Models of Murine Vaginal Colonization by Anaerobically Grown Bacteria

All animal experiments were approved and conducted in accordance with institutional guidelines and the Guide for the Care and Use of Laboratory Animals of the University of California San Diego (UCSD, Protocol Number: S20057, 2020-onward) and before that at Washington University, St. Louis (Protocol 20110149, up to 2020).

NOTE: The protocol below utilizes conventionally raised female C57Bl/ mice, age 6-11 weeks at the time of inoculation. Please note vendor information and specific age ranges used previously in the relevant cited work.

1. Preparation and administration of β-estradiol 17-valerate

NOTE: β-estradiol 17-valerate is a carcinogen and a reproductive toxin that can be absorbed through the skin^27,28. Wear proper PPE during the handling of powder and liquid solution. It is also an aquatic toxin²⁹; dispose of it properly in an appropriately sealed and labeled container.

1. Filter sterilize up to 50 mL of sesame oil through a 0.22 µm filter using a 50 mL conical tube vacuum filter system. Open only in a biosafety cabinet to maintain sterility.
2. Calculate the volume of β-estradiol valerate solution needed for the experiment: 200 µL per mouse plus 2-3 mL extra to account for sample loss during syringe filling (e.g., a 10-mouse experiment requires 4 mL in total). Calculate the amount of β-estradiol valerate needed to make a 5 mg/mL solution and weigh it directly in a 14 mL round-bottomed tube.
3. Cap the 14 mL tube tightly and vortex to break up clumps in the powder (this will allow the β-estradiol valerate to dissolve more quickly). Add sterile-filtered sesame oil to the 14 mL tube so that the final concentration of β-estradiol valerate is 5 mg/mL.
4. Place the tightly capped 14 mL tube on a rotator at 37 °C for 30-40 min, until the solid powder completely dissolves. Visually confirm the homogeneity of the solution before injection into the mice. Incubation at 37 °C decreases the viscosity of the sesame oil, making it easier to inject.
5. Draw up β-estradiol valerate solution into a 1 mL syringe (without a needle). To do this without introducing bubbles, first, draw up a little of the solution, then expel gently while keeping the tip of the syringe in the sesame oil. Draw up the solution again slowly, slightly past the 100 µL mark.
6. Place a 25 G x 5/8 in needle onto the syringe. Then, prime the needle by slowly expelling the sesame oil solution until it starts to come out of the needle tip. Expel the solution until the syringe is at the 100 µL mark. Prepare one syringe per mouse.
7. Administer the β-estradiol 17-valerate by intraperitoneal injection at 48 h or 72 h prior to inoculation. Inject each mouse with 100 µL of the 5 mg/mL β-estradiol valerate solution (0.5 mg β-estradiol valerate/mouse), as previously described^22,30. Store the remaining solution at 4 °C for 72 h until the next injection.
8. Administer the β-estradiol valerate solution again on the day of inoculation, immediately prior to vaginal inoculation of the mice. Remove the solution from 4 °C and place it on a rotator at 37 °C for 30 min before injection to allow the sesame oil to warm and become less viscous. Proceed with injections as described in step 1.7.

2. Preparation of inoculum

NOTE: This section contains the general steps for the preparation of inocula from anaerobically grown streptomycin-resistant Gardnerella vaginalis JCP8151B-SmR^24,25,31,32. All steps in this section should be done in an anaerobic chamber.

