We acknowledge Lynne Foster for technical assistance during the development of these models. We appreciate startup funds from the Department of Molecular Microbiology and the Center for Women&#39;s Infectious Disease Research (to AL), and the March of Dimes (Basil O&#39;Connor award to AL) which helped support early-stage experiments. The National Institute of Allergy and Infectious Diseases (R01 AI114635) also supported&#160;the development of these models.

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 &#946;-estradiol 17-valerate
NOTE: &#946;-estradiol 17-valerate is a carcinogen and a reproductive toxin that can be absorbed through the skin27,28. 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.
<ol>
	<li>Filter sterilize up to 50 mL of sesame oil through a 0.22 &#181;m filter using a 50 mL conical tube vacuum filter system. Open only in a biosafety cabinet to maintain sterility.</li>
	<li>Calculate the volume of &#946;-estradiol valerate solution needed for the experiment: 200 &#181;L 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 &#946;-estradiol valerate needed to make a 5 mg/mL solution and weigh it directly in a 14 mL round-bottomed tube.</li>
	<li>Cap the 14 mL tube tightly and vortex to break up clumps in the powder (this will allow the &#946;-estradiol valerate to dissolve more quickly). Add sterile-filtered sesame oil to the 14 mL tube so that the final concentration of &#946;-estradiol valerate is 5 mg/mL.</li>
	<li>Place the tightly capped 14 mL tube on a rotator at 37 &#176;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 &#176;C decreases the viscosity of the sesame oil, making it easier to inject.</li>
	<li>Draw up &#946;-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 &#181;L mark.</li>
	<li>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 &#181;L mark. Prepare one syringe per mouse.</li>
	<li>Administer the &#946;-estradiol 17-valerate by intraperitoneal injection at 48 h or 72 h prior to inoculation. Inject each mouse with 100 &#181;L of the 5 mg/mL &#946;-estradiol valerate solution (0.5 mg &#946;-estradiol valerate/mouse), as previously described22,30. Store the remaining solution at 4 &#176;C for 72 h until the next injection.</li>
	<li>Administer the &#946;-estradiol valerate solution again on the day of inoculation, immediately prior to vaginal inoculation of the mice. Remove the solution from 4 &#176;C and place it on a rotator at 37 &#176;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.</li>
</ol>
2. Preparation of inoculum
NOTE: This section contains the general steps for the preparation of inocula from anaerobically grown streptomycin-resistant&#160;Gardnerella vaginalis JCP8151B-SmR24,25,31,32. All steps in this section should be done in an anaerobic chamber.
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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 &#176;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.</li>
	<li>Prepare the inoculum from liquid culture as described below. Perform the inoculum preparation in the anaerobic chamber as much as possible.
	<ol>
		<li>Determine the culture optical density (OD) by adding 200 &#181;L of culture to 800 &#181;L 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.</li>
		<li>Calculate the volume of inoculum needed for the experiment. For example, to infect 15 mice with an inoculum of 20 &#181;L per mouse, 15 x 20 &#181;L = 300 &#181;L of inoculum is needed. Prepare approximately 25% more inoculum than needed, so for 15 mice, prepare 300 &#181;L + (0.25 x 300 &#181;L) = 375 &#181;L of inoculum.</li>
		<li>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 &#181;L, then the volume of culture to be spun down is (375 &#181;L x 5)/1.5 = 1250 &#181;L.</li>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Prior to removing the inoculum tube from the anaerobic chamber, prepare a serial dilution series from 1:10 through 1:10&#160;x 106&#160;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.</li>
</ol>
3. Vaginal pre-lavage and inoculation with Gardnerella
<ol>
	<li>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 &#181;L 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.</li>
	<li>Perform a vaginal lavage on each mouse with 50 &#181;L 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.
	<ol>
		<li>After administration of the second &#946;-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.</li>
		<li>Using a pipette and p-200 filter tip, draw up 50 &#181;L 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 &#181;L volume in and out gently, 3x-4x.</li>
		<li>Transfer the collected wash material (~50 &#181;L) back into the same wash tube it came from, pipetting up and down in the wash tube that still contains the remaining 20 &#181;L 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.</li>
	</ol>
	</li>
	<li>Vaginally administer 20 &#181;L 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 &#181;L for lavages and the other to 20 &#181;L for inoculations.
	<ol>
		<li>Restrain the mouse as described in step 3.2.1. Using a p-200 tip, pipette 20 &#181;L of bacterial suspension into the vagina. For co-inoculation experiments using two different bacterial species/strains, use 10 &#181;L of each bacterial suspension and avoid mixing the two strains prior to inoculation. 
		NOTE: The total inoculation volume should not exceed 20 &#181;L to avoid spillover out of the vagina.</li>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>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.</li>
	<li>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 &#176;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.</li>
</ol>
4. Collection of vaginal lavages to determine viable Gardnerella colonization
<ol>
	<li>Collect the vaginal washes at select time points to analyze vaginal colonization. Collect as described in step 3.2.</li>
	<li>Immediately after collection, cycle the vaginal lavages into an anaerobic chamber. Use 10 &#181;L 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).</li>
</ol>
5. Sacrifice, dissection, and tissue homogenization
<ol>
	<li>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 &#181;L for tubes used to collect uterine horns.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.
	<ol>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>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 &#181;L 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 &#181;L of undiluted homogenate.</li>
</ol>

