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. Author manuscript; available in PMC: 2020 Jun 2.
Published in final edited form as: Curr Opin Infect Dis. 2020 Feb;33(1):59–65. doi: 10.1097/QCO.0000000000000620

Host-Vaginal Microbiota Interactions in the Pathogenesis of Bacterial Vaginosis

Christina A Muzny 1,*, Paweł Łaniewski 2, Jane R Schwebke 1, Melissa M Herbst-Kralovetz 2
PMCID: PMC7265982  NIHMSID: NIHMS1590758  PMID: 31789672

Abstract

Purpose of Review

The etiology of BV, the most common cause of vaginal discharge in women, remains controversial. We recently published an updated conceptual model on BV pathogenesis, focusing on the roles of G. vaginalis and Prevotella bivia as early colonizers and Atopobium vaginae and other BVAB as secondary colonizers in this infection. In this paper, we extend the description of our model to include a discussion on the role of host-vaginal microbiota interactions in BV pathogenesis.

Recent Findings

Although G. vaginalis and P. bivia are highly abundant in women with BV, neither induce a robust inflammatory response from vaginal epithelial cells. These early colonizers may be evading the immune system while establishing the BV biofilm. Secondary colonizers, including A. vaginae, Sneathia spp., and potentially other BVAB are more potent stimulators of the host immune response to BV and likely contribute to its signs and symptoms as well as its adverse outcomes.

Summary

Elucidating the etiology of BV has important implications for diagnosis and treatment. Our current BV pathogenesis model provides a framework for key elements that should be considered when designing and testing novel BV diagnostics and therapeutics.

Keywords: Bacterial vaginosis, biofilm, Gardnerella vaginalis, Prevotella bivia, Atopobium vaginae, Sneathia species, host immune response

Introduction

Bacterial vaginosis (BV) is a vaginal dysbiosis resulting from displacement of lactic-acid producing Lactobacillus spp. with high concentrations of facultative and strict anaerobic bacteria including Gardnerella vaginalis, Prevotella spp., Atopobium vaginae, Sneathia spp., and other BV-associated bacteria (BVAB). A notable feature is the appearance of a polymicrobial biofilm on vaginal epithelial cells (1). BV is the most common cause of vaginal discharge globally, with an estimated annual economic burden of $4.8 billion dollars (2). Women with BV are at increased risk for adverse birth outcomes, gynecologic sequelae, and acquisition of HIV and other sexually transmitted infections (STIs) (3). BV is difficult to treat and has a high recurrence rate (4).

The epidemiology of BV strongly favors sexual transmission (5). However, the etiology remains controversial. G. vaginalis, a facultative anaerobe, is present in 95–100% of women with BV (6) and originally thought to be the single etiologic agent (7). G. vaginalis has unique characteristics compared to other BVAB that could allow it to function as an early colonizer on the vaginal epithelium (8, 9). G. vaginalis is able to effectively displace lactobacilli and adhere to vaginal epithelial cells (9) and has an increased propensity for biofilm formation compared to other BVAB (8). However, recent data suggest that G. vaginalis may be necessary, but not sufficient, for BV development (10), as colonization does not always lead to BV (11). Thus, other BVAB may also play an important role in BV pathogenesis.

Earlier genomic studies of G. vaginalis found that this genus consists of four non-recombining groups/clades of organisms with distinct gene pools and genomic properties (12). More recently, whole genome sequence analysis indicated 13 species exist within the genus Gardnerella (13). Correspondingly, genomic studies coupled with phenotypic characterizations have shown differences in pathogenic potential between G. vaginalis strains (14, 15). Healthy women may be colonized by G. vaginalis strains with lower pathogenic potential, while strains with higher pathogenic potential may be involved in BV development. This hypothesis is supported by data from a study investigating the association of behavioral practices and Nugent score with G. vaginalis clade distribution in women-who-have-sex-with-women (16). In this study, clades 1–3 and multi-clade (>2) communities were associated with BV by Nugent score. Clade 4 was neither associated with BV nor Lactobacillus-deficient microbiota. Clade 1 was associated with increasing number of recent sexual partners and clade 2 was associated with specific sexual behaviors. Overall, these data suggest that G. vaginalis clades have varying levels of pathogenicity, with acquisition through sexual activity.