1. Cycle any reagents, including prepared NYC III broth, agar, and PBS, into an anaerobic chamber to equilibrate at least 24 h prior to use.
   NOTE: Here, a vinyl anaerobic chamber equilibrated with 5% hydrogen gas mix was used. The internal oxygen concentration typically rests at 0 ppm, although there can be low-level transient increases (10-25 ppm) when cycling materials into the chamber.
2. Two days prior to vaginal inoculation, streak out Gardnerella from a frozen glycerol stock onto an NYC III agar plate in the anaerobic chamber. Use a small container (such as an empty pipette tip box) filled with dry ice to keep the glycerol stock frozen during transport in and out of the chamber.
3. 1 day prior to vaginal inoculation, use 5-10 individual Gardnerella colonies from the plate to inoculate 10 mL of NYC III broth. Allow the liquid culture to grow overnight for 16 h at 37 °C. Include a second 1 mL aliquot of the culture medium that is left uninoculated and incubate it alongside the inoculated culture to confirm that the medium is uncontaminated.
4. Prepare the inoculum from liquid culture as described below. Perform the inoculum preparation in the anaerobic chamber as much as possible.
   1. Determine the culture optical density (OD) by adding 200 µL of culture to 800 µL of fresh media in a 1.5 mL cuvette (1:5 dilution). Use a spectrophotometer to measure the density (OD) of the diluted culture at a wavelength of 600 nm (use a separate cuvette with fresh media alone as the blank). Multiply the OD reading by 5 to get the actual OD₆₀₀ of the 16 h culture.
   2. Calculate the volume of inoculum needed for the experiment. For example, to infect 15 mice with an inoculum of 20 µL per mouse, 15 x 20 µL = 300 µL of inoculum is needed. Prepare approximately 25% more inoculum than needed, so for 15 mice, prepare 300 µL + (0.25 x 300 µL) = 375 µL of inoculum.
   3. Using the OD₆₀₀ determined in step 2.4.1., calculate the volume of the 16 h culture that needs to be spun down and resuspended to get an OD₆₀₀ = 5.0 inoculum in the volume calculated in step 2.4.2. For example, if the OD₆₀₀ of the culture is 1.5, and the inoculum volume needed is 375 µL, then the volume of culture to be spun down is (375 µL x 5)/1.5 = 1250 µL.
   4. Pipette the volume of culture calculated in step 2.4.3. into a sterile microcentrifuge tube. Pellet the bacteria by centrifuging at 16,000 x g for 1-3 min.
   5. Remove the supernatant and resuspend the pellet in anaerobic PBS at the volume calculated in step 2.4.2. The inoculum will now be at a calculated OD₆₀₀ of 5.0. If applicable, also prepare a tube of PBS to serve as a vehicle control for mice in the uninfected control group.
5. Prior to removing the inoculum tube from the anaerobic chamber, prepare a serial dilution series from 1:10 through 1:10 x 10⁶ in PBS. Spot plate 5 replicates of each dilution onto an agar plate using a multichannel pipet to determine the CFU of the inoculum. This is the pre-inoculation CFU.