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E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estradiol-treated+female+mice+as+surrogate+hosts+for+Neisseria+gonorrhoeae+genital+tract+infections.">Estradiol-treated female mice as surrogate hosts for Neisseria gonorrhoeae genital tract infections.</a> Frontiers in Microbiology. 2, 107 (2011).</li><li>Gilbert, N. M., Lewis, W. G., Lewis, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+features+of+bacterial+vaginosis+in+a+murine+model+of+vaginal+infection+with+Gardnerella+vaginalis.">Clinical features of bacterial vaginosis in a murine model of vaginal infection with Gardnerella vaginalis.</a> PLoS One. 8 (3), 59539 (2013).</li><li>Gilbert, N. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gardnerella+vaginalis+and+Prevotella+bivia+trigger+distinct+and+overlapping+phenotypes+in+a+mouse+model+of+bacterial+vaginosis.">Gardnerella vaginalis and Prevotella bivia trigger distinct and overlapping phenotypes in a mouse model of bacterial vaginosis.</a> The Journal of Infectious Diseases. 220 (7), 1099-1108 (2019).</li><li>Agarwal, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycan+cross-feeding+supports+mutualism+between+Fusobacterium+and+the+vaginal+microbiota.">Glycan cross-feeding supports mutualism between Fusobacterium and the vaginal microbiota.</a> PLOS Biology. 18 (8), 3000788 (2020).</li><li>Liehr, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+estradiol+a+genotoxic+mutagenic+carcinogen.">Is estradiol a genotoxic mutagenic carcinogen.</a> Endocrine Reviews. 21 (1), 40-54 (2000).</li><li>Yager, J. D., Davidson, N. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estrogen+carcinogenesis+in+breast+cancer.">Estrogen carcinogenesis in breast cancer.</a> The New England Journal of Medicine. 354 (3), 270-282 (2006).</li><li>Adeel, M., Song, X., Wang, Y., Francis, D., Yang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environmental+impact+of+estrogens+on+human,+animal+and+plant+life:+A+critical+review.">Environmental impact of estrogens on human, animal and plant life: A critical review.</a> Environment International. 99, 107-119 (2017).</li><li>Miner, N. A., Koehler, J., Greenaway, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intraperitoneal+injection+of+mice.">Intraperitoneal injection of mice.</a> Journal of Applied Microbiology. 17 (2), 250-251 (1969).</li><li>Lewis, W. G., Robinson, L. S., Gilbert, N. M., Perry, J. C., Lewis, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Degradation,+foraging,+and+depletion+of+mucus+sialoglycans+by+the+vagina-adapted+Actinobacterium+Gardnerella+vaginalis.">Degradation, foraging, and depletion of mucus sialoglycans by the vagina-adapted Actinobacterium Gardnerella vaginalis.</a> Journal of Biological Chemistry. 288 (17), 12067-12079 (2013).</li><li>Robinson, L. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+sequences+of+15+Gardnerella+vaginalis+strains+isolated+from+the+vaginas+of+women+with+and+without+bacterial+vaginosis.">Genome sequences of 15 Gardnerella vaginalis strains isolated from the vaginas of women with and without bacterial vaginosis.</a> Genome Announcements. 4 (5), 00879 (2016).</li><li>Adapted from &amp;#34;mouse posterior turn&amp;#34; by BioRender.com. BioRender.com Available from: <a target="_blank" href="https://app.biorender.com/biorender-templates">https://app.biorender.com/biorender-templates</a> (2022)</li><li>Machholz, E., Mulder, G., Ruiz, C., Corning, B. F., Pritchett-Corning, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manual+restraint+and+common+compound+administration+routes+in+mice+and+rats.">Manual restraint and common compound administration routes in mice and rats.</a> Journal of Visualized Experiments: JoVE. (67), e2771 (2012).</li><li>Verollet, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+major+step+towards+efficient+sample+preparation+with+bead-beating.">A major step towards efficient sample preparation with bead-beating.</a> Biotechniques. 44 (6), 832-833 (2008).</li></ol>

The authors declare no conflict of interest.

The most significant way the model described here differs from previously published vaginal inoculation models is the use of anaerobically cultured bacteria, which require special considerations for the preparation of viable inoculum and the recovery of bacteria from the mice. Specific steps in the protocol above may vary slightly depending on the species/strain of bacteria used, as suggested in Supplementary File 1. Additionally, this manuscript describes a new procedural method for the administration of bacteria and vaginal washes that requires less experience on the part of the investigator and less restraint for the mice. If the experimenter is not comfortable with the form of restraint described in step 3.2. and step 3.3., the mice can instead be scruffed as previously described34 with or without anesthesia according to the experimental design and institutional IACUC guidelines.
An important caveat to the use of anaerobically cultured bacteria in murine models is that certain steps (such as any involving live animals) must be done outside of the anaerobic chamber. Therefore, the most critical steps in this protocol are those in which the bacteria of interest will be exposed to an aerobic environment, such as during inoculation of the mice. The use of any new anaerobe in this model will require preliminary investigation into its ability to maintain viability upon exposure to oxygen (such as the comparison of pre- and post-inoculation CFUs as described in step 2.5. and step 3.4.). Depending on the degree of oxygen and PBS sensitivity of the organism, different methods of inoculum preparation should be considered (Supplementary File 1, step 2.4.5.). For inoculations using G. vaginalis JCP8151B, we have found that a single tube of inoculum can be prepared in PBS and used to infect all of the mice. If a different anaerobic bacterium is being used that is more sensitive to oxygen, the inoculum can instead be prepared in separate tubes for each mouse so that repeated opening/closing of the tube is not required. Likewise, if the bacterial species of interest is more fastidious/less capable of surviving in PBS than G. vaginalis, inoculation can instead be prepared in culture media. If the medium used contains reducing agents, such as the CDC anaerobe medium used to culture Prevotella bivia (Supplementary File 1, step 2.1. and step 2.2.), then the bacteria will also have increased protection from oxygen exposure.
Alternative methods for homogenization can also be considered, such as bead beating22,35, which is done with the tube cap closed and may, therefore, introduce less oxygen than the handheld homogenizer. Additionally, more oxygen-sensitive organisms may require a longer equilibrium time for media. An oxygen indicator such as resazurin can be used to check that anaerobic conditions have been reached (step 2.1.). Alternative methods such as anaerobic jars may affect results and should be vetted for bacterial viability prior to use.
The oxygen sensitivity and fastidiousness of the experimental organism may also play a role in the successful recovery of viable bacteria from the mouse during lavages/homogenization (Supplementary File 1, step 3.2. and step 5.1.). For highly sensitive organisms, the time between the lavage being taken and plated may lead to a loss of viability and cause an artificially low estimate of bacterial vaginal colonization. To mitigate this effect, collected lavages can immediately be diluted into fresh anaerobic culture media (with reducing agent). Similarly, organ homogenization can be done in fresh culture media rather than PBS to increase bacterial viability and can also be done in the anaerobic chamber itself to minimize oxygen exposure.
Depending on the purpose of a given experiment, the experimenter must pay attention to control groups. To test hypotheses, baseline measures of uninfected control mice that are mock-treated with vehicle alone will help establish if there is induction of chemokines/cytokines or other specific outcomes of infection. The control vehicle used must be the same as the vehicle used for inoculation, so if the bacteria are inoculated in culture media, then uninoculated culture media must be used as the control (Supplementary File 1, step 2.4.5.).
The methods described in this protocol could also be applied to facultative bacteria capable of growing anaerobically but that are traditionally studied using aerobic culture conditions (such as Escherichia coli or Group B Streptococcus). This could be especially relevant for infections by these organisms in body sites that are believed to contain low levels of oxygen, such as the cervix. It may be that a facultative organism more successfully infects sites with less oxygen when the inoculum is prepared anaerobically as compared to aerobically. In such an experiment, the recovery of viable bacteria for CFU enumeration may not require anaerobic conditions.

