA, and 1x PBS in a 37 °C water bath.
   2. Obtain a flask of Vero cells from the cell culture incubator (37 °C and 5% CO₂) and place it in the sterile cell culture laminar flow hood. Use 70% ethanol to wipe down the hood and equipment prior to use. Using a serologic pipette, aspirate the media from the tissue culture flask to remove it from the Vero cells and discard it into a waste container.
   3. Rinse the Vero cells with 5 ml of 1x PBS (preheated) in the tissue culture flask. Aspirate the PBS and discard it into a waste container.
   4. Add 5 ml of 0.25% Trypsin-EDTA to the tissue culture flask. Allow a contact time of 2 – 3 min. During this time, observe the cells begin to come off the flask surface. Gently rock the tissue culture flask side to side to loosen the Vero cells.
   5. After contact time with Trypsin-EDTA, immediately add 5 ml of DMEM 1x media into the tissue culture flask. This will neutralize the Trypsin-EDTA. Use this solution to gently wash any unattached Vero cells off the back of the flask. Then, use a serological pipette to transfer this mixture into a 15-ml conical tube.
   6. Centrifuge the Vero cells in the conical tube for 5 min at 180 x g and 25 °C. Ensure that the centrifuge is properly balanced. After centrifuging, observe a visible pellet of Vero cells at the bottom of the conical tube. Be careful not to disrupt this pellet. Aspirate off the supernatant from the conical tube and discard it.
   7. Resuspend the Vero cells in 3 ml of DMEM 1x media.
2. Counting the Vero cells
   1. Add 100 µl of the Vero cell suspension (made in Step 5.1.7) to 100 µl of 0.4% Trypan Blue. Gently mix by pipetting the solution up and down.
   2. Add 10 µl of this mixture to each chamber of a hemocytometer.
   3. Count the number of viable cells within the four quadrants of the hemocytometer. Dead Vero cells will take up the Trypan Blue and thus will be stained blue (these cells should not be counted). Record these numbers in the "Vero Cell Worksheet," provided.
   4. Sum the total number of viable Vero cells in all four quadrants and calculate the average. Multiply by the dilution factor, which is 2 since 100 µl of cells are in a total of 200 µl of liquid.
   5. Multiply by the correction factor to give the "number of cells/ml." When using a hemocytometer, the correction factor is always 10⁴. Multiply by the total volume to give the "total number of cells."
3. Determining the Number of Wells Required for Each Experiment
   NOTE: A single sample (either feces or intestinal content) requires 24 separate wells to be performed in duplicate. A concentration of 10⁵ Vero cells per well (total volume of 90 µl) is required.
   NOTE: If one desires to incubate the Vero cell 96-well plates overnight at 37 °C, a concentration of 10³ Vero cells per well is recommended to account for the extended incubation time. Calculations within the Vero Cell Worksheet would need to be adjusted to account for this change.
   1. Determine the number of wells that can be utilized based on the amount of Vero cells available (from Step 5.2; use the Vero Cell Worksheet, provided).
   2. Determine the volume of Vero cell suspension needed to fill the number of desired wells (based on the number of samples being tested). Ensure that any additional volume is added in to account for pipetting error (use the provided Vero Cell Worksheet).
   3. Dilute the Vero cell suspension (obtained in Step 5.1.7) in DMEM 1x media to obtain the volume required for the experiment.
4. Seeding Vero Cells into a 96-well Cell Culture Plate
   1. Using the Vero cell resuspension from Step 5.3.2, use a multi-channel pipette to add 90 µl of the Vero cell suspension to each well of a 96-well cell culture flat-bottom plate.
   2. Incubate the plate with the Vero cells in a cell culture incubator at 37 °C and 5% CO₂ for a minimum of 4 hr before adding toxin (samples) to the wells. This incubation time will allow the Vero cells to adhere to the bottom of each well.
5. Creating Stock Solutions from the Samples (Feces or Intestinal Content)
   NOTE: All samples should have the "tube weight" and "final tube weight" measured via an analytical scale. Use Vero Cell Worksheet for calculations.
   1. Calculate the weight of the contents. Convert g to mg, then mg to µl. Calculate the final total number of µl of solution needed to make a 1:10 dilution in 1x PBS. Calculate the total amount of 1x PBS to add to the sample.
   2. Add the total volume of 1x PBS (calculated above) to the sample. Perform this aerobically, and using aseptic technique. For fecal pellets, use the tip of the pipette to gently disrupt the pellet into the solution.
   3. Vortex the samples and then centrifuge them at 3,522 x g for 5 min at room temperature. Ensure that the centrifuge is properly balanced. Be careful not to disrupt the pellet and resuspend the sample into solution.
      NOTE: During Step 5.5.3, if only a few samples are being processed, contents can be sterilized by passing them through a single 0.22-µm filter instead of using a 96-well filter plate. Some samples may require multiple filters to sterilize the entire volume content using this technique.
