Bacterial vaginosis biofilms: a target for therapeutic innovation

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BACKGROUND

Bacterial vaginosis (BV) is one of the most common gynecological conditions that does not directly affect a woman’s health, but significantly impairs her quality of life [1]. Complications in patients with BV can be both gynecological (pelvic inflammatory disease, infertility of various origins, etc.) and obstetric (in women with BV, rates of miscarriage and preterm delivery are 3–5 and 2–7 fold higher, respectively, depending on gestational age) [2–4]. These complications can seriously affect women’s reproductive health and therefore require attention of the medical community.

The global literature estimates the prevalence of BV in the female population to be approximately 29%, or more than 21 million diagnosed patients [5]. The prevalence of BV varies by geographic region and ethnicity. For example, a systematic review by Kenyon et al. [6] showed that the highest BV prevalence was reported in some parts of Africa (Botswana, Central African Republic, Gambia, Ghana, etc.), while most countries in Asia and Europe had low levels of the syndrome. A systematic review and meta-analysis by Peebles et al. found both regional and racial differences in the global prevalence of BV; rates of BV in African American and Hispanic women (33% and 31%, respectively) are significantly higher than in European (23%) and Asian (11%) women [7]. BV is a serious issue for women of childbearing age (23%–29%), which may affect the overall demographics, as the health of this population is directly related to the birth rate and the health of future generations [8]. In addition, the incidence of chronic and/or recurrent BV is increasing and the cost of diagnosis and treatment is a significant burden on the global economy [4]. For example, a meta-analysis by Peebles et al. [9] showed that annual cost burden of BV treatment is high, at an estimated US $4.8 billion.

BV can present in various clinical forms; one in four patients is asymptomatic, while symptomatic BV is often accompanied by a homogeneous gray or grayish-white foul-smelling (fishy odor) vaginal discharge, itching, burning, and other vaginal discomfort [5, 10]. Diagnosis of BV includes conventional modalities: vaginal pH, which is usually elevated in BV; the amine test (addition of 10% potassium hydroxide to vaginal discharge will produce a characteristic fishy odor); microscopy to detect key cells relevant to BV; microbiology to determine the composition of the microflora. New modalities are also being used, such as polymerase chain reaction for qualitative and quantitative assessment of vaginal bacterial composition, biochemical or molecular biotyping, and high-throughput sequencing, which provide a more comprehensive picture of microbiome composition [11].

HISTORY OF BACTERIAL VAGINOSIS RESEARCH

In practice, BV is usually characterized as a polymicrobial non-inflammatory condition caused by the decreased proportion of Lactobacilli or their complete absence in the vaginal microbiome and increased counts of obligate and facultative anaerobic opportunistic pathogens [12]. The vaginal microbiome is a complex, dynamic ecosystem that fluctuates throughout a menstrual period and an entire woman’s life. Some authors report the bacterial count in young women of childbearing age to be 10¹⁰–10¹¹ CFU/mL [13]. The vaginal microbiome of a healthy woman is maintained in homeostasis. However, exogenous and endogenous factors may affect this balance. Endogenous factors include alterations in hormone levels, a woman›s age, weight, pregnancy, immune status, etc. Exogenous factors include the use of antibacterial agents, genital/extragenital infections, and inflammation [14]. Lactobacilli are the main bacteria that maintain a stable and healthy vaginal microbiocenosis. Albert Döderlein [15] was the first who discovered rod-shaped bacilli in the vagina and cultured them in a nutrient broth. He also found that these microorganisms are capable of producing lactic acid in the vagina, making it acidic, and proposed the concept of “vaginal acidity.”

The history of vaginal microbiome research dates back to the mid-19th century, when clinicians noticed increasing rates of obstetrical and gynecological conditions caused by non-compliance with aseptic and antiseptic practice. The clinical use of light microscopy allowed in-depth research into the vaginal microflora composition and the search for causes of infectious complications in women. The identification of an etiologic agent and the development of a taxonomic classification of microbes associated with BV have been a challenge for many years due to the high selectivity of Gardnerella vaginalis to growth media, its unique physical and chemical structure and molecular architecture of the cell wall, genome diversity, and unclear pathogenic factors. For example, in 1953, Leopold published a study evaluating cervical discharge and urine in women with cervicitis and men with prostatitis, respectively. He detected a rod-shaped bacterium, which he described as small, gram-negative, non-motile, unencapsulated. Leopold could not identify the species of this bacterium, but based on its morphological characteristics, he proposed that it belonged to Haemophilus [16]. The microorganism previously described by Leopold was later discovered in 1955 by Gardner and Dukes [17] in women with non-specific vaginitis. Due to its tropism for blood-containing media, this rod-shaped bacterium was also classified as a member of the genus Haemophilus and named Haemophilus vaginalis due to its location. Since then, the condition associated with this pathogen has been classified as Haemophilus vaginalis vaginitis.

