Quantitative Polymerase Chain Reaction-based Analyses of Murine Intestinal Microbiota After Oral Antibiotic Treatment

This protocol provides a reliable method to quantify changes is intestinal microbiota of mice following oral antibiotic administration. This can help research on cross microbiota interactions providing insight in the pathologies associated with microbial dysbiosis such as inflammatory bowel disease. The main advantage of these antibiotic treatment regimes is that they prevent the characteristic weight loss normally associated with oral antibiotic administration to mice.

To preform antibiotic treatment through oral gavage prepare a cocktail of antibiotics by mixing stock solutions that have been prepped as described in the tech protocol. For a volume of milliliter mix 50 microliters of ampicillin, 50 microliters of gentamicin, 50 microliters of neomycin, 500 microliters of metronidazole, 25 microliters of vancomycin and 325 microliters of water. Load antibiotic mix onto a one milliliter syringe while eliminating any bubbles and attach an appropriate gage gavage needle.

Grab the skin over the mouse shoulder firmly and stretch the head and neck to straighten the esophagus. Direct the ball tip of the feeding needle along the roof of the mouth and towards the back of the pharynx. Then gently pass it down into the esophagus and inject the 200 microliters of solution.

Administer the antibiotic cocktail once daily for the duration of the experiment. Alternatively to administer antibiotics through the drinking water, prepare a cocktail of antibiotics as described in the tech protocol. It's important to include sweetener into the antibiotic mixture as this is a crucial factor to prevent mice dehydration.

Fill the antibiotic cocktail into a water bottle at approximately 100 milliliters per bottle. Protect the bottle from light and place in a mouse cage. Replace the antibiotic cocktail with fresh stock twice a week for the duration of the experiment.

Careful monitoring of mice weight and general health status should be preformed daily throughout antibiotic administration. Weigh and label two milliliter autoclave tubes for sample collection. For collection of fresh stool samples place each mouse in a restrainer.

Then collect fecal pellets directly from the anus in a collection tube. After performing euthanasia as described in the tech protocol lay a mouse carcass with the abdomen fully exposed and spray the abdominal area with 70%ethanol. Using sterilized forceps and scissors make a transverse incision in the abdomen to expose the peratenium without damaging any internal tissues.

Lift the peratenium and make an incision to expose the intestines. Remove the intestines with the forceps and scissors and place it in a steril Petri dish. Carefully use forceps to tease the small intestine away from the mesenteric arteries and fat.

Extend the intestine and place it on a clean lab wipe. With a ruler measure and cut four centimeters of the distal ileum of the small intestine. Cut and discard the one centimeter of intestine proximal to the cecum.

There will be a three centimeter portion of ileum left which will be used to collect intestinal bacteria. Hold the ileum portion over a two milliliter sterile tube. Collect the intestinal content by directly extruding the intestine and collecting the sample in the tube.

This sample will have bacteria from the ileum content. Prepare a 20 milliliter syringe with cold phosphate buffered saline and flush the ileum portion discarding the flow through. Place the ileum portion on a sterile Petri dish and open it longitudinally with scissors.

Scrape the inside of the ileum wall with a scalpel. Collect any bacteria on the scalpel by washing it with one milliliter of PBS over a clean centrifuge tube. Spin at eight thousand times G for five minutes to pellet the bacteria.

Following centrifugation discard the supernatant. This sample will contain bacteria from the ileum wall. Weigh the tubes containing the samples from stool and ileum.

Subtract the weight from the empty tubes to obtain the fecal weight in each sample. Extract bacterial DNA from stool, ileum content, and ileum wall samples using commercially available kits. Store the DNA samples at minus 20 degrees Celsius until use.

Thaw the qPCR standard, fecal sample DNA and qPCR reagents from a commercially available kit on ice. Dilute the standard in sterile DNA free water in a range from 10 to the seven to 10 to the two copies per microliter. Dilute the fecal sample DNA to one half, one fifth and one tenth concentrations.

Then make a master mix reaction for the total number of the reactions plus one. Mix 30 microliters of the master mix and 5 microliters of the template, which is either the standard, sample or water for the negative control. Then add 10 microliters of this mix to each well in a 384 well optical qPCR plate.

Perform each reaction in triplicate. Seal the qPCR plate and centrifuge briefly. Then load the plate on the qPCR machine that is programed as listed in the tech protocol.

Finally obtain the cycle threshold values for the standard and samples before calculating the 16S rDNA copy numbers for fecal samples. Representative results show weight loss in mice after administration of antibiotics by oral gavage or through the drinking water. No noticeable weight loss is detected in mice that receive antibiotics by oral gavage.

When mice are treated with antibiotics in the drinking water they loose about 10%of their body weight within the first few days of treatment but recover normal weight gain thereafter. The quantification of bacteria in fecal samples requires the use of a standard curve obtained by plotting the logarithm of copy number for the standard versus the C-T values obtained in the qPCR. Within the linear range the regression analysis equation enables quantification of the 16S rDNA abundance within the fecal samples.

As shown here for stool, small intestine content and small intestine wall samples. Following five or 10 days of oral antibiotic administration there is a strong decrease in the density of bacteria in stool samples from antibiotic treated mice. However the density of bacteria in feces recovered to normal levels one week after antibiotic administration is stopped.

Both oral gavage and administration of antibiotics in the drinking water briefly decrease the fecal bacterial load in treated mice. Researchers who select the individual method that better fits the experimental requirements considering factors such as the length of treatment. The qPCR method for quantification of bacteria can be modified to quantify individual bacterial taxa using specific primers.

This will provide both quantitative and qualitive information about the microbiome size and composition.