In mammals, the vagina is home to a consortium of bacterial species. The human vaginal microbiome is unique among mammals in its abundance of members of the genus Lactobacillus and corresponding low vaginal pH (3.8-4.5)1,2,3,4. Disruption of this Lactobacillus dominance is associated with a variety of negative health outcomes.
In bacterial vaginosis (BV) there are fewer lactobacilli and an increased abundance of diverse anaerobic bacteria, such as Gardnerella vaginalis and Prevotella bivia5,6. Women with BV are at increased risk of&#160;sexually transmitted infections7,8,9, infertility10, pregnancy losses11, preterm birth12,13,14, intrauterine infections15, cervical infections16, and cancer16,17,18. BV is also associated with a higher likelihood of vaginal colonization by potentially pathogenic bacteria, such as Fusobacterium nucleatum1,19,20, a common isolate from amniotic fluid infections21.
Due to the importance of anaerobic bacteria in human vaginal health, there is a need for animal models that can be used to investigate the biology and pathogenesis of these organisms. Here, we describe methods for vaginal inoculation and viable recovery of Gardnerella vaginalis in estrogenized mice and suggest additional strategies for Prevotella bivia and Fusobacterium nucleatum. Other models of murine vaginal colonization have previously been described, but these have focused on the inoculation and recovery of facultative anaerobic bacteria such as Group B Streptococcus22 and Neisseria gonorrhoeae23 that were cultured aerobically. Inoculation and recovery of obligate anaerobes can be successfully accomplished with appropriate experimental strategies. We discuss approaches for the viable recovery of several bacterial taxa and suggest empirical evaluation of conditions for the viability of additional species/strains of interest.
The classical method of estimating colonization by live bacteria is the recovery of colony-forming units (CFUs). In conventionally raised mice with their own endogenous microbiota, this requires recovery on agar media that selects against members of the endogenous vaginal microbiota while still allowing the inoculated strain of bacteria to grow. Here, we use a streptomycin-resistant isolate of G. vaginalis24 that can be selectively recovered on a streptomycin-containing agar medium. To ensure the medium is sufficiently selective and, conversely, that streptomycin-resistant bacteria are not present in the endogenous microbiome, vaginal lavages collected just prior to inoculation should be plated on selective (streptomycin-containing) agar.
The best method for inoculum preparation may vary among species and strains of bacteria. Prior to the start of experiments in mice, preliminary work should be performed to determine preferable conditions for the culture medium, growth endpoint, and preparation of inoculum, as well as the susceptibility to oxygen and viability in PBS. In the case of more oxygen-sensitive bacteria, alternate preparations of the inoculum can be considered (e.g., in an anaerobic culture medium with an appropriate vehicle control group)25,26.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>&#946;-estradiol 17-valerate</td>
			<td>Sigma</td>
			<td>E1631</td>
			<td>estrogenization of mice</td>
		</tr>
		<tr>
			<td>0.22 &#181;m, 50 mL Steriflip vacuum filter</td>
			<td>Fisher</td>
			<td>&#160;SCGP00525</td>
			<td>sterile filtering sesame oil</td>
		</tr>
		<tr>
			<td>14 mL tube</td>
			<td>Falcon</td>
			<td>352059</td>
			<td>estrogenization of mice</td>
		</tr>
		<tr>
			<td>190-proof ethanol</td>
			<td>Koptec</td>
			<td>&#160;V1105M</td>
			<td>dissection, tissue homogenization, CDC and Columbia media</td>
		</tr>
		<tr>
			<td>1x PBS</td>
			<td>Fisher</td>
			<td>MT21040CM</td>
			<td>vaginal lavage, G. vaginlalis inoculum prep, tissue collection and homogenization</td>
		</tr>
		<tr>
			<td>25 G &#215; 5/8 -in needle</td>
			<td>BD</td>
			<td>305122</td>
			<td>estrogenization of mice</td>
		</tr>
		<tr>
			<td>5 mL tube</td>
			<td>Falcon</td>
			<td>352063</td>
			<td>Tissue collection and homogenization</td>
		</tr>
		<tr>
			<td>Anaerobic chamber</td>
			<td>Coy</td>
			<td>7200000</td>
