Method Article

Murine Model of Candida albicans and Streptococcus agalactiae Vaginal Co-Colonization

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DOI:

10.3791/72557

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August 21st, 2026

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1Department of Immunology and Microbiology, University of Colorado Anschutz Medical Campus

Corresponding Authors: Kelly S. Doran <kelly.doran@cuanschutz.edu>

In This Article

Summary

This protocol details a method to vaginally co-colonize mice with Candida albicans and Streptococcus agalactiae. It may be used to study polymicrobial interactions, host-microbe interactions, and host responses to these organisms.

Abstract

Diverse microbes are isolated from the human vaginal tract, and many of the organisms that inhabit this niche can switch between commensal and pathogenic lifestyles. Factors, including community composition and the host environment, influence disease outcomes; however, the mechanisms determining these outcomes are not well understood. In this manuscript, we describe a murine model to investigate interkingdom interactions and the host response to Candida albicans and Streptococcus agalactiae (Group B Streptococcus, GBS), pathobionts that colonize the female genital tract (FGT). Emerging evidence suggests that C. albicans colonization is a risk factor for GBS carriage due to their frequent co-isolation. During pregnancy, GBS colonization can lead to adverse outcomes including chorioamnionitis and stillbirth. GBS can also be transmitted to a fetus in utero or a neonate during birth and is a leading cause of neonatal meningitis. Colonization of the vaginal tract is a critical precursor to GBS disease, and this niche exerts numerous selective pressures, some of which may be influenced by C. albicans colonization. This protocol describes a model of concurrent C. albicans-GBS vaginal co-colonization using antibiotic treatment and a single dose of 17β-estradiol to establish C. albicans colonization, followed by the removal of antibiotics and inoculation with GBS. Microbial burdens in the vaginal lumen are quantified over the course of the experiment by lavage, and burdens throughout the FGT are quantified by dissection, homogenization, and plating of the vagina, cervix, and uterus. This method will enable investigation of the mechanisms by which direct and indirect polymicrobial interactions influence the lifestyles and pathogenesis of these two organisms, as well as the host immune landscape in the FGT.

Introduction

Streptococcus agalactiae (Group B Streptococcus, GBS) is a Gram-positive bacterial pathobiont that colonizes multiple polymicrobial sites, including the gastrointestinal and vaginal tracts. While vaginal colonization is typically asymptomatic, during pregnancy, GBS can ascend from the vaginal tract into the uterus and traverse intact membranes, causing infection of the amniotic fluid and placenta. Thus, it is a primary risk factor for adverse pregnancy outcomes, fetal infection, and neonatal disease1,2. As a leading cause of invasive infection in neonates, it is critical to characterize factors that influence the risk of transmission and infection. Intrapartum antibiotic prophylaxis can reduce vertical transmission and early-onset GBS disease in neonates3,4; however, it has been associated with rising rates of antibiotic resistance and potential long-term effects related to prenatal or early-life antibiotic administration5,6,7,8. These factors necessitate the development of more targeted preventative and therapeutic interventions.

Emerging evidence has demonstrated that microbiome composition and polymicrobial interactions influence homeostasis and the likelihood of disease9. GBS dominance in the human vaginal microbiome has been associated with a decrease in Lactobacillus abundance, and species- and strain-specific interactions have been observed which can have diverse effects on colonization in vitro and in vivo10,11. Previously, our research group has developed a murine model of GBS vaginal colonization to study host and bacterial correlates of GBS fitness12. This involves treating mice with a single dose of 17β-estradiol, followed by intravaginal inoculation with GBS on the following day. This model has been utilized to characterize the importance of GBS surface proteins, regulatory signaling networks, and toxin secretion in maintaining colonization and invading FGT tissues. Further, we have found that GBS exhibits complex interactions with other bacteria in the vaginal tract, including Akkermansia muciniphila and Enterococcus faecalis13,14,15, demonstrating how GBS lifestyle and persistence are influenced by the presence of neighboring microbes.

