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.
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Method Article
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.
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.
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.
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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.
2. Synchronize mouse estrus cycles (day -3).
3. Inoculate mice with C. albicans (day -2).
4. Replace drinking water (day -1).
5. Quantify C. albicans burden in the vaginal lumen (day -1).
6. Inoculate mice with S. agalactiae (day 0).
7. Quantify C. albicans and S. agalactiae burdens in the vaginal lumen (beginning day +1).
8. Quantify C. albicans and S. agalactiae burdens in the female genital tract tissues (experimental endpoint).
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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 timeli...
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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 in...
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The authors have no conflicts of interest to report.
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.
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| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| 0.22 µm filter | Sigma-Aldrich | SLGSR33SB | |
| 0.45 µm filter | Sigma-Aldrich | SLHAR33SB | |
| 1 mL luer lock syringe | McKesson | 16-S1C | |
| 1.0 mm zirconia beads | Research Products International | 9835 | |
| 10 mL luer lock syringe | McKesson | 16-S10C | |
| 17β-estradiol | Sigma-Aldrich | E8875 | CAUTION: 17β-estradiol can be absorbed through skin and mucosal surfaces. Wear appropriate personal protective equipment (PPE) and exercise caution while using. |
| 18G 1.5" needle | BD | 305196 | |
| 2 mL screw-cap tube | Fisher Scientific | 02-681-374 | |
| 26G 0.5" needle | McKesson | 16-N2605 | |
| Agar | Alpha Biosciences | A01-102 | |
| Ampicillin | Research Products International | A40040 | |
| CHROMagar Candida | CHROMagar | CA222 | |
| CHROMagar StrepB | CHROMagar | SB282 | |
| Gentamycin | VWR Life Science | 0304 | |
| Neomycin | Research Products International | N20040 | |
| Phosphate-buffered saline | Fisher Scientific | BP2944 | |
| Sesame oil | Sigma-Aldrich | S3547 | |
| Todd Hewitt Broth | Research Products International | T47500 | |
| Yeast Peptone Dextrose Broth | BD | 242810 |
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