The human vagina is a dynamic ecosystem in which homeostasis depends on mutually beneficial interactions between the host and their microorganisms. However, the vaginal ecosystem can be thrown off balance by a wide variety of factors. Bacterial vaginosis (BV) is the most common vaginal infection in women of childbearing age, but its etiology is not yet fully understood, with different controversial theories being raised over the years. What is generally accepted is that BV is often characterized by a shift in the composition of the normal vaginal microbiota, from a Lactobacillus species dominated microbiota to a mixture of anaerobic and facultative anaerobic bacteria. During BV, a polymicrobial biofilm develops in the vaginal microenvironment, being mainly composed of Gardnerella species. The interactions between vaginal microorganisms are thought to play a pivotal role in the shift from health to disease and might also increase the risk of sexually transmitted infections acquisition. Here we review the current knowledge regarding the specific interactions that occur in the vaginal niche and discuss mechanisms by which these interactions might be mediated. Furthermore, we discuss the importance of novel strategies to fight chronic vaginal infections.

Bacterial vaginosis (BV) is the most common vaginal infection affecting women worldwide. This infection is characterized by the loss of the dominant Lactobacillus community in the vaginal microbiota and an increase of anaerobic bacteria, that leads to the formation of a polymicrobial biofilm, mostly composed of Gardnerella spp. Treatment of BV is normally performed using broad-spectrum antibiotics, such as metronidazole and clindamycin. However, the high levels of recurrence of infection after treatment cessation have led to a demand for new therapeutic alternatives. Thymbra capitata essential oils (EOs) are known to have a wide spectrum of biological properties, including antibacterial activity. Thus, herein, we characterized two EOs of T. capitata and tested their antimicrobial activity as well as some of their main components, aiming to assess possible synergistic effects. Our findings showed that carvacrol and ρ-cymene established a strong synergistic antimicrobial effect against planktonic cultures of Gardnerella spp. On biofilm, carvacrol and linalool at sub-MIC concentrations proved more efficient in eliminating biofilm cells, while showing no cytotoxicity observed in a reconstituted human vaginal epithelium. The antibiofilm potential of the EOs and compounds was highlighted by the fact cells were not able to recover culturability after exposure to fresh medium.

Bacterial Vaginosis (BV) involves the presence of a multi-species biofilm adhered to vaginal epithelial cells, but its in-depth study has been limited due to the complexity of the bacterial community, which makes the design of in vitro models challenging. Perhaps the most common experimental technique to quantify biofilms is the crystal violet (CV) staining method. Despite its widespread utilization, the CV method is not without flaws. While biofilm CV quantification within the same strain in different conditions is normally accepted, assessing multi-species biofilms formation by CV staining might provide significant bias. For BV research, determining possible synergism or antagonism between species is a fundamental step for assessing the roles of individual species in BV development. Herein, we provide our perspective on how CV fails to properly quantify an in vitro triple-species biofilm composed of Gardnerella vaginalis, Fannyhessea (Atopobium) vaginae, and Prevotella bivia, three common BV-associated bacteria thought to play key roles in incident BV pathogenesis. We compared the CV method with total colony forming units (CFU) and fluorescence microscopy cell count methods. Not surprisingly, when comparing single-species biofilms, the relationship between biofilm biomass, total number of cells, and total cultivable cells was very different between each tested method, and also varied with the time of incubation. Thus, despite its wide utilization for single-species biofilm quantification, the CV method should not be considered for accurate quantification of multi-species biofilms in BV pathogenesis research.

Bacterial vaginosis (BV) is associated with serious gynaecologic and obstetric complications. The hallmark of BV is the presence of a polymicrobial biofilm on the vaginal epithelium, but BV aetiology is still a matter of debate. We have previously developed an in vitro biofilm model that included three BV-associated species, but, up to now, no studies are available whereby more bacterial species are grown together to better mimic the in vivo situation. Herein, we characterized the first polymicrobial BV biofilm consisting of six cultivable BV-associated species by using both in vitro and ex vivo vaginal tissue models. Both models revealed that the six species were able to incorporate the polymicrobial biofilm, at different bacterial concentrations. As it has been thought that this polymicrobial biofilm may increase the survival of BV-associated species when exposed to antibiotics, we also assessed if the Thymbra capitata essential oil (EO), which has recently been shown to be highly bactericidal against several Gardnerella species, could maintain its anti-biofilm activity against this polymicrobial biofilm. Under our experimental conditions, T. capitata EO exhibited a high antibacterial effect against polymicrobial biofilms, in both tested models, with a significant reduction in the biofilm biomass and the number of culturable cells. Overall, this study shows that six BV-associated species can grow together and form a biofilm both in vitro and when using an ex vivo model. Moreover, the data obtained herein should be considered in further applications of T. capitata EO as an antimicrobial agent fighting BV.