			<td>Growth of anaerobic bacteria</td>
		</tr>
		<tr>
			<td>Bacto agar</td>
			<td>Fisher</td>
			<td>DF0140074</td>
			<td>G. vaginalis, F. nucleatum, and P. bivia agar plates</td>
		</tr>
		<tr>
			<td>Bacto peptone</td>
			<td>Fisher</td>
			<td>DF0118170</td>
			<td>CDC anaerobic media for P. bivia growth</td>
		</tr>
		<tr>
			<td>Columbia broth</td>
			<td>Fisher</td>
			<td>DF0944170</td>
			<td>Columbia media for F. nucleatum growth</td>
		</tr>
		<tr>
			<td>Defibrinated sheep blood</td>
			<td>Hemostat</td>
			<td>DSB500</td>
			<td>CDC anaerobic media for P. bivia growth</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Fine Science Tools</td>
			<td>&#160;11008-13</td>
			<td>mouse/tissue dissection</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma</td>
			<td>G7528</td>
			<td>NYCIII media for G. vaginalis growth</td>
		</tr>
		<tr>
			<td>Handheld homogenizer</td>
			<td>ISC Bio</td>
			<td>3305500</td>
			<td>Tissue homogenization</td>
		</tr>
		<tr>
			<td>Hemin</td>
			<td>Sigma</td>
			<td>51280</td>
			<td>CDC anaerobic media for P. bivia growth, Columbia media for F. nucleatum growth</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Cellgro</td>
			<td>25-060-Cl</td>
			<td>NYCIII media for G. vaginalis growth</td>
		</tr>
		<tr>
			<td>Horse serum</td>
			<td>Hemostat</td>
			<td>SHS500</td>
			<td>NYCIII media for G. vaginalis growth</td>
		</tr>
		<tr>
			<td>Hydrogen gas mix (5% hydrogen, 10% CO2, 85% nitrogen)</td>
			<td>Matheson</td>
			<td></td>
			<td>Gas for anaerobic chamber</td>
		</tr>
		<tr>
			<td>Isofluorane</td>
			<td>VetOne</td>
			<td>502017</td>
			<td>mouse anaethesia at sacrifice</td>
		</tr>
		<tr>
			<td>L-cysteine</td>
			<td>Sigma</td>
			<td>C1276</td>
			<td>CDC anaerobic media for P. bivia growth</td>
		</tr>
		<tr>
			<td>L-glutamate</td>
			<td>Sigma</td>
			<td>G1626</td>
			<td>CDC anaerobic media for P. bivia growth</td>
		</tr>
		<tr>
			<td>Milli-Q Water Purifier</td>
			<td>Millipore</td>
			<td>IQ-7000</td>
			<td>for making media and homogenization</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S3014</td>
			<td>NYCIII media for G. vaginalis growth</td>
		</tr>
		<tr>
			<td>NaOH tablets</td>
			<td>Sigma</td>
			<td>S5881</td>
			<td>CDC anaerobic media for P. bivia growth</td>
		</tr>
		<tr>
			<td>Nitrogen gas</td>
			<td>&#160;Airgas</td>
			<td></td>
			<td>&#160;Gas for anaerobic chamber</td>
		</tr>
		<tr>
			<td>p-200 filter tips</td>
			<td>ISC Bio</td>
			<td>&#160;P-1237-200</td>
			<td>vaginal lavages and bacterial inoculations</td>
		</tr>
		<tr>
			<td>pancreatic digest of casein</td>
			<td>Sigma</td>
			<td>70169</td>
			<td>CDC anaerobic media for P. bivia growth</td>
		</tr>
		<tr>
			<td>Proteose peptone #3</td>
			<td>Fisher</td>
			<td>DF-122-17-4</td>
			<td>NYCIII media for G. vaginalis growth</td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>Fine Science Tools</td>
			<td>&#160;14084-08</td>
			<td>mouse/tissue dissection</td>
		</tr>
		<tr>
			<td>Sesame oil</td>
			<td>Fisher</td>
			<td>&#160;ICN15662191</td>
			<td>estrogenization of mice</td>
		</tr>
		<tr>
			<td>Single edge surgical carbon steel razor blades</td>
			<td>VWR</td>
			<td>55411-050</td>
			<td>tissue dissection</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Sigma</td>
			<td>S3014</td>
			<td>CDC anaerobic media for P. bivia growth</td>
		</tr>
		<tr>
			<td>Spectrophotometer</td>
			<td>BioChrom</td>
			<td>80-3000-45</td>
			<td>measuring bacterial OD600</td>
		</tr>
		<tr>
			<td>Streptomycin</td>
			<td>Gibco</td>
			<td>11860038</td>
			<td>add to agar media to make selective plates</td>
		</tr>
		<tr>
			<td>Tuberculin slip tip 1ml syringe</td>
			<td>BD</td>
			<td>309659</td>
			<td>estrogenization of mice</td>
		</tr>
		<tr>
			<td>Vitaim K3</td>
			<td>Sigma</td>
			<td>M5625</td>
			<td>Columbia media for F. nucleatum growth</td>
		</tr>
		<tr>
			<td>Vitamin K1</td>
			<td>Sigma</td>
			<td>95271</td>
			<td>CDC anaerobic media for P. bivia growth</td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>Fisher</td>
			<td>DF0127-17-9</td>
			<td>NYCIII media for G. vaginalis growth</td>
		</tr>
	</tbody>
</table>