Based upon our prospective study (10), a mouse model study (17), and an updated literature review (9, 18–20), we recently modified our prior conceptual model on BV pathogenesis which mainly involved G. vaginalis (21), to now focus on the roles of G. vaginalis and Prevotella bivia as early colonizers and Atopobium vaginae and other BVAB as secondary colonizers in BV (22). The proposed steps of BV development in this model include: (1) strains of G. vaginalis with higher pathogenic potential displace lactobacilli and initiate biofilm formation on the vaginal epithelium, (2) synergy between G. vaginalis and P. bivia (normally present in very low concentrations, acquired from maternal and environmental sources) occurs on the vaginal epithelium with production of metabolites facilitating their growth, (3) vaginal sialidase and other enzymes, produced by G. vaginalis and P. bivia, promote breakdown of the mucous layer of the vaginal epithelium, and (4) loss of the protective mucous layer leads to increased adherence of secondary colonizers, including A. vaginae, to the mature, polymicrobial BV biofilm (22).

Interestingly, although G. vaginalis and P. bivia are highly abundant in women with BV (10), neither induce a robust inflammatory response from vaginal epithelial cells (17, 23). Other BVAB, including A. vaginae (24, 25), may be more potent stimulators of the host immune response to BV and contribute to its signs and symptoms in addition to adverse outcomes (26). Here we provide a discussion of our current opinions regarding the role of the host immune response in the pathogenesis of BV. We extend the description of our BV conceptual model to include a discussion of the immune barrier of an optimal vaginal microbiota and the role of host-vaginal microbiota interactions in BV pathogenesis, focusing on G. vaginalis, P. bivia, A. vaginae, and Sneathia spp. (Figure 1). Next, we discuss implications for BV diagnosis and treatment, outlining areas requiring additional research.

Figure 1. Depiction of putative model for the establishment of BV and immunologic/physiological changes related to host-microbe interactions.

Figure 1.

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A. In a healthy state the vaginal microbiome is dominated by Lactobacillus species. Lactobacilli produce lactic acid, which lowers the vaginal pH and protects against invading pathogens and pathobionts. Epithelial cells constitutively produce low levels of antimicrobial peptides (e.g. SLPI) and cytokines. Additionally, epithelial cells and immune cells contribute to homeostasis by producing anti-inflammatory cytokines (e.g. IL-1RA). B. Vaginal dysbiosis begins with initial colonization with the facultative anaerobe, Gardnerella vaginalis, usually following a sexual exposure. G. vaginalis colonizes the vaginal epithelial cells, replaces lactobacilli, and provides scaffolds for biofilm formation. Following G. vaginalis colonization, the strict anaerobe, Prevotella bivia, is recruited to the biofilm. G. vaginalis and P. bivia support each other’s growth through ammonia and amino acid metabolism. Both G. vaginalis and P. bivia are capable of producing enzymes, e. g. sialidase, which may contribute to mucus degradation and barrier disruption. No overt inflammation is observed, which suggest that these bacterial species are able to evade host immune response through unknown mechanisms. C. Other secondary colonizers, e.g. Atopobium vaginae and Sneathia spp., are recruited to the biofilm. At this stage, exfoliation of epithelial cells coated with the polymicrobial biofilm occurs. These “clue cells” can be detected in wet mounts of vaginal fluid and are included in the Amsel criteria. Production of biogenic amines and other metabolites produced by BV-associated bacteria contribute to elevated vaginal pH and BV symptoms such as fishy odor. Epithelial cells and recruited immune cells produce pro-inflammatory cytokines and chemokines, which could lead to genital inflammation.

The Immune Barrier of an Optimal Vaginal Microbiota

Lactobacilli colonizing the lower female reproductive tract (FRT) play an important role in protection against invading pathogens through direct (production of bactericidal compounds and metabolites) and indirect (modulation of vaginal immune barrier) mechanisms (27). However, not all vaginal Lactobacillus species benefit the host equally. Data suggest that Lactobacillis crispatus is the optimal species associated with vaginal health whereas L. iners may be associated with dysbiosis and colonization with BVAB (25). The role of other common vaginal lactobacilli such as L. jensenii and L. gasseri is less studied and requires additional research.

