The Role of Prevotella Species in Female Genital Tract Infections.

Female genital tract infections (FGTIs) include vaginal infections (e.g., bacterial vaginosis [BV]), endometritis, pelvic inflammatory disease [PID], and chorioamnionitis [amniotic fluid infection]. They commonly occur in women of reproductive age and are strongly associated with multiple adverse health outcomes including increased risk of HIV/sexually transmitted infection acquisition and transmission, infertility, and adverse birth outcomes such as preterm birth. These FGTIs are characterized by a disruption of the cervicovaginal microbiota which largely affects host immunity through the loss of protective, lactic acid-producing Lactobacillus spp. and the overgrowth of facultative and strict anaerobic bacteria. Prevotella species (spp.), anaerobic Gram-negative rods, are implicated in the pathogenesis of multiple bacterial FGTIs. Specifically, P. bivia, P. amnii, and P. timonensis have unique virulence factors in this setting, including resistance to antibiotics commonly used in treatment. Additionally, evidence suggests that the presence of Prevotella spp. in untreated BV cases can lead to infections of the upper female genital tract by ascension into the uterus. This narrative review aims to explore the most common Prevotella spp. in FGTIs, highlight their important role in the pathogenesis of FGTIs, and propose future research in this area.

Microbial interactions among Gardnerella, Prevotella and Fannyhessea prior to incident bacterial vaginosis: protocol for a prospective, observational study.

INTRODUCTION: The aetiology of bacterial vaginosis (BV), a biofilm-associated vaginal infection, remains unknown. Epidemiologic data suggest that it is sexually transmitted. BV is characterised by loss of lactic acid-producing lactobacilli and an increase in facultative and strict anaerobic bacteria. Gardnerella spp are present in 95%-100% of cases; Gardnerella vaginalis has been found to be more virulent than other BV-associated bacteria (BVAB) in vitro. However, G. vaginalis is found in women with normal vaginal microbiota and colonisation is not sufficient for BV development. We hypothesise that Gardnerella spp initiate BV biofilm formation, but incident BV (iBV) requires incorporation of other key BVAB (ie, Prevotella bivia, Fannyhessea vaginae) into the biofilm that alter the transcriptome of the polymicrobial consortium. This study will investigate the sequence of microbiologic events preceding iBV. METHODS AND ANALYSIS: This study will enrol 150 women aged 18-45 years with normal vaginal microbiota and no sexually transmitted infections at a sexual health research clinic in Birmingham, Alabama. Women will self-collect twice daily vaginal specimens up to 60 days. A combination of 16S rRNA gene sequencing, qPCR for Gardnerella spp, P. bivia and F. vaginae, and broad range 16S rRNA gene qPCR will be performed on twice daily vaginal specimens from women with iBV (Nugent score 7-10 on at least 2 consecutive days) and controls (with comparable age, race, contraceptive method and menstrual cycle days) maintaining normal vaginal microbiota to investigate changes in the vaginal microbiota over time for women with iBV. Participants will complete daily diaries on multiple factors including sexual activity. ETHICS AND DISSEMINATION: This protocol is approved by the University of Alabama at Birmingham Institutional Review Board (IRB-300004547) and written informed consent will be obtained from all participants. Findings will be presented at scientific conferences and published in peer-reviewed journals as well as disseminated to providers and patients in communities of interest.

Accurate Absolute Quantification of Bacterial Populations in Mixed Cultures by qPCR.

Quantitative PCR (qPCR) is a well-established technique that allows to accurately quantify nucleic acids or proteins, being widely used in several types of biological samples for bacterial load quantification. However, there are many recent studies that do not consider the potential pitfalls involved in key experimental qPCR stages, namely, those related to the extraction and purification of genomic DNA and to the thermal amplification process, that can lead to biased results in mixed cultures. Herein, we outline a proper protocol for bacterial quantification by qPCR, addressing how to overcome the main issues in that methodology.

Development of a Prevotella bivia PNA probe and a multiplex approach to detect three relevant species in bacterial vaginosis-associated biofilms.

Bacterial vaginosis (BV) is the most common vaginal infection worldwide. We developed a peptide nucleic acid (PNA) probe targeting Prevotella bivia, a common BV-associated bacteria, and optimized a multiplex approach for detection of Gardnerella spp., P. bivia and Fannyhessea vaginae. Our P. bivia PNA probe specifically detected the target species, and the optimized multiplex approach was able to detect the presence of the three species in multi-species BV biofilms.

Fighting polymicrobial biofilms in bacterial vaginosis.

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.

Vaginal Sheets with Thymbra capitata Essential Oil for the Treatment of Bacterial Vaginosis: Design, Characterization and In Vitro Evaluation of Efficacy and Safety.

We aimed to incorporate Thymbra capitata essential oil (TCEO), a potent antimicrobial natural product against bacterial vaginosis (BV)-related bacteria, in a suitable drug delivery system. We used vaginal sheets as dosage form to promote immediate relief of the typical abundant vaginal discharge with unpleasant odour. Excipients were selected to promote the healthy vaginal environment reestablishment and bioadhesion of formulations, while the TCEO acts directly on BV pathogens. We characterized vaginal sheets with TCEO in regard to technological characterization, predictable in vivo performance, in vitro efficacy and safety. Vaginal sheet D.O (acid lactic buffer, gelatine, glycerine, chitosan coated with TCEO 1% w/w) presented a higher buffer capacity and ability to absorb vaginal fluid simulant (VFS) among all vaginal sheets with EO, showing one of the most promising bioadhesive profiles, an excellent flexibility and structure that allow it to be easily rolled for application. Vaginal sheet D.O with 0.32 µL/mL TCEO was able to significantly reduce the bacterial load of all in vitro tested Gardnerella species. Although vaginal sheet D.O presented toxicity at some concentrations, this product was developed for a short time period of treatment, so this toxicity can probably be limited or even reversed when the treatment ends.

Assessing recovery rates of distinct exogenous controls for gDNA extraction efficiency using phenol-chloroform or silica-column based extractions.

Assessment of genomic DNA (gDNA) extraction efficiency is required for accurate bacterial quantification by qPCR. Exogenous DNA molecules are often added after bacterial cultures are lysed, but before DNA purification steps, to determine extraction efficiency. Herein we found that different exogenous DNA controls have different recovery rates, suggesting distinct DNA extraction efficiencies. Recovery rates are also affected by the gDNA extraction method being more affected in silica-based columns than in phenol-chloroform extraction. Overall, we determined that the use of long DNA fragments, such as gDNA, as exogenous controls have a higher recovery rate than use of smaller size DNA molecules.

Exploiting the Anti-Biofilm Effect of the Engineered Phage Endolysin PM-477 to Disrupt In Vitro Single- and Dual-Species Biofilms of Vaginal Pathogens Associated with Bacterial Vaginosis.

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.

Six Bacterial Vaginosis-Associated Species Can Form an In Vitro and Ex Vivo Polymicrobial Biofilm That Is Susceptible to Thymbra capitata Essential Oil.

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.

In vitro interactions within a biofilm containing three species found in bacterial vaginosis (BV) support the higher antimicrobial tolerance associated with BV recurrence.

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.

Synergistic effects of carvacrol, α-terpinene, γ-terpinene, ρ-cymene and linalool against Gardnerella species.

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.

A New PNA-FISH Probe Targeting Fannyhessea vaginae.

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.

Crystal Violet Staining Alone Is Not Adequate to Assess Synergism or Antagonism in Multi-Species Biofilms of Bacteria Associated With Bacterial Vaginosis.

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.

Gardnerella and vaginal health: the truth is out there.

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.