The species identification for the microorganism described by Leopold, Gardner, and Dukes has been the focus of research for many scientists since the 1960s, as subsequent studies provided conflicting results. In 1963, Zinnemann and Turnerg proposed to classify Haemophilus vaginalis as a member of the genus Corynebacterium and named it Corynebacterium vaginale [18]. The taxonomic classification did not include the microorganism in Corynebacteria. Gardnerella vaginalis, formerly known as Corynebacterium vaginale and Haemophilus vaginalis, was named after Hermann L. Gardner, who discovered and classified it as the only member of the genus Gardnerella [19, 20]. Later, some features of Gardnerella vaginalis were identified: it was a gram-variable, pleomorphic, non-motile, non-sporeforming, acapsular, aflagellar bacterium ranging from 0.4–1.5 µm to 2–3 µm [21]. In addition, the size and morphology of the cells vary significantly depending on growth conditions and physiological status [22]. Due to the unique structure, Gardnerella vaginalis has attracted not only clinicians but also basic scientists, driving ongoing research in the field. Electron microscopy revealed fimbriae on the Gardnerella vaginalis surface. The fimbriae mediate adhesion to vaginal epithelium in vivo and to desquamated epithelium, which are the key cells [23]. Microscopy and cultivation of Gardnerella vaginalis often found that its cells were often linked together, which was explained by the production of exopolysaccharide with bacteria binding its filaments together [24]. It is now recognized that this is a biofilm consisting of microorganisms bound by a polymer matrix [25].

BIOFILMS: PATHOGENESIS OF BACTERIAL VAGINOSIS

Currently, biofilm-associated BV is detected in 90% of patients [8]. Of note that this term, as well as the term “biofilm-associated gardnerellosis” does not exist officially, but it is proposed by some authors to clarify the etiopathogenesis of the disease. Biofilms are a structured community of one or more species embedded in a polymer matrix consisting of proteins, carbohydrates, and nucleic acids [26, 27]. The biofilm concentration of some microbes can reach 10¹¹ CFU/mL [28]. The American researcher John William Costerton described this phenomenon and introduced the term “biofilm” [29]. The formation of biofilms is a complex, stepwise, and dynamic process, as well as an effective survival strategy of microorganisms under unfavorable conditions [30]. Biofilm microorganisms are capable of regulating the production of virulence factors through an identified type of communication called quorum sensing. The biofilm cycle includes three stages: adhesion to the surface of the vaginal epithelium, secretion of the polymer matrix, and aggregation of microbes on the surface of the mature biofilm [27]. Biofilms have a higher tolerance to the aggressive factors compared to free-living microorganisms in the vaginal tract. This is possible due to the unique multi-component biofilm structure and the layered organization of microorganisms within the biofilm [31]. This structure contributes to the impaired diffusion of antimicrobial agents through the biofilm, reduced metabolic activity of the cells, and the emergence of antibiotic-resistant bacteria [32, 33].

