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

Experiments described here were performed using 6-8 weeks old wild-type (C57BL6/J) mice maintained in a specific pathogen free (SPF) facility. All animal experiments were approved by the King’s College London and the Francis Crick Institute Animal Welfare and Ethical Review Body and the United Kingdom Home Office. Prior to beginning any animal procedure, ensure that the appropriate permissions are obtained through the local institution/organization.

1. Administration of Antibiotics

NOTE: Two alternative methods for antibiotic treatment are provided: oral gavage (step 1.1) and administration of antibiotics via drinking water (step 1.2).

1. Oral gavage
   1. Prepare stocks of individual antibiotics by dissolving them in autoclaved water at the following concentrations: Ampicillin at 100 mg/mL, Gentamicin at 100 mg/mL, Neomycin at 100 mg/mL, Metronidazole at 10 mg/mL and Vancomycin at 100 mg/mL. Filter sterilize using a 0.45 µm filter. Aliquot and store at -20 °C.
   2. Prepare a cocktail of antibiotics by mixing the stocks prepared above. For a volume of 1 mL mix 50 µL of Ampicillin (5 mg/mL final), 50 µL of Gentamicin (5 mg/mL final), 50 µL of Neomycin (5 mg/mL final), 500 µL of Metronidazole (5 mg/mL final), 25 µL of Vancomycin (2.5 mg/mL final) and 325 µL of water. Prepare the cocktail fresh before use.
   3. Load the antibiotic mix into a sterile 1 mL syringe and fix an appropriate gauge gavage needle (20 G for 15–20 g mice) while eliminating any bubbles.
   4. Gavage mice with 200 µL of antibiotic mix.
      1. Grab the skin over the mouse shoulder firmly, stretch the head and neck to straighten the esophagus. Direct the ball-tip of the feeding needle along the roof of the mouth and toward the back of the pharynx. Then, gently pass it down into the esophagus and inject the 200 μL solution.
   5. Administer the antibiotic cocktail once daily for the duration of the experiment.
2. Antibiotics in the Drinking Water
   CAUTION: Carefully monitor mouse weight daily for the first two weeks of antibiotic administration in the drinking water.
   1. Prepare a cocktail of antibiotics by dissolving the following in 1 L of autoclaved water: 1 g of Ampicillin (1 g/L final), 1 g of Neomycin (1 g/L final), 1 g of Metronidazole (1 g/L final), 0.5 g of Vancomycin (0.5 g/L final) and 8 sachets (0.75 g each) of artificial sweetener (60 g/L final). Shake until dissolved. Store at 4 °C.
      NOTE: Sweetener is added to hide the flavor of antibiotics and prevent mice dehydration. While several sweetener brands may work, the final concentration required may be different for each specific brand.
   2. Fill the antibiotic cocktail into a water bottle (~100 mL/bottle) and place the bottle on a mouse cage.
      NOTE: Use brown bottles or cover the bottles with foil to protect antibiotics from light.
   3. Replace the antibiotic cocktail with fresh stock twice a week for the duration of the experiment.

2. Collection of Fecal Samples from Stool, Ileum Content, and Ileum Wall

1. Weigh and label 2 mL autoclaved tubes for sample collection.
2. For collection of fresh stool samples, place each mouse in a restrainer and collect fecal pellets directly from the anus in a collection tube.
   NOTE: Samples can also be obtained by placing mice in a clean autoclaved cage, and collecting stool samples with clean sterilized forceps.
3. Euthanize the mice with CO₂ asphyxiation followed by cervical dislocation.
4. Lay a mouse carcass with the abdomen fully exposed and spray the abdominal area with 70% ethanol.
5. Using sterilized forceps and scissors, make a transverse incision in the abdomen to expose the peritoneum without damaging any internal tissues. Lift the peritoneum and make an incision to expose the intestines.
6. Remove the intestines (from colon to stomach) with the forceps and scissors and place it in a sterile Petri dish.
7. Carefully use forceps to tease the small intestine (SI) away from the mesenteric arteries and fat. Extend the intestine and place it on a clean lab wipe.
8. With a ruler, measure and cut 4 cm of the distal ileum of the SI (the closest part to the cecum). Cut and discard the 1 cm of intestine proximal to the cecum. There will be a 3 cm portion of ileum left which will be used to collect intestinal bacteria.
9. Hold the ileum portion (3 cm) over a 2 mL sterile tube. Collect the intestinal content by directly extruding the intestine and colleting the sample in the tube. This sample will have bacteria from the ileum content.
10. Prepare a 20 mL syringe with cold phosphate buffered saline (PBS) and flush the ileum portion (discard the flow-through).
11. Place the ileum portion on a clean paper towel and open it longitudinally with scissors.
12. Scrape the inside of the ileum wall with a scalpel.
13. Collect any bacteria on the scalpel by washing the scalpel with 1 mL of PBS over a clean centrifuge tube. Spin at 8,000 × g for 5 min to pellet the bacteria and discard the supernatant. This sample will contain bacteria from the ileum wall.
    NOTE: Fecal samples from stool, ileum content and wall can be frozen and stored at -80 °C until use.
14. Weigh the tubes containing the samples from stool and ileum content, and subtract the weight from the empty tubes (from step 2.1) to obtain the fecal weight in each sample.
15. Extract bacterial DNA from stool, ileum content and ileum wall samples using commercially available kits. Store DNA samples at -20 °C until use.