While this existing model has been adapted to study interbacterial interactions, additional modifications were required to study other microbes. Fungi are an understudied component of the vaginal microbiome, and with the model presented herein, we sought to develop a tool to study fungal colonization alongside fungal-bacterial interactions in vivo. The most commonly isolated fungus from the human vaginal tract is Candida albicans, which colonizes upwards of 70% of individuals16. C. albicans is polymorphic and can undergo morphogenesis to switch between budding yeast and filamentous morphotypes. Filamentation is associated with a distinct gene expression repertoire17,18. Some of these hyphal-specific genes have recently been reported to provide a fitness advantage in the context of polymicrobial colonization19,20,21.

Several studies have reported the co-isolation of C. albicans and GBS in human vaginal swabs, and our research group has recently found that individuals colonized with C. albicans have significantly higher rates of GBS carriage22. We have developed an in vivo system to study the tripartite interactions between commensal fungi, bacteria, and the host. Fungal studies in conventional mice typically require antibiotic treatment to overcome bacterial-mediated colonization resistance and establish persistent colonization23. To accomplish this without disrupting the ability of GBS to colonize, we treat mice with an antibiotic cocktail, inoculate them with C. albicans, and after colonization is established, we remove the antibiotic exposure to allow GBS to subsequently colonize. This has been optimized so that both C. albicans and GBS colonize the vaginal tract at similar burdens and on similar timelines, enabling direct interactions to occur within the vaginal tract. This protocol describes a method to prime mice with antibiotics, deliver 17β-estradiol, establish colonization with both fungi and bacteria, and collect lavage and tissue samples for subsequent analysis.

Protocol

The animal work presented in this manuscript was approved by and performed in accordance with the Institutional Animal Care and Use Committee (IACUC) of the University of Colorado Anschutz under protocol #00316. In the timeline below, day 0 refers to the day of S. agalactiae inoculation.

1. Administer antibiotics before inoculation.

  1. Make a concentrated solution of 50 mg/mL each of ampicillin, neomycin, and gentamycin dissolved in mouse drinking water. Use a 0.22 µm filter to sterilize the antibiotic solution.
  2. Add the antibiotic solution to the mouse drinking water at a 1:100 dilution by adding 1 mL of the solution for every 99 g of water.
  3. After 7 days, replace the water by repeating steps 1.1 and 1.2.
  4. Maintain the antibiotics for at least 7–10 days prior to beginning the experiment, but do not exceed 14 total days of antibiotic treatment.

2. Synchronize mouse estrus cycles (day -3).

  1. Add 0.5 mg per mouse of 17β-estradiol, plus a small excess to account for adherence of the suspension to tube walls, to a sterile 50 mL conical tube. Tightly cap the conical tube and briefly vortex to break up clumps.
    CAUTION: Because 17β-estradiol may enter the body through dermal or mucosal contact, handle this reagent carefully and use appropriate personal protective equipment.
  2. Use a 0.45 µm filter to sterilize 100 µL of sesame oil per 0.5 mg of 17β-estradiol directly into the conical tube containing 17β-estradiol. The concentration is 5 mg/mL. Tightly cap and vortex the solution thoroughly to create a homogeneous suspension.
  3. Prepare sterile 1 mL syringes (one per mouse) by pulling the plunger down to the 0.15 mL marker and propping them up such that the plunger faces down, and the luer lock tip faces up.
  4. Draw up the 17β-estradiol suspension into a new sterile 10 mL syringe with a sterile 18G 1.5” needle and use this to fill the prepared 1 mL syringes with 100 µL of the suspension.
  5. Cap each 1 mL syringe with a sterile 26G 0.5” needle.
  6. Prepare a 100 µL sesame oil suspension containing 0.5 mg of 17β-estradiol, and administer one dose to each mouse by intraperitoneal injection one day prior to C. albicans inoculation.