For a long time, the role of Gardnerella in the biofilm formation was unclear: does Gardnerella vaginalis trigger this process or does it support the matrix of the biofilm formed by the association of bacteria? However, current literature suggests that G. vaginalis is a major colonizer that can provide a matrix for attachment of other BV-associated microorganisms, thereby enabling the formation of polymicrobial biofilms [27]. Some researchers, such as Bonnardel et al. [34], linked the mechanism of biofilm formation to bacterial lectins of Gardnerella, which are tropic to glycosylated components of the mucosa, thus forming a vaginal cell–Gardnerella complex. A study by Martin reported a role of a collagen-binding protein produced by Gardnerella, which may also contribute to the biofilm formation and possibly immune evasion through interaction with complement proteins [35]. In addition, regarding the microbiological features of Gardnerella, its significant adhesion to vaginal cells and ability to form biofilms confirm the trigger role of Gardnerella in the colonization of the vaginal epithelium, acting as a frame for the adherence of other species. Modern molecular diagnostic modalities show that G. vaginalis constitutes 60–90% of the biofilm. Other common biofilm members include Sneathia sanguinegens, Porphyromonas assaccharolytica, Megasphera spp. and the difficult-to-culture Atopobium vaginae, which may comprise up to 40% of the biofilm [5]. Their role in vaginal biofilm formation and pathogenicity of G. vaginalis has not been adequately evaluated [36]. Some studies reported that some, but not all, types of vaginal microflora can participate in biofilm formation and enhance G. vaginalis virulence [37]. Other studies found that microorganisms other than G. vaginalis may increase total biofilm biovolume [38]. Therefore, biofilm formation and development depend on both the triggering effect of Gardnerella and other vaginal microorganisms that form a kind of social network and thereby regulate the pathogenetic process of biofilm-associated BV. As a result, BV involves a complex interaction between opportunistic pathogens, endogenous vaginal microbiota, and the vaginal epithelium [39].

The main biofilm component is an extracellular polysaccharide matrix (exopolysaccharide), which constitutes 85% of its volume [25]. The matrix is produced by, and is composed of, the bacteria associated with BV, and it organizes the biofilm structurally and protects it from physical, chemical, and immune factors [40–42]. Nowadays, biofilms are thought to contribute to recurrent BV, although the term “recurrent BV” does not currently have a generally accepted definition [41]. There is no classification of BV in the relevant clinical guidelines published in 2022. The literature describes recurrent BV in a variety of ways. For example, Marshall et al. [43] considered this diagnosis in patients who “recur one or more times after completion of an episodic regimen,” Letyaeva [1] diagnosed recurrent BV in patients with more than four episodes per year, and Pestrikova et al. used the criterion of 3–4 episodes per year [28].

CONCLUSION

There is an increasing research focus on biofilm as a pathogenetic factor of BV. Most treatment options focus on the etiologic agent of a disease, i.e. the direct mechanism of action. However, the ability of microorganisms to adapt to the effects of aggressive agents makes many treatment regimens ineffective, as evidenced by increased recurrence rates. The data on the structure of the biofilm confirmed the importance of the development and clinical use of agents that disrupt the polysaccharide matrix of the biofilm and release the bacteria present in this matrix (dispersants), and this is the focus of our research. This will both improve treatment outcomes and enable the practical use of previously effective and established treatment options.

ADDITIONAL INFO

Authors' contributions. K.A. Rossolovskaya performed literature review, collected and analyzed literature sources, prepared, wrote and edited the manuscript; N.S. Trifonova reviewed and edited the manuscript; I.V. Gadaeva reviewed and edited the manuscript; L.G. Spivak reviewed and edited the manuscript. All authors confirm that their authorship meets the international ICMJE criteria (all authors have made a significant contribution to the development of the concept, research and preparation of the article, read and approved the final version before publication).

Funding source. This study was not supported by any external sources of funding.

Competing interests. The authors declares that there are no obvious and potential conflicts of interest associated with the publication of this article.

About the authors

Kseniya A. Rossolovskaya

I.M. Sechenov First Moscow State Medical University

Author for correspondence.
Email: dr.rossolovskaya@yandex.ru
ORCID iD: 0000-0002-7026-1607
SPIN-code: 4432-5748

Graduate Student

Russian Federation, Moscow

Natalia S. Trifonova

I.M. Sechenov First Moscow State Medical University

Email: trifonova.nataly@mail.ru
ORCID iD: 0000-0002-2891-3421
SPIN-code: 4753-5430

MD, Dr. Sci. (Medicine), Professor

Russian Federation, Moscow

Irina V. Gadaeva

I.M. Sechenov First Moscow State Medical University

Email: irina090765@gmail.com
ORCID iD: 0000-0003-0144-4984
SPIN-code: 9593-1990

MD, Cand. Sci. (Medicine)

Russian Federation, Moscow

Leonid G. Spivak

I.M. Sechenov First Moscow State Medical University

Email: leonid.spivak@gmail.com
ORCID iD: 0000-0003-1575-6268
SPIN-code: 5230-8811

MD, Dr. Sci. (Medicine), Professor

Russian Federation, Moscow

References

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