A key protective mechanism of vaginal lactobacilli includes the production of lactic acid through fermentation of glycogen by-products, which acidify the cervicovaginal micro-environment (pH<4.5) (Figure 1A). Several in vitro studies have demonstrated that lactic acid is a potent antiviral and bactericidal compound, inhibiting replication/growth of genital STI pathogens and pathobionts, including Chlamydia trachomatis (28), Neisseria gonorrhoeae (29), group B Streptococcus (30), and HIV (31). These micro-organisms are more sensitive to lactic acid than hydrochloric acid, indicating that a low pH environment is necessary, but not sufficient, for this inhibitory effect. In the lower FRT, lactic acid exists in both D- and L-forms and the ratio of D- and L-isomers depends on the predominant Lactobacillus spp. (32). A recent study demonstrated that D-lactic acid, produced by L. crispatus but not L. iners, was more protective against chlamydia (28). This suggests that production of specific lactic acid isoforms may contribute to differential protective capacities of vaginal Lactobacillus spp. against STIs, including BVAB (e.g. G. vaginalis) that may be sexually transmitted. Other postulated protective mechanisms include hydrogen peroxide production by L. crispatus; however, it is still controversial whether this antimicrobial compound can be produced in vivo at inhibitory concentrations (33). Lastly, the protective effect of lactobacilli can be attributed to competitive exclusion, which is the ability to effectively compete with other micro-organisms for resources in the local micro-environment (34).

Colonization of the lower FRT with L. crispatus and other protective Lactobacillus spp. also results in low levels of vaginal epithelial cell activation. Multiple epidemiological studies have demonstrated that women with a L. crispatus-dominated vaginal microbiota exhibit low levels of pro-inflammatory cytokines [e.g. interleukin (IL)-1α, IL-1β] and chemokines [e.g. IL-8, interferon gamma inducible protein (IP-10), macrophage inflammatory protein (MIP)-3α] in their cervicovaginal secretions (35, 36). In a cross-sectional study investigating the role of the vaginal microbiota in cervical carcinogenesis, women with a Lactobacillus-dominant vaginal microbiota showed less evidence of genital inflammation measured by genital inflammatory scores (e.g. elevated levels IL-1α, IL-1β, IL-8, MIP-1β, MIP-3α, RANTES, TNFα) compared to women with a non-Lactobacillus-dominant vaginal microbiota (37).

Interestingly, colonization with L. iners does not provide the same mucosal immune quiescence as L. crispatus. Two independent studies have shown that L. iners dominance is associated with elevated levels of chemokines, including IP-10, MIP-3α, and monokine induced by gamma interferon (MIG), which may result in mucosal recruitment of CD4+ cells and increased risk of HIV (35, 38). In vitro studies, employing robust organotypic human three-dimensional (3-D) female reproductive tract epithelial cell models, validated these epidemiological findings and showed that L. crispatus does not significantly alter levels of pro-inflammatory cytokines (IL-1β, IL-6, TNFα), chemokines (IL-8, MIP-3α), or antimicrobial peptides [human β-defensin (hBD)-2 and secretory leukocyte protease inhibitor (SLPI)], resulting in low epithelial cell activation (25, 39, 40). Others have demonstrated that L. crispatus does not significantly change IL-8, RANTES, or SLPI levels in cervicovaginal monolayer cultures (41). In addition, metabolites produced by lactobacilli can modulate epithelial cell responses and stimulate secretion of anti-inflammatory cytokines, including IL-1 receptor antagonist (IL-1Ra), and low-level production of antimicrobial peptides (e.g. hBD-1, SLPI), resulting in mucosal homeostasis (42, 43).