3. Inoculate mice with C. albicans (day -2).

  1. One day prior to inoculation (the same day as 17β-estradiol administration), grow a 5 mL overnight culture of C. albicans in Yeast Peptone Dextrose Broth in a 30 °C incubator shaking at 250 rpm.
  2. On the day of inoculation, transfer an aliquot of the overnight culture into a sterile microcentrifuge tube and centrifuge at ≤10,000 × g for 7–8 s or until a pellet forms.
  3. Remove the supernatant and resuspend the pellet in sterile phosphate-buffered saline (PBS).
  4. Use a hemocytometer to calculate the concentration of the suspension.
  5. Transfer an aliquot of the suspension to a new sterile microcentrifuge tube, re-pellet, and suspend in fresh sterile PBS to reach a final concentration of 109 C. albicans cells/mL in a final volume of at least 10 µL per mouse. For example, if the concentration is 2 × 108 cells/mL and there are 10 mice, pellet 500 µL and resuspend in 100 µL.
  6. Intravaginally inoculate each mouse with 10 µL of C. albicans suspended in PBS at 109 cells/mL (107 cells/mouse).
    1. Restrain each mouse by securing the skin on the scruff of the neck between the thumb and index finger of the non-dominant hand, then rotating the wrist such that the back of the mouse rests in the palm of the hand. Use the ring and pinky fingers to immobilize the tail.
    2. Draw up 10 µL of the C. albicans suspension into a sterile pipette tip. Insert the tip approximately 5–10 mm into the vaginal lumen, adjusting the depth based on the anatomy of the mouse.
    3. Dispense the C. albicans inoculum into the vagina and withdraw the pipette tip.
    4. After releasing the scruff, hold the mouse by the tail to elevate the hindquarters. Walk the mouse on its two front paws for approximately 1 min or until the C. albicans inoculum is no longer visible.

4. Replace drinking water (day -1).

  1. One day following C. albicans inoculation, which coincides with the day prior to S. agalactiae inoculation, move the mice into clean cages.
  2. Ensure that the mice are provided with fresh water without antibiotics.

5. Quantify C. albicans burden in the vaginal lumen (day -1).

  1. On day -1, prepare one sterile microcentrifuge tube and 100 µL of sterile PBS per mouse.
  2. Restrain each mouse as described in step 3.6.1.
  3. Draw up 50 µL of PBS into a sterile pipette tip. Insert the tip approximately 1–5 mm into the vaginal lumen and pipette the entire volume of PBS up and down 3–5 times. Dispense the PBS into a sterile microcentrifuge tube.
  4. Repeat step 5.3 to generate a total of 100 µL of lavage fluid.
  5. Vortex the microcentrifuge tube, then perform 10-fold serial dilutions through a 1:10,000 dilution in sterile PBS.
  6. Plate 10 µL of the diluted suspensions and 25 µL of the undiluted lavage fluid on Candida-selective chromogenic agar plates.
  7. Incubate the plates at 30 °C for 48 h. C. albicans colonies will appear smooth and teal or green in color.

6. Inoculate mice with S. agalactiae (day 0).

  1. One day prior to inoculation (the same day as drinking water replacement), grow a 3 mL overnight culture of S. agalactiae in Todd Hewitt Broth (THB) in a static 37 °C incubator.
  2. On the day of inoculation, dilute the overnight culture 1:10 in 4 mL of THB and incubate in a static 37 °C incubator until it reaches mid-log phase (optical density at 600 nm [OD600] of 0.4–0.5) in approximately 1.5–2 h.
  3. Transfer the mid-log culture to a sterile 15 mL conical tube and centrifuge at 3,000 × g for 5 min or until a pellet forms.
  4. Remove the supernatant by aspiration, then suspend the pellet in 300 µL of sterile PBS.
  5. Use the resuspended pellet to prepare at least 100 µL of S. agalactiae suspension per mouse, plus an additional 500 µL, in sterile PBS at OD600 0.4 (approximately 108 colony-forming units [CFU]/mL; ≥1.5 mL total for 10 mice).
    NOTE: Some S. agalactiae strains may differ in their OD600-to-CFU/mL ratio. Determine which OD600 corresponds to 108 CFU/mL in advance.
  6. Transfer the suspension to a new sterile 15 mL conical tube and centrifuge at 3,000 × g for 5 min or until a pellet forms.
  7. Aspirate the supernatant and resuspend the pellet in 1/10th of the initial volume of sterile PBS (if 1.5 mL of the suspension was pelleted, resuspend in 150 µL). This suspension will be at 109 CFU/mL and serve as the bacterial inoculum.
  8. Serially dilute and plate 50 µL of the inoculum on Todd Hewitt Agar, then incubate the plates at 37 °C for 24 h to determine the exact inoculum concentration.
  9. Intravaginally inoculate each mouse with 10 µL of S. agalactiae suspended in PBS at 109 CFU/mL (107 CFU/mouse) using the same technique as in steps 3.6.1–3.6.4.

