Bacterial vaginosis (or BV for short) is the most common vaginal infection among women of childbearing age. In fact, it is estimated that one in three women worldwide will get BV at some time in their life. Common signs of BV include unusual vaginal discharge, malodor, and vaginal irritation; however, some women may have no symptoms. Treatment options for women with BV are still limited and frequently ineffective in the long-term with a high rate of disease recurrence. Certainly, BV remains a great enigma in women’s health.

What is BV and why is it important we study it? Bacterial vaginosis results from imbalance in bacteria residing in a woman’s vagina and cervix¹. In healthy reproductive-age women, these microbial communities (referred to as the cervicovaginal microbiota) are typically dominated by one or few Lactobacillus species (with Lactobacillus crispatus being associated with optimal health). In women with BV, these protective lactobacilli are depleted and overgrown by multiple anaerobes (bacteria that do not require oxygen to live), such as Gardnerella, Prevotella, Atopobium, Sneathia, Megasphaera, Mobiluncus, and others. Risk factors for BV include unprotected sex, multiple sex partners or a new partner, intrauterine device usage, smoking, and douching. Still, we do not fully understand the exact cause of this disease and how it progresses over time in an individual. Remarkably, multiple clinical studies have demonstrated a strong association of BV with several adverse gynecologic and reproductive health outcomes. Overall, women with BV are at higher risk of sexually transmitted infections (STIs), such as HIV, gonorrhea, chlamydia, trichomoniasis), as well as pelvic inflammatory disease, cervical cancer², and preterm birth, if pregnant. However, our understanding of this connection between BV and theses conditions is still limited.

In order to fill in this gap, we tested how key vaginal microbes change the local microenvironment and, in consequence, make women more susceptible to STIs, preterm birth and other adverse outcomes, using our three-dimensional (3D) cell culture model of the human cervix³ and state-of-art “omics” technologies. The Herbst-Kralovetz laboratory pioneered the development of female reproductive tract models to study host-microbe interactions. These advanced 3D tissue models are based on the rotating wall vessel bioreactor technology⁴, originally invented by scientists and engineers at NASA. Importantly, our 3D human cervical model³ and other genital models^5,6 demonstrate a remarkable resemblance to human tissue and provide a powerful tool to study host-microbe interactions within the reproductive tract^7-10.

In our study, we colonized 3D human cervical models with Lactobacillus crispatus as representative health-associated commensals and four key bacterial species frequently isolated from women with BV: Gardnerella vaginalis, Prevotella bivia, Atopobium vaginae (recently renamed Fannyhessea vaginae), and Sneathia amnii (recently renamed Sneathia vaginalis). Since BV is referred to as a polymicrobial infection (infection with more than one organism), we mimicked it by creating a “cocktail” consisting of a mix of all four tested BV anaerobes and infecting our models with this polymicrobial community (Figure 1). Following infections, we used Luminex-based immunoassays and global metabolomics approach to define how tested bacteria, alone or in polymicrobial mixtures, shape the immunometabolic landscape of the human cervix. These high-throughput technologies allowed us to quantify key proteins and small molecules (metabolites) that regulate immune responses, inflammation, and other processes in the human body that contribute to health or disease.

We found that members of cervicovaginal microbiota play unique functions in the cervix that relate to a healthy physiological balance or pathological changes. Regarding health-associated lactobacilli, our study showed that Lactobacillus crispatus reinforces a protective microenvironment (Figure 2). We showed that colonization with this bacterium does not result in inflammation or any other tissue-damaging responses. In addition, we demonstrated that Lactobacillus crispatus contributes to production of antimicrobial compounds, for example PLA (short for phenyllactic acid). This biochemical has been previously found in honey and fermented food and can inhibit the growth of other bacteria and fungi. This suggests that vaginal lactobacilli produce PLA to outcompete other vaginal bacteria or fungi for their own benefit. At the same time, PLA likely inhibits invading sexually transmitted pathogens, which benefits a woman. This mutualistic relationship between vaginal lactobacilli and human host warrants further investigations.