3. Vaginal pre-lavage and inoculation with Gardnerella

1. Prepare vaginal lavage/wash tubes in the anaerobic chamber (this can be done at the same time as inoculum preparation in step 2.4.). Pipette 70 µL of sterile, anaerobic PBS into 1.5 mL microcentrifuge tubes labeled with mouse numbers. Prepare one wash tube per mouse. Tightly close the tubes and cycle them out of the anaerobic chamber.
2. Perform a vaginal lavage on each mouse with 50 µL anaerobic PBS from the individual tubes prepared in step 3.1. These vaginal lavages are the pre-inoculation washes and can be used for downstream measurements and assays.
   1. After administration of the second β-estradiol valerate injection (step 1.8), place each mouse on top of a cage or a clean hard surface that it can grip with its forepaws. Gently grip the middle part of the tail and lift so that the hindquarters are slightly elevated and the vaginal opening is exposed.
   2. Using a pipette and p-200 filter tip, draw up 50 µL of PBS from the wash tube corresponding to the appropriate mouse. Gently insert the pipette tip into the vaginal lumen ~2-3 mm, and pipette the 50 µL volume in and out gently, 3x-4x.
   3. Transfer the collected wash material (~50 µL) back into the same wash tube it came from, pipetting up and down in the wash tube that still contains the remaining 20 µL of PBS. If there is excessive mucus and the tip gets clogged, use a clean p-200 tip to collect the remaining material and place it into the same wash tube.
3. Vaginally administer 20 µL of the concentrated bacterial suspension or vehicle control into the vagina of each mouse. Inoculate each mouse immediately following the vaginal lavage for that mouse (i.e., perform lavage and inoculation in sequence for each mouse before moving on to the next). To simplify this process, use two p200 pipettes-one set to 50 µL for lavages and the other to 20 µL for inoculations.
   1. Restrain the mouse as described in step 3.2.1. Using a p-200 tip, pipette 20 µL of bacterial suspension into the vagina. For co-inoculation experiments using two different bacterial species/strains, use 10 µL of each bacterial suspension and avoid mixing the two strains prior to inoculation.
      NOTE: The total inoculation volume should not exceed 20 µL to avoid spillover out of the vagina.
   2. Continue to hold up the hind end of each mouse for 10-20 s to prevent the administered volume from immediately pooling at the vaginal introitus. Then, place the mouse in a new cage/receptacle or with cage mates that have already been inoculated.
   3. Repeat step 3.2. and step 3.3. for each mouse. Never place mice back in a cage with animals that have not yet been inoculated. This prevents inadvertent exposure of mice to streptomycin-resistant bacteria prior to inoculation.
4. After all the mice have been inoculated, cycle the inoculum tube back into the anaerobic chamber. Prepare and plate another serial dilution as described in step 2.5. This is the post-inoculation CFU. Compare the CFUs from the post-inoculation and pre-inoculation plates to determine if the viability of the inoculum decreased while outside of the anaerobic chamber during the inoculation of the animals.
5. Plate the vaginal lavages collected in step 3.2. on selective agar (in this case, 1 mg/mL streptomycin). Incubate the plates for 1-2 days in an anaerobic chamber at 37 °C and examine for growth.
   NOTE: Growth indicates that endogenous members of the microbiome are resistant to the selection marker and may, therefore, obscure CFU enumeration of the target organism.

4. Collection of vaginal lavages to determine viable Gardnerella colonization

1. Collect the vaginal washes at select time points to analyze vaginal colonization. Collect as described in step 3.2.
2. Immediately after collection, cycle the vaginal lavages into an anaerobic chamber. Use 10 µL of each lavage to perform serial dilution and plating onto selective agar as described in step 2.5. Use the CFU counts as a measure of vaginal colonization by Gardnerella (or another anaerobe of interest).