   4. Sterilize the sample/PBS mixture by taking 150 µl of supernatant and placing it into separate wells on a 96-well filter plate. Place the filter plate securely on top of a 96-well cell culture flat-bottom plate so that the contents will filter into this plate.
   5. Centrifuge the filter plate/cell culture plate stack at 431 x g for 5 min at room temperature. Ensure that the centrifuge is properly balanced and that the filter plate/cell culture plate stack are tightly aligned and will not shift during centrifugation.
      NOTE: These filtered contents are the 10^-1 stock that will be utilized in the dilution plates.
   6. Place plate with filtered samples onto ice.
6. Creating Dilution Plate #1
   NOTE: Dilution Plate #1 (DP#1) will require 6 wells per sample, including positive (C. difficile Toxin A) and negative (1x PBS) controls (see the DP#1 layout on the Vero Cell Worksheet).
   1. To prepare the positive control (C. difficile Toxin A) for this assay, obtain 3-µl aliquots (1 µg/µl) from -80 °C and add 297 µl of ultrapure water, yielding a final concentration of 0.01 µg/µl. Place on ice.
   2. Label the wells with their dilutions (10^-1 to 10^-6) for each sample and indicate the positive and negative controls (see the DP#1 layout).
   3. Add appropriate amounts of 1x PBS to each well. In the 10^-1 wells, add NO PBS. For the 10^-2 to 10^-6 wells, add 90 µl of 1x PBS.
   4. Add appropriate amounts of the samples to each well. In the 10^-1 wells, add 100 µl of the 10^-1 stock for each sample (see Step 5.5.7 from the filter plate). For the 10^-2 to 10^-6 wells, make serial dilutions; start by taking 10 µl from the 10^-1 well and adding it to the 10^-2 well. Continue the serial dilutions out to the 10^-6 dilution.
   5. Cover the DP#1 plate with a clear plastic adhesive seal and place it on ice.
7. Creating Dilution Plate #2
   NOTE: Dilution Plate (DP#2) will require 2 lanes of 6 wells for each sample, including positive and negative controls (see the DP#2 layout on the Vero Cell Worksheet).
   1. Label the wells with their dilutions (10^-1 to 10^-6) for each sample and indicate the positive and negative controls (see the DP#2 layout). Label the wells with the sample name (denoted as "sample wells") or the sample name with antitoxin (denoted as "antitoxin wells").
   2. Calculate the amount of antitoxin required for this assay. NOTE: The antitoxin is a 25x stock solution that needs to be diluted 1:25 in 1x PBS. Place the prepared antitoxin 1x solution on ice.
   3. For "sample wells," add 20 µl of 1x PBS to each well for all dilutions (10^-1 to 10^-6) of that sample. For "antitoxin wells," add 20 µl of 1x Antitoxin to each well for all dilutions (10^-1 to 10⁶) of that sample.
   4. Transfer 20 µl of the diluted sample in wells from DP#1 into the designated wells on DP#2 for rows 1 – 6 and rows 7 – 12 (see the DP#2 layout on the Vero Cell Worksheet). Mix the solution gently by pipetting up and down.
   5. Cover DP#2 with a clear plastic adhesive seal and allow 40 min of incubation at room temperature. This incubation is to allow the antitoxin to bind and thus inactivate and neutralize the C. difficile toxin.
   6. After the incubation, add 10 µl of mixture from DP#2 to the appropriate Vero cell wells (see the Vero Cell Plate layout on the Vero Cell Worksheet). Gently mix the solution by pipetting up and down, taking care to not disrupt the Vero cells that are adhered to the bottom surface of the wells.
   7. Incubate the Vero cell plate overnight in a cell culture incubator at 37 °C and 5% CO₂.
8. Assess Vero Cells for Rounding using a Light Microscope (day 2)
   NOTE: Recall that the dilution factor changes by a factor of 10 when adding the sample mixtures to the Vero cell plate (e.g., 10^-3 is now 10^-4). Wells that have antitoxin present should have little to no Vero cell rounding noted. Vero cell rounding (cytotoxicity) will be noted in samples that contain C. difficile toxin. If the cytotoxic activity is neutralized in a dilution containing the sample and the antitoxin, the presence of C. difficile toxin resulting in Vero cell cytotoxicity is confirmed.
   1. Examine for Vero cell rounding using a light microscope at 200X magnification. For sample wells (including the positive control), record the dilution of the well that is the last to have 80% Vero cell rounding noted.
   2. Calculate the cytotoxic titer, which is defined as the reciprocal of the highest dilution that produced 80% Vero cell rounding per g of sample. For example, if the highest dilution is 10^-5, then the dilution factor would be 10^-6 for this sample. Thus, the log[final dilution factor]/g of sample would be log[10⁶], which yields a reciprocal titer of 6.
      NOTE: Vero cells need to have undergone at least 3 – 4 passages and no more than 30 passages prior to their use in this assay²⁶.