In contrast to lactobacilli, BV anaerobes created potentially harmful microenvironments (Figure 2). Yet, the contributions of each tested bacterial species to those detrimental alterations of 3D models differed. Atopobium vaginae, followed by Sneathia amnii, induced the highest level of proteins and metabolites related to inflammation. The inflammation associated with these bacteria might facilitate STIs, such as HIV, through recruitment and activation of specialized immune cells. In addition, infection with Sneathia amnii led to increased levels of biochemicals related to oxidative stress, a condition characterized by release of damaging oxygen free radicals, which might promote development or progression of cervical cancer. On the other hand, Gardnerella vaginalis and Prevotella bivia did not cause robust inflammatory responses but impacted the physical and chemical properties of the epithelial barrier that prevents harmful bacteria, toxins, and other unwanted substances from entering the body. We found evidence that Prevotella bivia produces ammonia, which impairs the normally acidic vaginal microenvironment and increases pH. BV anaerobes also uniquely impacted polyamines (chemicals responsible for malodor in women with BV) that also contribute to abnormal pH and might favor acquisition of STIs, such as gonorrhea. Finally, Gardnerella and Prevotella also showed an indication of collagen and mucus degradation in our models, which links BV anaerobes to preterm birth, ascension to the uterus, and pelvic inflammatory disease.

Fascinatingly, our “cocktail” of four BV anaerobes led to the most robust metabolic activity in our 3D models, which likely resulted from synergistic microbe-microbe interactions within the polymicrobial community. Infecting our models with this “synthetic” microbial community created biomarker profiles similar to profiles of cervical and vaginal specimens collected from women with BV in previous clinical studies. Thus, our well-characterized system can successfully be used as a preclinical model to study BV, host-microbe interactions, the vaginal microbiome, as well as STIs. Overall, this study identified mechanisms by which key vaginal bacteria contribute to adverse women’s health outcomes and paves the way for future studies aiming to unravel the enigma of BV.

To learn more about our study, read the full article in NPJ Biofilms and Microbiomes: Bacterial vaginosis and health-associated bacteria modulate the immunometabolic landscape in 3D model of human cervix.

References

1          Muzny, C. A., Łaniewski, P., Schwebke, J. R. & Herbst-Kralovetz, M. M. Host–vaginal microbiota interactions in the pathogenesis of bacterial vaginosis. Curr Opin in Infect Dis 33, 59-65, doi:10.1097/QCO.0000000000000620 (2020).

2          Łaniewski, P., Ilhan, Z. E. & Herbst-Kralovetz, M. M. The microbiome and gynaecological cancer development, prevention and therapy. Nat Rev Urol 17, 232-250, doi:10.1038/s41585-020-0286-z (2020).

3          Radtke, A. L., Quayle, A. J. & Herbst-Kralovetz, M. M. Microbial products alter the expression of membrane-associated mucin and antimicrobial peptides in a three-dimensional human endocervical epithelial cell model. Biol Reprod 87, 132, doi:10.1095/biolreprod.112.103366 (2012).

4          Radtke, A. L. & Herbst-Kralovetz, M. M. Culturing and applications of rotating wall vessel bioreactor derived 3D epithelial cell models. J Vis Exp, doi:10.3791/3868 (2012).

5          Hjelm, B. E., Berta, A. N., Nickerson, C. A., Arntzen, C. J. & Herbst-Kralovetz, M. M. Development and characterization of a three-dimensional organotypic human vaginal epithelial cell model. Biol Reprod 82, 617-627, doi:10.1095/biolreprod.109.080408 (2010).

6          Łaniewski, P., Gomez, A., Hire, G., So, M. & Herbst-Kralovetz, M. M. Human three-dimensional endometrial epithelial cell model to study host interactions with vaginal bacteria and Neisseria gonorrhoeae. Infect Immun 85, doi:10.1128/IAI.01049-16 (2017).

7          Doerflinger, S. Y., Throop, A. L. & Herbst-Kralovetz, M. M. Bacteria in the vaginal microbiome alter the innate immune response and barrier properties of the human vaginal epithelia in a species-specific manner. J Infect Dis 209, 1989-1999, doi:10.1093/infdis/jiu004 (2014).

8          Gardner, J. K. et al. Interleukin-36gamma is elevated in cervicovaginal epithelial cells in women with bacterial vaginosis and in vitro after infection with microbes associated with bacterial vaginosis. J Infect Dis 221, 983-988, doi:10.1093/infdis/jiz514 (2020).

9          Ilhan, Z. E., Łaniewski, P., Tonachio, A. & Herbst-Kralovetz, M. M. Members of Prevotella genus distinctively modulate innate immune and barrier functions in a human three-dimensional endometrial epithelial cell model. J Infect Dis, doi:10.1093/infdis/jiaa324 (2020).

10        Salliss, M. E., Maarsingh, J. D., Garza, C., Łaniewski, P. & Herbst-Kralovetz, M. M. Veillonellaceae family members uniquely alter the cervical metabolic microenvironment in a human three-dimensional epithelial model. NPJ Biofilms Microbiomes 7, 57, doi:10.1038/s41522-021-00229-0 (2021).