5. Sacrifice, dissection, and tissue homogenization

1. Prepare a 5 mL tube for each organ being collected from each mouse (for example, if the vagina, cervix, and uterus are all being collected, prepare three tubes per mouse). Fill each tube with sterile anaerobic 1x PBS (or other homogenization media) in the anaerobic chamber. Use 1 mL of PBS for tubes used to collect vaginas and cervices and 750 µL for tubes used to collect uterine horns.
2. Once PBS is added, weigh each individual tube (this is the pre-collection weight and will be used to determine the total weight of each harvested tissue). If a scale is not available in the anaerobic chamber, tightly cap the tubes and cycle them out of the chamber to be weighed, then cycle them back in.
   NOTE: Tube preparation and weight collection can be done 1 day in advance of sacrifice, but tubes should be kept in the anaerobic chamber with caps tightly sealed to prevent evaporation.
3. Prepare a set of vaginal lavage tubes as described in step 3.1. to collect the final lavage at sacrifice. Also, prepare a 50 mL beaker of deionized (DI) water and a second 50 mL beaker of 70%-90% ethanol to use for rinsing and sterilizing standard steel dissection instruments during the sacrifice.
4. Cycle out the PBS-filled organ collection tubes from the anaerobic chamber. Keep the tubes tightly closed until the tissues are ready to be added.
5. Sacrifice each mouse using IACUC-approved methods. Perform the final lavage as described in steps 3.2.2. and 3.2.3. either immediately prior to sacrifice or immediately thereafter.
6. Begin dissection and tissue collection. Spray down the abdomen and back of each mouse with 70% ethanol. Sterilize the scissors in a beaker of ethanol, allow them to dry, and then make a single vertical cut up the mouse abdominopelvic cavity.
7. Use sterile forceps to pull back the skin and gently push the intestines out of the body cavity and to the side of the open field, which will expose the reproductive tract.
   NOTE: Be careful not to puncture or pierce the intestines as they may harbor streptomycin-resistant bacteria that can confound the experimental results.
8. To collect the maximum vaginal tissue, break the pubic bone during the necropsy. Using sterile scissors, clip the center of the pubic bone (the pubic symphysis) while being careful not to cut into the vagina. Using two sets of sterile forceps, grab each side of the pelvis and pull the pelvis open laterally, exposing the lower portion of the vagina underneath.
9. Use forceps to gently grip the cervix (a harder structure compared to the surrounding tissue). Gently pull up on the cervix to expose the colon, hidden behind and closely connected to the vagina.
10. Use small scissors to release the vagina from the external connective tissues. Carefully separate the vagina from the colon and avoid puncturing the intestinal tract. When fully released, flank the vagina at the introitus with scissors and snip to release from the mouse body.
11. Maintain a gentle grip on the cervix while pulling up slightly to make the uterine horns more visible. With the other hand, cut directly below the ovaries on the right and left sides to free the uterine horns. Remove the now-disconnected reproductive tract from the body cavity and place it into a sterile Petri plate.
12. Using a razor, separate the uterine horns, cervix, and vagina. Place each tissue in its respective labeled collection tube. If cytokines are going to be collected, place the tubes on ice after the addition of tissue.
13. Weigh each tube again after the tissue has been added. This is the post-collection weight. Subtract the pre-collection weight from the post-collection weight to get the total weight of the tissue.
14. Use a handheld homogenizer to homogenize the organs in their collection tubes. For Gardnerella JCP8151B, perform this step in the biosafety cabinet (aerobically) or the anaerobic chamber.
    1. Prepare several sets of tubes for washing and aseptic treatment of the handheld homogenizer between samples. Ensure that each set has three 50 mL conical tubes: one filled with DI water, one with 70% ethanol, and the third with 1x PBS. Prepare a separate set of wash tubes for each mouse treatment group.
    2. To clean the homogenizer, lower the blades into the liquid in each wash tube and turn the instrument to max speed. Hold the homogenizer in each of the three tubes for about 3 s. The order of washing is DI water (to remove physical debris), then ethanol (to sanitize), and finally 1x PBS (to neutralize). Shake the probe onto a paper towel or disposable bench pad to remove excess fluid between each rinse.
    3. Following initial rinsing, homogenize the tissues by lowering the homogenizer probe to the bottom of the collection tube, trapping the tissue underneath. Turn on the homogenizer to grind the tissue, gently moving the probe up and down about 5x while remaining submerged. Homogenize each organ for about 15-30 s.
    4. After turning off and removing the homogenizer from the tube, visually inspect the contents to ensure complete tissue disruption. This is especially important for the cervix and vagina, which sometimes fail to be caught by the probe.
       NOTE: It is okay if some excess fat on the uterine horns does not fully homogenize.
    5. Repeat steps 5.14.1.-5.14.4., washing and sterilizing the probe between each sample. Switch to a new set of wash tubes for each experimental group.
15. Move the tubes with homogenized samples into the anaerobic chamber. To determine the bacterial burden of each tissue, perform brief gentle vortexing of the sample to mix, then remove 100 µL of tissue homogenate for serial dilution and plating on selective agar for CFU determination (the first dilution is 1 x 10⁰). If a lower limit of detection is required, perform spread-plating of 200-250 µL of undiluted homogenate.