models of murine vaginal colonization by anaerobically grown bacteria

The mammalian vagina can be colonized by many bacterial taxa. The human vaginal microbiome is often dominated by Lactobacillus species, but one-in-four women experience bacterial vaginosis, in which a low level of lactobacilli is accompanied by an overgrowth of diverse anaerobic bacteria. This condition has been associated with many health complications, including risks to reproductive and sexual health. While there is growing evidence showing the complex nature of microbial interactions in human vaginal health, the individual roles of these different anaerobic bacteria are not fully understood. This is complicated by the lack of adequate models to study anaerobically grown vaginal bacteria. Mouse models allow us to investigate the biology and virulence of these organisms in vivo. Other mouse models of vaginal bacterial inoculation have previously been described. Here, we describe methods for the inoculation of anaerobically grown bacteria and their viable recovery in conventionally raised C57Bl/6 mice. A new, less stressful procedural method for vaginal inoculation and washing is also described. Inoculation and viable recovery of Gardnerella are outlined in detail, and strategies for additional anaerobes such as Prevotella bivia and Fusobacterium nucleatum are discussed.

A schematic representation of an example experiment shows some of the ways one might carry out the described protocol (Figure 1).
Some animals will carry endogenous microbes in their vaginal microbiomes that will grow on nonselective media. The methods described here rely on streptomycin-resistant isolates of the studied bacterial strains, together with selective media containing streptomycin, to select against endogenous bacteria (Figure 2A,B). It is possible for streptomycin resistance to develop spontaneously, so checking mice prior to infection (day 0 washes) can help establish if this may be a problem.
Recovery of viable bacteria from vaginal washes is accomplished by serial dilution and plating on agar media. A standard petri dish can hold up to a 6 x 6 array of 5 &#181;L spots, allowing enumeration of bacterial titers from six replica spots across six orders of magnitude for each sample if&#160;using 10-fold serial dilutions (Figure 3A). This method can be used to enumerate titers of bacteria ranging from Gardnerella to Prevotella and Fusobacterium (Figure 3B-D).
Together, these methods allow one to test hypotheses and make interpretations about bacterial colonization/infection of the female reproductive tract. For example, a comparison of Gardnerella titers in vaginal washes and vaginal tissue homogenates demonstrated concordance between these methods (Figure 4A). Likewise, in a vaginal co-inoculation model, titers of Prevotella in uterine tissue were significantly higher (approximately 20-fold) during Gardnerella co-infection compared to Prevotella mono-infection (Figure 4B). Finally, wild-type versus mutant bacterial strains can be compared in these models to establish the roles played by specific genes and their products in the processes of reproductive tract colonization/infection. For example, a mutant strain of Fusobacterium lacking the ability to transport and consume the carbohydrate sialic acid led to lower levels of colonization by 3 days post-inoculation (Figure 4C).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64032/64032fig01.jpg" /> 
Figure 1: Schematic of an example experiment33. <a href="https://www.jove.com/files/ftp_upload/64032/64032fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64032/64032fig02.jpg" /> 
Figure 2: Growth of endogenous murine vaginal microbes on selective versus nonselective agar. Vaginal lavages from five female C57Bl/6 mice (1-5) were streaked onto two different NYC III agar plates and incubated anaerobically. (A)&#160;The plate on the left does not contain any antibiotics and permits the growth of endogenous anaerobic bacteria from the murine vaginal tract. (B)&#160;The plate on the right contains 1 mg/mL streptomycin and selects against the growth of endogenous bacteria. <a href="https://www.jove.com/files/ftp_upload/64032/64032fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64032/64032fig03.jpg" /> 
Figure 3: Recovery of viable G. vaginalis, P. bivia, and F. nucleatum CFUs from vaginal washes.&#160;(A)&#160;CFUs of G. vaginalis JCP8151B-SmR inoculum, replica-plated as a dilution series on NYC III agar. (B-D)&#160;Recovery of viable (B)&#160;G. vaginalis24, (C) P. bivia25, and (D)&#160;F. nucleatum26 from the vaginal lavages of mono-infected animals. For each graph (B-D), the data is combined from two independent experiments, with 10-15 mice per experiment24,25,26. <a href="https://www.jove.com/files/ftp_upload/64032/64032fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64032/64032fig04v2.jpg" /> 
Figure 4: Further analysis of anaerobic bacterial colonization in a murine vaginal inoculation model. (A) G. vaginalis titers in vaginal washes correlate with G. vaginalis titers recovered from vaginal tissue homogenates at 24 h and 72 h post-inoculation (combination of two independent experiments, n = 10 each. Significance determined by Spearman&#39;s rank correlation test)24.&#160;(B) Co-infection with G. vaginalis leads to increased P. bivia titers in uterine horn tissue when compared to P. bivia mono-infected animals at 48 h post-inoculation (combination of two independent experiments, each with 6-10 mice per group. Significance determined by Mann-Whitney U-test, **p = 0.0017)25. (C)&#160;F. nucleatum mutant with disrupted sialic acid transporter (&#937; SiaT) has decreased titers in vaginal lavages of mono-infected mice as compared to F. nucleatum wild-type at 72 h post-inoculation (combination of two independent experiments, each with 10 mice per group. Significance determined by Mann-Whitney U-test, **p &#60; 0.01)26. <a href="https://www.jove.com/files/ftp_upload/64032/64032fig04largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary File 1: Examples of methods used for different vaginal bacteria grown under anaerobic conditions. <a href="https://www.jove.com/files/ftp_upload/64032/64032_SupplementalFile.zip" target="_blank">Please click here to download this File.</a>

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Models of Murine Vaginal Colonization by Anaerobically Grown Bacteria

The protocol presents a mouse model of vaginal colonization with anaerobically cultured human vaginal bacteria. We focus on Gardnerella vaginalis,&#160;while including suggestions for Prevotella bivia and Fusobacterium nucleatum. This protocol can also be used as a guide for vaginal inoculations and viable recovery of other anaerobically grown bacteria.

The mammalian vagina can be colonized by many bacterial taxa. The human vaginal microbiome is often dominated by Lactobacillus species, but one-in-four ...