7. Quantify C. albicans and S. agalactiae burdens in the vaginal lumen (beginning day +1).

  1. Beginning one day after S. agalactiae inoculation, collect 100 µL of vaginal lavage fluid and serially dilute it using the same technique as in steps 5.1–5.5.
  2. Duplicate plate 10 µL of the diluted suspensions and 25 µL of the undiluted lavage fluid on both Candida-selective and StrepB-selective chromogenic agar plates.
  3. Incubate the Candida plates at 30 °C for 48 h. C. albicans colonies will appear smooth and teal or green in color.
  4. Incubate the StrepB plates at 37 °C for 24 h. S. agalactiae colonies will appear smooth and pink or mauve in color.
  5. Repeat steps 7.1–7.4 daily, or at an appropriate time interval, to monitor the microbial burdens in the vaginal lumen.

8. Quantify C. albicans and S. agalactiae burdens in the female genital tract tissues (experimental endpoint).

  1. Prepare three 2 mL screw-cap tubes filled with approximately 0.4–0.5 g of 1 mm zirconia beads and 500 µL of sterile PBS per mouse to collect tissues (vagina, cervix, and uterus). Also prepare one sterile microcentrifuge tube and 100 µL of sterile PBS per mouse to collect lavage. Record the weight of each screw-cap tube to use as a reference when calculating the tissue weights.
  2. Euthanize the mouse using an institutionally approved method such as CO2 asphyxiation and cervical dislocation. Use 70% ethanol to sterilize scissors and forceps.
  3. Lift the hind end of the mouse by the tail and collect vaginal lavage using the technique in steps 5.3–5.4.
  4. Spray the abdomen of the mouse with 70% ethanol and use the sterilized scissors to open the abdominal cavity. Use forceps to lift the skin back and displace the gastrointestinal tract to expose the genital tract tissues.
  5. Use the sterilized scissors to remove the bladder as well as the visceral fat and membranes that obscure the uterus. Separate the uterine horns from the ovaries by cutting between the uterine body and each ovary.
  6. Using sterilized forceps, grip the vaginal tract at the point inferior to the cervix. Lift until the entire vaginal tract is visible and use sterilized scissors to make a transverse cut as close to the vulva as possible, avoiding any skin tissue. Remove the entire genital tract and place in a sterile 10 cm Petri dish.
  7. Use a sterile razor blade to make two transverse cuts inferior and superior to the cervix, separating the vagina, cervix, and uterus. Mince each of the tissues by making an additional 2–5 sagittal and transverse cuts across each. Place each tissue into its respective prepared screw-cap tube.
  8. Weigh each screw-cap tube and calculate the weight of each tissue by subtracting the value from step 8.1. Tightly cap each tube, bead-beat at 3,450 rpm for 1 min, place the tubes on ice for 1 min, then bead-beat at 3,450 rpm for 1 min again.
  9. Serially dilute and plate each tissue homogenate and lavage sample using the method described in steps 5.5–5.6. Incubate the plates as described in steps 7.3–7.4.

Results

To characterize GBS and C. albicans vaginal colonization, mice were intravaginally inoculated with C. albicans strain SC5314 or a PBS vehicle control, followed by inoculation with GBS strain COH1. This strain represents hypervirulent capsular serotype III, sequence type 17 isolates that are most frequently associated with neonatal invasive disease and meningitis1. Mice not colonized with GBS one day post inoculation were excluded from analysis. Over a four-day experimental timeline, mice colonized with GBS alone progressively clear GBS from the vaginal lumen. The majority of mice co-colonized with C. albicans retain high GBS burdens (Figure 1A,B). C. albicans also colonizes the mouse vaginal lumen for approximately four days before it is cleared (Figure 1C). Previous work utilizing this model has demonstrated that GBS burdens in the FGT tissues, including the vagina, cervix, and uterus, are also higher in co-colonized mice compared to mono-colonized animals22.

