Modeling Murine Vaginal Colonization by Anaerobic Bacteria

Samenvatting

Source: Morrill, S. R., et al. Models of Murine Vaginal Colonization by Anaerobically Grown Bacteria. J. Vis. Exp. (2022)

This video demonstrates a protocol for modeling vaginal colonization in mice using antibiotic-resistant anaerobic bacteria. It begins with hormonal treatment to increase epithelial susceptibility, followed by vaginal inoculation and co-inoculation under anaerobic conditions. The procedure concludes with post-inoculation sampling and culture to evaluate bacterial colonization and biofilm formation.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Preparation of inoculum

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

1. Cycle any reagents, including prepared New York City broth III (NYCIII), agar, and Phosphate-Buffered Saline (PBS), into an anaerobic chamber to equilibrate for at least 24 h prior to use.
   NOTE: Here, a vinyl anaerobic chamber equilibrated with a 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 1.4.1, 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 1.4.2. 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 1.4.3. 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 1.4.2. 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 Colony-Forming Units or CFU of the inoculum. This is the pre-inoculation CFU.
6. Vaginal pre-lavage and inoculation with Gardnerella
   1. Prepare vaginal lavage/wash tubes in the anaerobic chamber. 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 2.1. 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, 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 p200 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 2.2.1. 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 2.2. and step 2.3. 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 1.5. 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 2.2. 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.
7. 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 2.2.
   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 1.5. Use the CFU counts as a measure of vaginal colonization by Gardnerella (or another anaerobe of interest).

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
β-estradiol 17-valerate	Sigma	E1631	estrogenization of mice
1x PBS	Fisher	MT21040CM	vaginal lavage, G. vaginlalis inoculum prep, tissue collection and homogenization
Anaerobic chamber	Coy	7200000	Growth of anaerobic bacteria
Bacto agar	Fisher	DF0140074	G. vaginalis, F. nucleatum, and P. bivia agar plates
Glucose	Sigma	G7528	NYCIII media for G. vaginalis growth
HEPES	Cellgro	25-060-Cl	NYCIII media for G. vaginalis growth
Horse serum	Hemostat	SHS500	NYCIII media for G. vaginalis growth
Hydrogen gas mix (5% hydrogen, 10% CO₂, 85% nitrogen)	Matheson		Gas for anaerobic chamber
Milli-Q Water Purifier	Millipore	IQ-7000	for making media and homogenization
NaCl	Sigma	S3014	NYCIII media for G. vaginalis growth
Nitrogen gas	Airgas		Gas for anaerobic chamber
p-200 filter tips	ISC Bio	P-1237-200	vaginal lavages and bacterial inoculations
Sesame oil	Fisher	ICN15662191	estrogenization of mice
Spectrophotometer	BioChrom	80-3000-45	measuring bacterial OD₆₀₀
Streptomycin	Gibco	11860038	add to agar media to make selective plates
Yeast extract	Fisher	DF0127-17-9	NYCIII media for G. vaginalis growth