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JoVE Journal

Immunology and Infection

3.4K Views.  Washington University School of Medicine. The protocol presents a mouse model of vaginal colonization with anaerobically cultured human vaginal bacteria. We focus on Gardnerella vaginalis, while including suggestions for Prevotella bivia and Fusobacterium nucleatum. This protocol can also be used as a guide for vaginal inoculations and viable recovery of other anaerobically grown bacteria.

Video: Models of Murine Vaginal Colonization by Anaerobically Grown Bacteria

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living surfaces. In this paper, we describe protocols for forming Pseudomonas aeruginosa biofilms on human airway epithelial cells (CFBE cells) grown in culture. In the first method (termed the Static Co-culture Biofilm Model), P. aeruginosa is incubated with CFBE cells grown as confluent monolayers on standard tissue culture plates. Although the bacterium is quite toxic to epithelial cells, the addition of arginine delays the destruction of the monolayer long enough for biofilms to form on the CFBE cells. The second method (termed the Flow Cell Co-culture Biofilm Model), involves adaptation of a biofilm flow cell apparatus, which is often used in biofilm research, to accommodate a glass coverslip supporting a confluent monolayer of CFBE cells. This monolayer is inoculated with P. aeruginosa and a peristaltic pump then flows fresh medium across the cells. In both systems, bacterial biofilms form within 6-8 hours after inoculation. Visualization of the biofilm is enhanced by the use of P. aeruginosa strains constitutively expressing green fluorescent protein (GFP). The Static and Flow Cell Co-culture Biofilm assays are model systems for early P. aeruginosa infection of the Cystic Fibrosis (CF) lung, and these techniques allow different aspects of P. aeruginosa biofilm formation and virulence to be studied, including biofilm cytotoxicity, measurement of biofilm CFU, and staining and visualizing the biofilm.

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

This paper describes different methods of growing Pseudomonas aeruginosa biofilms on cultured human airway epithelial cells. These protocols can be adapted to study different aspects of biofilm formation, including visualization of the biofilm, staining of the biofilm, measuring the colony forming units (CFU) of the biofilm, and studying biofilm cytotoxicity.

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living ...

Assessing Hepatic Metabolic Changes During Progressive Colonization of Germ-free Mouse by 1H NMR Spectroscopy

It is well known that gut bacteria contribute significantly to the host homeostasis, providing a range of benefits such as immune protection and vitamin synthesis. They also supply the host with a considerable amount of nutrients, making this ecosystem an essential metabolic organ. In the context of increasing evidence of the link between the gut flora and the metabolic syndrome, understanding the metabolic interaction between the host and its gut microbiota is becoming an important challenge of modern biology.1-4
 Colonization (also referred to as normalization process) designates the establishment of micro-organisms in a former germ-free animal. While it is a natural process occurring at birth, it is also used in adult germ-free animals to control the gut floral ecosystem and further determine its impact on the host metabolism. A common procedure to control the colonization process is to use the gavage method with a single or a mixture of micro-organisms. This method results in a very quick colonization and presents the disadvantage of being extremely stressful5. It is therefore useful to minimize the stress and to obtain a slower colonization process to observe gradually the impact of bacterial establishment on the host metabolism.
 In this manuscript, we describe a procedure to assess the modification of hepatic metabolism during a gradual colonization process using a non-destructive metabolic profiling technique. We propose to monitor gut microbial colonization by assessing the gut microbial metabolic activity reflected by the urinary excretion of microbial co-metabolites by 1H NMR-based metabolic profiling. This allows an appreciation of the stability of gut microbial activity beyond the stable establishment of the gut microbial ecosystem usually assessed by monitoring fecal bacteria by DGGE (denaturing gradient gel electrophoresis).6 The colonization takes place in a conventional open environment and is initiated by a dirty litter soiled by conventional animals, which will serve as controls. Rodents being coprophagous animals, this ensures a homogenous colonization as previously described.7
 Hepatic metabolic profiling is measured directly from an intact liver biopsy using 1H High Resolution Magic Angle Spinning NMR spectroscopy. This semi-quantitative technique offers a quick way to assess, without damaging the cell structure, the major metabolites such as triglycerides, glucose and glycogen in order to further estimate the complex interaction between the colonization process and the hepatic metabolism7-10. This method can also be applied to any tissue biopsy11,12.

Assessing Hepatic Metabolic Changes During Progressive Colonization of Germ-free Mouse by 1H NMR Spectroscopy

A progressive colonization procedure is described to further assess its impact on the host hepatic metabolism. Colonization is monitored non invasively by evaluating the urinary excretion of microbial co-metabolites by NMR-based metabolic profiling while hepatic metabolism is assessed by High Resolution Magic Angle Spinning (HR MAS) NMR profiling of intact biopsy.

It is well known that gut bacteria contribute significantly to the host homeostasis, providing a range of benefits such as immune protection and vitamin ...

Colonization of Euprymna scolopes Squid by Vibrio fischeri

Specific bacteria are found in association with animal tissue1-5. Such host-bacterial associations (symbioses) can be detrimental (pathogenic), have no fitness consequence (commensal), or be beneficial (mutualistic). While much attention has been given to pathogenic interactions, little is known about the processes that dictate the reproducible acquisition of beneficial/commensal bacteria from the environment. The light-organ mutualism between the marine Gram-negative bacterium V. fischeri and the Hawaiian bobtail squid, E. scolopes, represents a highly specific interaction in which one host (E. scolopes) establishes a symbiotic relationship with only one bacterial species (V. fischeri) throughout the course of its lifetime6,7. Bioluminescence produced by V. fischeri during this interaction provides an anti-predatory benefit to E. scolopes during nocturnal activities8,9, while the nutrient-rich host tissue provides V. fischeri with a protected niche10. During each host generation, this relationship is recapitulated, thus representing a predictable process that can be assessed in detail at various stages of symbiotic development. In the laboratory, the juvenile squid hatch aposymbiotically (uncolonized), and, if collected within the first 30-60 minutes and transferred to symbiont-free water, cannot be colonized except by the experimental inoculum6. This interaction thus provides a useful model system in which to assess the individual steps that lead to specific acquisition of a symbiotic microbe from the environment11,12.
Here we describe a method to assess the degree of colonization that occurs when newly hatched aposymbiotic E. scolopes are exposed to (artificial) seawater containing V. fischeri. This simple assay describes inoculation, natural infection, and recovery of the bacterial symbiont from the nascent light organ of E. scolopes. Care is taken to provide a consistent environment for the animals during symbiotic development, especially with regard to water quality and light cues. Methods to characterize the symbiotic population described include (1) measurement of bacterially-derived bioluminescence, and (2) direct colony counting of recovered symbionts.

