Colonization with Murine pks+ Escherichia coli under Non-Inflammatory Conditions

This protocol describing PKS positive E.coli colonization and quantification in a mice allows for the study of host microbiota interactions in evolving the key pathobionts implicated in colorectal cancer development. In comparison to other methods that utilize genetically or chemically induced models of colon inflammation, this protocol describes colonization under non-inflammatory condition. To begin add ampicillin, colistin, and streptomycin to a container.

And then pour sterile drinking water to dilute the antibiotics. Provide the antibiotic containing drinking water to the eight to 12 week old female C57 BL6 mouse for three days. Then remove the antibiotic containing drinking water and restore access to sterile drinking water for a 24 hour washout period before the gavage.

Using a sterile inoculating loop, pick a single colony of Escherichia coli NC101 from the agar plate and inoculate five milliliters of sterile lysogeny broth. Incubate the culture overnight at 37 degrees Celsius in a benchtop incubation shaker at 150 revolutions per minute. Then centrifuge the culture tube at 1500 G four five minutes at room temperature.

Remove and discard the supernatant while avoiding disturbance of the bacterial pellet. The bacterial pellet appears as an off-white deposit at the base of the tube. Resuspend the bacterial pellet in five milliliters of 0.9%sterile saline.

Repeat the centrifugation step and wash the pellet again in 0.9%sterile saline. After the second wash, measure the optical density of the suspension at 600 nanometers using a cell density meter. Adjust the optical density at 600 nanometers to 0.625, corresponding to approximately one times 10 to the power of eight colony forming units per mouse using sterile saline.

Select an appropriate gavage needle based on the size of the animal. Gently agitate the bacterial inoculum to ensure equal distribution of the bacteria. Fill the syringe with 0.2 milliliters of the bacterial inoculum and remove any air bubbles.

Remove the mouse from the cage. Scruff the mouse by grasping the skin just behind the ears to the shoulder blades using the thumb pad and the side of the index finger. Secure the tail with the middle or ring finger of the same hand.

Hold the mouse in an upright position. Insert the gavage needle into the side of the mouth. Glide the tip of the gavage needle along the roof of the mouth into the esophagus and stomach while gently pushing the mouse head back to extend the neck.

Slowly inject 0.2 milliliters of the bacterial inoculum and withdraw the gavage needle following the same angle as insertion. Monitor the mouse five minutes after gavage for signs of respiratory distress and discomfort. Return the mouse to the cage.

And place the animal on a diet supplemented with inulin or a no fiber control diet. Collect a fecal pellet from each mouse in a 1.5 milliliter micro centrifuge tube by scruffing the mouse as previously demonstrated. Alternatively, place the mouse in a clean, empty cage and wait up to 30 minutes for defecation to occur.

Snap freeze the sample by placing the micro centrifuge tube in liquid nitrogen. If required, freeze the sample at minus 80 degrees Celsius until further analysis. Next, weigh an empty 1.5 milliliter micro centrifuge tube.

Add approximately 20 milligrams of stool to the 1.5 milliliter micro centrifuge tube and record the weight. Add 200 microliters of 0.9%sterile saline to the tube and incubate at room temperature to soften the stool. After 10 to 15 minutes, vortex the tube for five minutes at maximum speed to produce a homogeneous suspension.

Small particles of undigested food material may remain visible. Transfer 100 microliters of each suspension onto a MacConkey agar plate and evenly spread the homogenate using an L-shaped spreader. Incubate the plates overnight at 37 degrees Celsius.

On the following day, count the number of pink colonies on each plate. Normalize the colon accounts by the weight of each fecal pellet and the plated volume. Extract genomic DNA from the fecal samples using a DNA extraction kit.

Quantify the CLB genes and enterobacteriaceae abundance using quantitative PCR with gene specific and family specific primers. Endpoint, PCR and gel electrophoresis detected both Colibactin A and E.coli 16S genes in feces from E.coli NC101 colonized mice. In control mice, only the E.coli 16S gene was detected, supporting the absence of PKS plus E.coli colonization.

After one week, the E.coli NC101 colonized mice that received the inulin supplemented diet showed greater colony forming unit recovery from the fecal homogenates cultured on MacConkey agar than the mice that received the no fiber control diet. At the same time point, the inulin fed mice also showed increased Colibactin P gene levels. And a higher enterobacteriaceae abundance than the no fiber control mice.

This protocol describes several methods for the validation of PKS positive E.coli colonization and the quantification of PKS positive E.coli growth by bacterial culturing and PCR amplification. This protocol can be combined with one of several colorectal cancer models, allowing for the assessment of PKS positive E.coli associated mutagenesis in colon tissue. This protocol can be used to study microbiota targeting methods to limit PKS positive E.coli expansion, and mutagenic potential in healthy hosts.