GBS colonization graphs; statistical comparison, survival curve, CFU/mL log scale data analysis.
Figure 1. Quantifying Group B Streptococcus colonization. CD-1 mice were colonized with C. albicans (SC5314) or treated with phosphate-buffered saline as a control, followed by vaginal inoculation with GBS (COH1). (A) GBS burdens in vaginal lavage fluid, medians shown. (B) Percentage of mice colonized over time assessed by GBS detection in vaginal lavage fluid. (C) C. albicans burdens in vaginal lavage fluid, medians shown. Three independent experiments. n = 26-27 mice per group. * p < 0.05; ** p ≤ 0.01; *** p ≤ 0.001 by multiple t-tests with Holm-Sidak’s multiple comparisons test (A) or log-rank test (B). Abbreviations: GBS = Group B Streptococcus, Ca = Candida albicans, CFU = colony-forming units. Please click here to view a larger version of this figure.

Discussion

GBS vaginal colonization poses a critical risk to maternal-fetal and neonatal health. In vivo models are imperative for understanding the determinants of colonization and for developing preventative therapeutics. In this manuscript, we describe a murine model to vaginally co-colonize mice with GBS and C. albicans, assess microbial burdens in the vaginal lumen and FGT, and collect relevant tissues for further downstream analyses. This model has been developed to study fungal-bacterial and host-microbe interactions in the context of polymicrobial vaginal colonization. Our research group has utilized this model to elucidate the mechanisms that contribute to C. albicans promoting GBS vaginal colonization and ascending infection22.

Animal models have been successfully used to understand host-microbe interactions; however, incorporating polymicrobial communities into these models can pose several unique challenges. The primary goals in the development of this model were to establish fungal colonization, allow time for C. albicans to form hyphae, and inoculate with GBS to monitor colonization dynamics before either microbe was cleared by the host. As a polymorphic fungus, C. albicans can respond to a number of host-related environmental cues by initiating hyphal morphogenesis17. Filamentation in vivo was prioritized in this model due to accumulating evidence that hyphal-specific interactions with GBS drive symbiosis in the FGT. Using a C. albicans strain expressing NRG1 under the control of the repressible TetO promoter, our research group has previously shown that GBS displays higher co-aggregation and higher epithelial adherence with hyphal-locked compared to yeast-locked C. albicans22. Co-aggregation with C. albicans and association with human vaginal epithelial cells are important determinants of GBS fitness in this host environment. Additional bacteria including Staphylococcus aureus, Pseudomonas aeruginosa, and diverse Streptococcus species have been shown to modulate morphogenesis-associated gene expression in C. albicans as well as interact preferentially with hyphae24. In the future, interrogating the implications of filamentation in vivo can be addressed by using this model to colonize mice with hyphal- or yeast-locked C. albicans strains.

Existing murine models involving C. albicans intravaginal inoculation have primarily focused on vulvovaginal candidiasis (VVC)25 and have been used to characterize the hyperinflammatory state associated with symptomatic infection26. Our model, described in this manuscript, more closely represents a state of colonization. Previous work from our research group has demonstrated that, using this model, co-colonization with C. albicans decreases the abundance of pro-inflammatory cytokines IL-6, CXCL1, CXCL2, and TNFα compared to colonization with GBS alone, and that the levels of these proteins in co-colonized mice are comparable to those in naïve animals22. This indicates that we do not observe the hyperinflammatory environment that is characteristic of VVC, despite fungal filamentation in the vaginal lumen. Outbred CD-1 mice have been reported to be resistant to VVC, possibly due to adequate immune-mediated control of fungal outgrowth27. Furthermore, CD-1 mice initiate robust immune signaling upon intravaginal GBS inoculation that is sufficient to control the bacterial population within approximately one week in the absence of C. albicans28,29,30, although we have previously investigated GBS vaginal colonization in other mouse strains12. The model described in this manuscript reflects the transient nature of both C. albicans and GBS colonization that is observed in humans. Additionally, there is high variability across humans in both pH and microbiome composition. This model has near-neutral pH conditions (~6.5)31. The CD-1 vaginal microbiome is dominated by Enterobacteriaceae and Proteus species13, which are associated with the same vaginal microbial community state types as GBS9,32.