Colonization of Euprymna scolopes Squid by Vibrio fischeri

The method outlines the procedure by which the Hawaiian bobtail squid, Euprymna scolopes and its bacterial symbiont, Vibrio fischeri, are raised separately and then introduced to allow for specific colonization of the squid light organ by the bacteria. Colonization detection by bacterially-derived luminescence and by direct colony counting are described.

Specific bacteria are found in association with animal tissue1-5. Such host-bacterial associations (symbioses) can be detrimental (pathogenic), have no ...

Purification and Visualization of Lipopolysaccharide from Gram-negative Bacteria by Hot Aqueous-phenol Extraction

Lipopolysaccharide (LPS) is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of lipid A, which is embedded in the outer membrane, a core oligosaccharide and repeating O-antigen units that extend outward from the surface of the cell1, 2. LPS is an immunodominant molecule that is important for the virulence and pathogenesis of many bacterial species, including Pseudomonas aeruginosa, Salmonella species, and Escherichia coli3-5, and differences in LPS O-antigen composition form the basis for serotyping of strains. LPS is involved in attachment to host cells at the initiation of infection and provides protection from complement-mediated killing; strains that lack LPS can be attenuated for virulence6-8. For these reasons, it is important to visualize LPS, particularly from clinical isolates. Visualizing LPS banding patterns and recognition by specific antibodies can be useful tools to identify strain lineages and to characterize various mutants. 
In this report, we describe a hot aqueous-phenol method for the isolation and purification of LPS from Gram-negative bacterial cells. This protocol allows for the extraction of LPS away from nucleic acids and proteins that can interfere with visualization of LPS that occurs with shorter, less intensive extraction methods9. LPS prepared this way can be separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and directly stained using carbohydrate/glycoprotein stains or standard silver staining methods. Many anti-sera to LPS contain antibodies that cross-react with outer membrane proteins or other antigenic targets that can hinder reactivity observed following Western immunoblot of SDS-PAGE-separated crude cell lysates. Protease treatment of crude cell lysates alone is not always an effective way of removing this background using this or other visualization methods. Further, extensive protease treatment in an attempt to remove this background can lead to poor quality LPS that is not well resolved by any of the aforementioned methods. For these reasons, we believe that the following protocol, adapted from Westpahl and Jann10, is ideal for LPS extraction.

We describe a modified hot aqueous-phenol extraction method for purifying lipopolysaccharide (LPS) from Gram-negative bacteria. Once extracted, the LPS can be subsequently analyzed by SDS-PAGE and visualized by direct staining or Western immunoblot.

Lipopolysaccharide (LPS)  is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of  lipid A, which is ...

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing the mucosal surface of the nasopharynx, where it can reside, multiply and eventually overcome host defences to invade to other tissues of the host. Establishment of an infection in the normally lower respiratory tract results in pneumonia. Alternatively, the bacteria can disseminate into the bloodstream causing bacteraemia, which is associated with high mortality rates2, or else lead directly to the development of pneumococcal meningitis. Understanding the kinetics of, and immune responses to, nasopharyngeal colonization is an important aspect of S. pneumoniae infection models.
Our mouse model of intranasal colonization is adapted from human models3 and has been used by multiple research groups in the study of host-pathogen responses in the nasopharynx4-7. In the first part of the model, we use a clinical isolate of S. pneumoniae to establish a self-limiting bacterial colonization that is similar to carriage events in human adults. The procedure detailed herein involves preparation of a bacterial inoculum, followed by the establishment of a colonization event through delivery of the inoculum via an intranasal route of administration. Resident macrophages are the predominant cell type in the nasopharynx during the steady state. Typically, there are few lymphocytes present in uninfected mice8, however mucosal colonization will lead to low- to high-grade inflammation (depending on the virulence of the bacterial species and strain) that will result in an immune response and the subsequent recruitment of host immune cells. These cells can be isolated by a lavage of the tracheal contents through the nares, and correlated to the density of colonization bacteria to better understand the kinetics of the infection.

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Colonization of the murine nasopharynx with Streptococcus pneumoniae and the subsequent extraction of adherent or recruited cells is described. This technique involves flushing the nasopharynx and collection of the fluid through the nares and is adaptable for various readouts, including differential cell quantification and analysis of mRNA expression in situ.

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing ...

Application of Fluorescent Nanoparticles to Study Remodeling of the Endo-lysosomal System by Intracellular Bacteria

Fluorescent nanoparticles (NPs) with desirable chemical, optical and mechanical properties are promising tools to label intracellular organelles. Here, we introduce a method using gold-BSA-rhodamine NPs to label the endo-lysosomal system of eukaryotic cells and monitor manipulations of host cellular pathways by the intracellular pathogen Salmonella enterica. The NPs were readily internalized by HeLa cells and localized in late endosomes/lysosomes. Salmonella infection induced rearrangement of the vesicles and accumulation of NPs in Salmonella-induced membrane structures. We deployed the Imaris software package for quantitative analyses of confocal microscopy images. The number of objects and their size distribution in non-infected cells were distinct from the ones in Salmonella-infected cells, indicating extremely remodeling of the endo-lysosomal system by WT Salmonella.