The Role of Host-Vaginal Microbiota Interactions in BV Pathogenesis

BV is not characterized as a neutrophilic disease (27). Vaginal white blood cells are uncommon in BV, unless concomitant vaginal (Trichomonas vaginalis or vulvovaginal candidiasis) and/or cervical infection (e.g. C. trachomatis) is present (44). BV is also not commonly associated with pain, redness, or swelling typical of gross tissue inflammation (27). However, studies have reported elevated cytokine and chemokine levels (e.g., IL-1β, TNFα, IL-6, and IL-8) in vaginal washes from women with BV (45). Early colonizers such as G. vaginalis and P. bivia may actively inhibit the host inflammatory response in the vaginal epithelium, evading the immune system while establishing the BV biofilm (Figure 1B). This is corroborated by data from a mouse model study where P. bivia alone or in combination with G. vaginalis did not cause an increase in histological inflammation in vaginal tissue (17). In contrast, secondary colonizers of the BV biofilm (e.g., A. vaginae and other BVAB) may stimulate the host immune response in vaginal epithelial cells and contribute to the symptoms (e.g., vaginal discharge and odor) and signs (e.g., homogeneous, white, vaginal discharge) of BV (22) (Figure 1C). Related to these symptoms of BV are the metabolites produced by BVAB, including biogenic amines (46, 47).

A limited number of studies have examined mechanisms of the host response and immunity to key BVAB using in vitro or animal models (17). A recent study utilizing EpiVaginal tissues demonstrated that apical infection with G. vaginalis does not induce significant changes in levels of pro-inflammatory immune mediators (48) in accordance with a previous study showing that G. vaginalis does not induce IL-1β, IL-6 MIP-3α or TNFα in a human 3-D endometrial epithelial cell model (39). Regarding P. bivia and A. vaginae, Doerflinger et al demonstrated in a human 3-D vaginal epithelial cell model that A. vaginae induces a broad range of pro-inflammatory cytokines, chemokines, and antimicrobial peptides including IL-1β, IL-6, IL-8, MIP-3α, TNFα and hBD-2; whereas P. bivia significantly only induced IL-1β and MIP-3α (25). Subsequent unsupervised hierarchical clustering analysis of these data suggests that A. vaginae clusters separately from P. bivia based on their immune mediator profiles and are distinct from both L. iners and L. crispatus (23). Other reports have demonstrated that A. vaginae induces hBD-4, MIP-1β, Gro-α, and G-CSF and that the host immune response was Toll-like receptor (TLR) 2-dependent (24, 49). Interestingly, G. vaginalis and A. vaginae have also been found to amplify pro-inflammatory responses to T. vaginalis, whereas P. bivia suppressed these responses (50).

Other secondary BVAB besides A. vaginae may also stimulate the host immune response in vaginal epithelial cells. One report examining Sneathia spp. found that two species (S. sanguinegens and S. amnii) induced IL-1α, IL-1β, and IL-8, but not TNFα, in vaginal epithelial monolayer cultures (51). With regard to these microbes, A. vaginae or S. amnii alone and a polymicrobial cocktail of BVAB (G. vaginalis + P. bivia + A. vaginae + S. amnii) also induced IL-36γ, a novel cytokine belonging to the IL-1 superfamily, at a higher magnitude than other BVAB alone (G. vaginalis or P. bivia) in a human 3-D cervical model (23). Hierarchical clustering analysis revealed two main clusters. G. vaginalis and P. bivia did not exhibit robust inflammatory profiles and clustered with L. crispatus, while A. vaginae, S. amnii, and the polymicrobial cocktail formed a separate cluster defined by higher levels of inflammatory mediators (40). IL-36γ was recently shown to play a significant role in genital HSV-2 pathogenesis (40) and may be a key immune factor in the lower FRT and in BV (23).

Overall, these data support the hypothesis that G. vaginalis and P. bivia do not induce robust epithelial cell activation and that secondary colonizers, including A. vaginae, Sneathia spp., and other BVAB, are required to induce pro-inflammatory responses observed in women with BV. However, G. vaginalis and P. bivia might impact other components of the cervicovaginal epithelial barrier. A mouse model study revealed that G. vaginalis facilitates ascension of P. bivia to uterine horns (17). In addition, both bacteria contributed to sialidase activity, which plays an important role in mucus degradation. Interestingly, we have observed that not all Prevotella spp., similar to G. vaginalis strains, exhibit sialidase activity and that selected Prevotella spp. induced mucin production in our human 3-D endometrial epithelial model (manuscript in preparation). Furthermore, vaginal metabolites, such as lipids (e.g. long chain polyunsaturated fatty acids), are associated with both BVAB and genital inflammation, whereas anti-inflammatory nucleotides are associated with lactobacilli dominance (52). Interestingly, elevated levels of biogenic amines produced by BVAB do not correlate with genital inflammation (52). Overall, these data suggest that the pathogenic potentials of many BVAB are strain-specific. Robust longitudinal studies coupled with mechanistic studies utilizing human 3-D models are needed to identify unique mechanisms of pathogenesis for key BVAB and polymicrobial mixtures.