Background Bacterial vaginosis (BV), the most common cause of vaginal discharge, is characterized by the presence of a polymicrobial biofilm on the vaginal epithelium, formed primarily by Gardnerella spp., but also other anaerobic species. Interactions between bacteria in multi-species biofilms are likely to contribute to increased virulence and to enhanced antimicrobial tolerance observed in vivo. However, functional studies addressing this question are lacking. Objectives To gain insights into the role that interactions between BV-associated species in multi-species BV biofilms might have on antimicrobial tolerance, single- and triple-species biofilms formed by Gardnerella vaginalis, Fannyhessea (Atopobium) vaginae and Peptostreptococcus anaerobius were characterized, before and after metronidazole or clindamycin treatment. Methods Total biofilm biomass, total cells and cfu counts prior to and after antibiotic treatment were first determined. In addition, bacterial populations in the triple-species biofilms were also quantified by quantitative PCR (qPCR) and peptide nucleic acid (PNA) fluorescence in situ hybridization (FISH). Results Despite the effect observed in single-species biofilms, neither metronidazole nor clindamycin was effective in reducing triple-species biofilm biomass. Similar results were obtained when evaluating the number of total or culturable cells. Interestingly, despite differences between strain susceptibilities to antibiotics, the composition of the triple-species biofilms was not strongly affected by antibiotics. Conclusions Taken together, these results strengthen the idea that, when co-incubated, bacteria can interact synergistically, leading to increased tolerance to antimicrobial therapy, which helps explain the observed clinically high BV recurrence rates.

Bacterial vaginosis (BV) is the most common vaginal infection in women of reproductive age and has been associated with serious health complications, mainly in pregnant women. It is characterized by a decrease in the number of Lactobacillus species in the healthy vaginal microbiota and an overgrowth of strict and facultative anaerobic bacteria that develop a polymicrobial biofilm. Despite over 60 years of research investigating BV, its etiology is not fully understood. Gardnerella spp. is a crucial microorganism that contributes to the formation of the biofilm and the development of BV, but the role of other BV-associated bacteria is not clear. Nevertheless, Fannyhessea vaginae (previously known as Atopobium vaginae) is a highly specific species for BV, and co-colonization with Gardnerella is thought to be a very specific diagnostic marker. The diagnosis of BV still presents some limitations, since currently used methods often fail to accurately detect BV. This work aims to develop a novel peptide nucleic acid (PNA) probe targeting F. vaginae. This probe was further validated in a multiplex assay, which included a Gardnerella-specific PNA probe, as a possible method for diagnosis of BV, and was compared with quantification by qPCR. The new PNA probe showed excellent sensitivity and specificity and could discriminate F. vaginae-Gardnerella biofilms, confirming the potential to be used for the detection of BV-associated pathogens.

Bacterial vaginosis (BV) is the most frequent vaginal infection in women of reproductive age. It is caused by the overgrowth of anaerobic vaginal pathogens, such as Gardnerella vaginalis, Fannyhessea vaginae, and Prevotella bivia, which are vaginal pathogens detected during the early stages of incident BV and have been found to form multi-species biofilms. Treatment of biofilm-associated infections, such as BV, is challenging. In this study, we tested the role of an investigational engineered phage endolysin, PM-477, in the eradication of dual-species biofilms composed of G. vaginalis–F. vaginae or G. vaginalis–P. bivia. Single-species biofilms formed by these species were also analysed as controls. The effect of PM-477 on biomass and culturability of single- and dual-species biofilms was assessed in vitro using a microtiter plate assay, epifluorescence microscopy, confocal laser scanning microscopy, and quantitative PCR. The results showed that PM-477 was particularly effective in the disruption and reduction of culturability of G. vaginalis biofilms. In dual-species biofilms, PM-477 exhibited lower efficiency but was still able to selectively and significantly eliminate G. vaginalis. Since polymicrobial interactions have been shown to strongly affect the activity of various antibiotics, the activity of PM-477 in dual-species biofilms is a potentially promising result that should be further explored, aiming to completely eradicate multi-species biofilms associated with BV.

Bacterial vaginosis (BV) is the most common cause of vaginal discharge and is often associated with other health consequences mainly in pregnant women. BV is described by an imbalance in the vaginal microbiota where strictly and facultative anaerobic bacteria outgrow the lactic acid‐ and hydrogen peroxide‐producing Lactobacillus species. The species involved in BV are capable to grow and form a polymicrobial biofilm in the vaginal epithelium. The treatment of BV is usually performed using broad‐spectrum antibiotics, including metronidazole and clindamycin. However, these conventional treatments are associated with high recurrence rates. The BV polymicrobial biofilm may have an important role on the treatment outcome and is accounted as one of the factors for treatment failure. Other possible reasons for treatment failure include the presence of species resistant to antibiotics or the chance of reinfection after treatment. Therefore, novel strategies to increase the rates of treatment have been studied namely the use of probiotics and prebiotics, acidifying agents, antiseptics, plant‐based products, vaginal microbiota transplantation, and phage endolysins. Although some of them are still in an initial phase of development with very preliminary results, they show great perspectives for application. In this review, we aimed to study the role of the polymicrobial nature of BV in treatment failure and explore a few alternatives for treatment.