3. Quantification of Intestinal Microbiota by qPCR

NOTE: This procedure includes the generation of a standard (step 3.1) and the method for qPCR set-up for standard and fecal samples (step 3.2)

1. Generation of a standard for qPCR
   1. Use polymerase chain reaction (PCR) to amplify the 16S rRNA gene from the genomic DNA extracted from a bacterial culture using the reagents¹³ and PCR conditions detailed in Table 1.
   2. Run the PCR product on a 1.5% agarose gel and purify the DNA band using a commercially available DNA extraction kit.
   3. Ligate the purified DNA fragment using a cloning kit (see the Table of Materials, use a vector containing antibiotic resistance markers and β-galactosidase (LacZ) gene fusion for blue/white colony selection) and transform DH10 competent E. coli following the manufacturer’s instructions. Plate transformation onto Ampicillin (100 µg/mL), X-gal (20 µg/mL) Luria Bertani (LB) agar plates (10 g/L Tryptone, 5 g/L Yeast Extract, 10 g/L NaCl, 15 g/L Agar). Incubate overnight (O/N) at 37 °C.
   4. Select a single positive (white) colony from the plate. Inoculate it in 5 mL LB broth containing 100 µg/mL Ampicillin and incubate O/N at 37 °C, shaking at 250 rpm.
   5. From the O/N culture, isolate and purify the plasmid using a commercial miniprep kit according to manufacturer's instructions.
      NOTE: It is important to sequence the plasmid insert at this stage, to ensure that the plasmid contains only one copy of the 16S rRNA gene and to determine its length in base-pairs (bp).
   6. Linearize the plasmid with a restriction enzyme which cuts the plasmid only once.
   7. Purify the linearized plasmid using a commercially available kit.
   8. Determine the concentration of the plasmid by measuring absorbance at 260 nm using a spectrophotometer.
   9. Calculate the number of plasmid copies/µL of sample using the following formula¹⁴:
      number of copies = (amount * 6.022 × 10²³) / (length * 1 × 10⁹ * 650)
      where amount is the DNA concentration obtained in step 3.1.8 (in ng/µL); and length is the total length of the plasmid (with the insert) in bp. The number of copies will be obtained as copies/µL.
      NOTE: There are online tools based on the above formula that enable easy calculation of the number of plasmid copies.
   10. Aliquot and store the standard as needed at -20 °C until use.
2. qPCR Set-up for Standard and Fecal Samples
   1. Thaw the qPCR standard (from step 3.1.10), fecal samples DNA (from step 2.15) and qPCR reagents (from a commercially available kit) on ice.
      NOTE: qPCR reagents used in this example include the SYBR Green qPCR reaction mix and forward and reverse primers (Table 2).
   2. Dilute the standard in sterile DNA-free water in a range from 10⁷ to 10² copies/µL (e.g., 1/5 serial dilutions from 10⁷ to 6.4 x 10² copies/µL). Dilute fecal sample DNA to 1/2, 1/5, 1/10.
   3. Make a master mix reaction for the total number of reactions plus 1 (Table 2).
   4. Mix 30 µL of the master mix and 5 µL of template (standard, sample or water for negative control)
   5. Add 10 µL of this mix to each well in a 384-well optical qPCR plate. Perform each reaction in triplicate.
   6. Seal the qPCR place, centrifuge briefly and load the plate on the qPCR machine programmed with the following cycling conditions: 95 °C for 20 min followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min.
   7. Obtain C_T values for standard and samples.
   8. Generate a standard curve by plotting the C_T values for the standards versus the logarithm of the copy number of the plasmid (as calculated in step 3.1.9). Perform lineal regression of the standard curve.
   9. Calculate the 16S rDNA copy numbers for fecal samples by interpolating C_T values (obtained in step 3.2.2) in the standard curve.
      NOTE: 16S rDNA copy numbers for each sample should be corrected considering the dilution factor of the sample (as prepared in step 3.2.2), the final volume nucleic acids that were eluted (in step 2.15) and the quantity of fecal sample (calculated in step 2.14) to obtain gene copies per gram of feces.