This article describes methods for the synthesis and fluorescent labeling of nanoparticles (NPs). The NPs were applied in pulse-chase experiments to label the endo-lysosomal system of eukaryotic cells. Manipulation of the endo-lysosomal system by activities of the intracellular pathogen Salmonella enterica were followed by live cell imaging and quantified.

Fluorescent nanoparticles (NPs) with desirable chemical, optical and mechanical properties are promising tools to label intracellular organelles. Here, we ...

T Cells Capture Bacteria by Transinfection from Dendritic Cells

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic cells (DCs) by a process called transinfection. Here, we describe the analysis of the transinfection process, which occurs during the course of antigen presentation. This process was unveiled by using CD4+ T cells from transgenic OTII mice, which bear a T cell receptor (TCR) specific for a peptide of ovoalbumin (OVAp), which therefore can form stable immune complexes with infected dendritic cells loaded with this specific OVAp. The dynamics of green fluorescent protein (GFP)-expressing bacteria during DC-T cell transmission can be monitored by live-cell imaging and the quantification of bacterial transinfection can be performed by flow cytometry. In addition, transinfection can be quantified by a more sensitive method based in the use of gentamicin, a non-permeable aminoglycoside antibiotic killing extracellular bacteria but not intracellular ones. This classical method has been used previously in microbiology to study the efficiency of bacterial infections. We hereby explain the protocol of the complete process, from the isolation of the primary cells to the quantification of transinfection.

Here a protocol is presented to measure bacterial capture by CD4+ T cells which occurs during antigen presentation via transinfection from pre-infected dendritic cells (DC). We show how to perform the necessary steps: isolation of primary cells, infection of DC, DC/T cell conjugate formation, and measurement of bacterial T cell transfection.

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic ...

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

Analysis of the transcriptome of bacterial pathogens during mammalian infection is a valuable tool for studying genes and factors that mediate infection. However, isolating bacterial RNA from infected cells or tissues is a challenging task, since mammalian RNA mostly dominates the lysates of infected cells. Here we describe an optimized method for RNA isolation of Listeria monocytogenes bacteria growing within bone marrow derived macrophage cells. Upon infection, cells are mildly lysed and rapidly filtered to discard most of the host proteins and RNA, while retaining intact bacteria. Next, bacterial RNA is isolated using hot phenol-SDS extraction followed by DNase treatment. The extracted RNA is suitable for gene transcription analysis by multiple techniques. This method is successfully employed in our studies of Listeria monocytogenes gene regulation during infection of macrophage cells 1-4. The protocol can be easily modified to study other bacterial pathogens and cell types.

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

Here we describe a method for bacterial RNA isolation from Listeria monocytogenes bacteria growing inside murine macrophages. This technique can be used with other intracellular pathogens and mammalian host cells.

Analysis of the transcriptome of bacterial pathogens during mammalian infection is a valuable tool for studying genes and factors that mediate infection. ...

Determination of Biofilm Initiation on Virus-infected Cells by Bacteria and Fungi

The study of polymicrobial interactions across the taxonomic kingdoms that include fungi, bacteria and virus have not been previously examined with respect to how viral members of the microbiome affect subsequent microbe interactions with these virus-infected host cells. The co-habitation of virus with bacteria and fungi is principally present on the mucosal surfaces of the oral cavity and genital tract. Mucosal cells, particularly those with persistent chronic or persistent latent viral infections, could have a significant impact on members of the microbiome through virus alteration in number and type of receptors expressed. Modification in host cell membrane architecture would result in altered ability of subsequent members of the normal flora and opportunistic pathogens to initiate the first step in biofilm formation, i.e., adherence. This study describes a method for quantitation and visual examination of HSV&#39;s effect on the initiation of biofilm formation (adherence) of S. aureus and C. albicans.

A method is described herein for the determination of inter-Kingdom association and competition (bacterial and fungal) for adherence to virus-infected HeLa cell monolayers. This protocol can be extended to multiple combinations of prokaryotes, eukaryotes, and viruses.

The study of polymicrobial interactions across the taxonomic kingdoms that include fungi, bacteria and virus have not been previously examined with ...

A Murine Model of Group B Streptococcus Vaginal Colonization

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of 10 - 30% of adults. In immune-compromised individuals, including neonates, pregnant women, and the elderly, GBS may switch to an invasive pathogen causing sepsis, arthritis, pneumonia, and meningitis. Because GBS is a leading bacterial pathogen of neonates, current prophylaxis is comprised of late gestation screening for GBS vaginal colonization and subsequent peripartum antibiotic treatment of GBS-positive mothers. Heavy GBS vaginal burden is a risk factor for both neonatal disease and colonization. Unfortunately, little is known about the host and bacterial factors that promote or permit GBS vaginal colonization. This protocol describes a technique for establishing persistent GBS vaginal colonization using a single &#946;-estradiol pre-treatment and daily sampling to determine bacterial load. It further details methods to administer additional therapies or reagents of interest and to collect vaginal lavage fluid and reproductive tract tissues. This mouse model will further the understanding of the GBS-host interaction within the vaginal environment, which will lead to potential therapeutic targets to control maternal vaginal colonization during pregnancy and to prevent transmission to the vulnerable newborn. It will also be of interest to increase our understanding of general bacterial-host interactions in the female vaginal tract.

A Murine Model of Group B Streptococcus Vaginal Colonization

The purpose of this protocol is to imitate human group B Streptococcus (GBS) vaginal colonization in a murine model. This method may be used to investigate host immune responses and bacterial factors contributing to GBS vaginal persistence, as well as to test therapeutic strategies.

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of ...