By treating mice with antibiotics and a single dose of 17β-estradiol prior to fungal inoculation, we prime the environment to be receptive to C. albicans, facilitating colonization. Estradiol treatment additionally promotes reproducibility across experiments by synchronizing the estrus stage, and thus the local immune environment, at the time of inoculation. We synchronize mice to the proestrus stage at C. albicans inoculation, which coincides with decreased neutrophil abundance and has been shown to promote fungal burdens in the FGT33,34. Removing the antibiotics and replacing them with fresh drinking water early in the experimental timeline not only facilitates GBS colonization but also allows native microbes to repopulate the vaginal tract. Furthermore, limiting the estradiol treatment to a single early dose allows mice to resume typical estrus cycle dynamics, although continuous estradiol administration has been shown to promote colonization12. The immune cell composition in the murine FGT varies throughout the estrus cycle, and mucosal immune responses are highly responsive to sex steroid hormones35,36,37. Thus, we circumvent the confounding variable of repeated estradiol administration to specifically interrogate the impacts of microbial colonization on the host environment.

The data presented in this study represent the C. albicans strain SC5314 and the GBS strain COH1. We selected SC5314 to establish the model due to its frequent use in mycology, the abundance of genetic tools available in this background, and an established body of knowledge on its biology and dynamics in vitro and in vivo. Our research group has also shown that C. albicans strain 529L can promote GBS burdens in the FGT22. GBS strain COH1 was utilized as a representative of the serotype III sequence type 17 lineage, which is associated with antimicrobial resistance and neonatal invasive disease1,2. This model uses selective and differential media to quantify the abundance of GBS and C. albicans in vaginal lavage and tissues. To adapt this model for use with other bacterial and fungal species, appropriate selective growth conditions would need to be optimized.

Fungal-bacterial interactions are emerging as important mediators of microbial lifestyles38,39. Several studies have demonstrated that interactions can considerably shape commensalism and virulence potential. Additionally, the host environment imposes unique pressures and can influence factors such as metabolic programming, transcription factor regulons, and antimicrobial resistance profiles. Thus, it is essential to use representative in vivo models to characterize how microbes interact with each other. The murine model described in this manuscript can be used to study how vaginal colonization with bacteria and fungi of interest can influence microbial burdens, gene expression, and the host response.

Disclosures

The authors have no conflicts of interest to report.

Acknowledgements

We thank the Office of Laboratory Animal Resources at the University of Colorado Anschutz, including the facility manager and the dedicated vivarium staff. This work is supported by NIH grants R01AI153332 and R21AI188719 to K.S.D.

Materials

List of materials used in this article
NameCompanyCatalog NumberComments
0.22 µm filterSigma-AldrichSLGSR33SB
0.45 µm filterSigma-AldrichSLHAR33SB
1 mL luer lock syringeMcKesson16-S1C
1.0 mm zirconia beadsResearch Products International9835
10 mL luer lock syringeMcKesson16-S10C
17β-estradiolSigma-AldrichE8875CAUTION: 17β-estradiol can be absorbed through skin and mucosal surfaces. Wear appropriate personal protective equipment (PPE) and exercise caution while using.
18G 1.5" needleBD305196
2 mL screw-cap tubeFisher Scientific02-681-374
26G 0.5" needleMcKesson16-N2605
AgarAlpha BiosciencesA01-102
AmpicillinResearch Products InternationalA40040
CHROMagar CandidaCHROMagarCA222
CHROMagar StrepBCHROMagarSB282
GentamycinVWR Life Science0304
NeomycinResearch Products InternationalN20040
Phosphate-buffered salineFisher ScientificBP2944
Sesame oilSigma-AldrichS3547
Todd Hewitt BrothResearch Products InternationalT47500
Yeast Peptone Dextrose BrothBD242810

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Group B StreptococcusFemale Genital TractInterkingdom InteractionsAntibiotic TreatmentMicrobial Burden QuantificationHost Immune Response
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