Conclusion: Implications for BV Diagnosis and Treatment

Elucidating the etiology of BV has important implications for diagnosis and treatment. In addition to the commonly used Amsel criteria and Nugent score, five highly sensitive and specific multiplex PCR tests are available for the diagnosis of BV in symptomatic women (BD Max™ Vaginal Panel (53), Hologic Aptima® BV (54), LabCorp NuSwab® VG (55), Quest Diagnostics™ SureSwab® Bacterial Vaginosis, and Medical Diagnostics Laboratory (MDL) OneSwab® (56)). These assays include various combinations of Lactobacillus spp. in addition G. vaginalis, A. vaginae, BVAB2, and Megasphaera-1 and −2. Based upon our conceptual model (Figure 1), it may be prudent to determine the level to which the addition of P. bivia and Sneathia spp. contributes to the sensitivity and specificity of these assays.

With regards to treatment, one aspect of the high rate of BV recurrence after therapy could be due to biofilm persistence (57). Biofilm-disrupting agents such as TOL-463 (intra-vaginal boric acid enhanced with ethylenediaminetetraacetic acid) (58) are being investigated to determine their role in BV treatment (NCT03930745). Use of biofilm-disrupting agents could increase the susceptibility of key BVAB to commonly used antibiotics when they are disassociated from the biofilm (4). Additionally, future studies focusing on interventions that modify or block the synergistic relationship between key BVAB and host response mechanisms are needed (22). Studies on the role of secondary colonizers in BV symptomatology should also be conducted. Finally, clinical trials evaluating the treatment of P. bivia or A. vaginae colonization in women without BV are needed. Of note, we are conducting a clinical trial of treatment of G. vaginalis colonization with amoxicillin among women without BV (NCT03211156). Vaginal microbiota transplant from a donor with optimal vaginal microbiota is a new provocative treatment option for women with BV. A universal donor screening approach was recently implemented in a pilot study (59). An inherent risk of this procedure, however, is transmission of a sexually transmitted pathogen. The baseline infection status of the recipient should be known in addition to that of the donor. Collectively, our model provides a framework for key elements that should be considered when designing and testing novel diagnostics and therapeutics.

Key Points.

  1. The etiology of BV, the most common vaginal infection, remains controversial.

  2. G. vaginalis and P. bivia (early colonizers) in addition to A. vaginae, Sneathia spp. (secondary colonizers) may be key bacteria in the pathogenesis of BV.

  3. G. vaginalis and P. bivia appear to actively inhibit the host inflammatory response in the vaginal epithelium, potentially evading the immune system while setting up infection and establishing the BV biofilm.

  4. Secondary colonizers, including A. vaginae, likely contribute to the signs and symptoms of BV, as they have been found to stimulate the host immune response in vaginal epithelial cells.

  5. Elucidating the exact etiology of BV has important implications for diagnosis and treatment moving forward.

Acknowledgments

Financial Support and Sponsorship

CAM is currently supported by the National Institute of Allergy and Infectious Diseases (NIAID) (K23AI106957-01A1). JRS is currently supported by NIAID (U19AI113212-01A1, U01AI108509, and HHSN27200011). PŁ and MMHK are supported by the National Cancer Institute (P30CA023074 and 2U54CA143924-11).

Conflicts of Interest

CAM has received research funding from Lupin Pharmaceuticals. She is also a consultant for Lupin Pharmaceuticals, BioFire Diagnostics, and Cepheid and has received honoraria from Roche Diagnostics and Becton Dickinson. JRS has received research funding from Becton Dickinson, Hologic, Talis, Toltec, Lupin Pharmaceuticals, and StarPharma. MMHK has been a consultant for Lupin Pharmaceuticals and Becton Dickinson. PŁ has no reported conflicts of interest.

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