Social interactions between Lactobacillus iners and Gardnerella vaginalis biofilms: an unexpected friendship in the bacterial vaginosis ecosystem

Microbiotec'17 - Congress of Microbiology and Biotechnology 2017Background Worldwide, bacterial vaginosis(BV) is the leading dysbiosis of the vaginal microbiome. BV is a complex polymicrobial condition characterized by a disruption of the vaginal econiche, normally resulting in a reduction of beneficial lactobacilli and an overgrowth of anaerobes. It is noteworthy that a hallmark feature of BV is the presence of a highly structured polymicrobial biofilm, primarily consisting of Gardnerella vaginalis, strongly adhered to the vaginal epithelium, and a variety of other bacteria. There are some observational studies that described the rapid fluctuation over time of the vaginal microflora, showing that Lactobacillus inersis a dominant part of the vaginal flora in a transitional stage between abnormal and normal flora. Compared to other Lactobacillus species, L. iners has more complex nutritional requirements, a Gram- variable morphology and an unusually small genome, indicative of a symbiotic or parasitic lifestyle. However, till date, the role of L. iners in the development of a BV- associated biofilm remains unclear. Method This study aimed to unravel the interactions between G. vaginalis and L. iners, both isolated from BV cases, using an in vitro dual - species biofilm assembly. Bacterial coaggregation ability was determined for single - or between dual - species community. Furthermore, the total biofilm biomass was also determined by the crystal violet method. Next, we discriminated the dual - species populations in the biofilm by using Peptide Nucleic Acid Fluorescence in situ Hybridization method. Additionally, biofilm structure was evaluated using a confocal laser scanning microscopy analysis. Finally, the transcripts levels of G. vaginalis virulence genes, in a dual-species consortium, were determined by quantitative PCR. Results & Conclusions This study pointed out that L. iners seems to be well adapted to BV dysbiosis. We observed that L. iners was able to incorporate a pre-established G. vaginalis biofilm. Confocal microscopy analysis revealed that both species can live in close proximity, forming clusters from the bottom to the biofilm top layer. Curiously, L. iners did not affect G. vaginalis virulence,as determined by the transcription levels of key virulence genes. Remarkably, one could argue that L. iners is capable of surviving and adapting to a metabolic stress - related conditions found in BV(FCT) by the strategic project of UID/BIO/04469/2013 and by BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by European Regional Development Fund under the scope of Norte2020. JC had an individual FCT fellowship (SFRH/BD/93963/2013). NC is an Investigador FCTinfo:eu-repo/semantics/publishedVersio

Adhesion of vaginal microorganisms to epithelial cells and its association with Bacterial Vaginosis

Dissertação de mestrado integrado em Engenharia Biomédica (área de especialização em Engenharia Clínica)A vaginose bacteriana (VB) é um distúrbio da flora vaginal normal e um importante problema de saúde pública em mulheres de idade reprodutiva. A VB é caracterizada pela substituição de lactobacilos vaginais por microrganismos, predominantemente, anaeróbios. Desconhecendo-se a etiologia da VB, duas hipóteses tentam explicar esta condição: a hipótese polimicrobiana, que infere que a VB é causada por uma mistura de bactérias patogénicas, principalmente anaeróbias; e outra hipótese que aponta para a Gardnerella vaginalis como o verdadeiro agente causador da VB. No entanto o isolamento frequente desta espécie em mulheres aparentemente saudáveis lançou dúvidas sobre essa afirmação. Assim, num esforço para se compreender a etiologia desta doença, foram realizados ensaios de adesão in vitro para comparar a capacidade de adesão de vários isolados vaginais provenientes de exsudados vaginais de mulheres que foram diagnosticadas como tendo VB e de mulheres saudáveis. No total, foram caracterizados 15 isolados vaginais quanto à sua capacidade de adesão inicial numa monocamada de células HeLa. Estes ensaios revelaram que os isolados de G. vaginalis apresentaram uma capacidade de adesão inicial mais forte do que os outros isolados analisados. Além disso, estirpes de G. vaginalis isoladas de pacientes com VB apresentaram uma maior capacidade de adesão inicial do que as estirpes de G. vaginalis que foram isoladas de mulheres saudáveis. Assim, a fim de compreender as diferenças verificadas, foi estudada a competição entre lactobacilos (Lactobacillus iners, Lactobacillus crispatus e Lactobacillus casei) e estirpes de G. vaginalis (não-patogénicas e patogénicas). Todos os ensaios de competição foram quantificados por microscopia de fluorescência, usando DAPI para contar as células totais e uma sonda de PNA-FISH para quantificar G. vaginalis. Os resultados mostraram que a adesão de L. iners não diminuiu na presença de estirpes patogénicas de G. vaginalis. Pelo contrário, o L. crispatus mostrou uma diminuição na capacidade de adesão às células epiteliais na presença de estirpes patogénicas de G. vaginalis. O L. crispatus mostrou, também, que tem uma grande capacidade de inibir a adesão de isolados patogénicos de G. vaginalis. Por sua vez, o L. casei foi o lactobacilos menos aderente de todos os utilizados no presente estudo. Como resultado, estes estudos de adesão ajudam a fornecer informações sobre a situação clínica na qual os lactobacilos vaginais indígenas podem interferir com a presença de G. vaginalis na microflora vaginal.Bacterial vaginosis (BV) is an unhealthy disturbance of the normal vaginal flora and an important public health problem in women in reproductive age. BV is characterized by the replacement of vaginal lactobacilli by predominantly anaerobic microorganisms. The lack of basic information about the etiology of BV has lead to the postulation of two hypotheses. The first is the polymicrobial hypothesis, which infers that BV is caused by a mixture of pathogenic bacteria, mainly anaerobes. The second is that a single pathogenic species, in many cases Gardnerella vaginalis is the causative agent of BV, but frequent isolation of this species from seemingly healthy women has cast doubt on this claim. So, in an effort to tease apart the aetiology of this disorder, in vitro adherence assays were performed to compare the initial adhesion, the first step of biofilm formation, of G. vaginalis relative to other microorganisms isolated from vaginal swabs from patients with BV and healthy women. In total, 15 unique vaginal isolates were characterized for their initial adhesion ability to a monolayer of the HeLa cells. These assays revealed that G. vaginalis isolates had a stronger initial adhesion capability than the other isolates recovered. Furthermore, G. vaginalis strains isolated from BV patients had stronger initial adhesion ability than G. vaginalis isolated from healthy women. In order to understand these differences, the competition between lactobacilli (Lactobacillus iners, Lactobacillus crispatus and Lactobacillus casei) and G. vaginalis strains (non-pathogenic and pathogenic) was studied. All competition assays were quantified by fluorescence microscopy, using DAPI for total cell count and PNA-FISH probe for G. vaginalis quantification. The results showed that L. iners did not decrease in presence of pathogenic G. vaginalis strains. In contrast, L. crispatus showed a decreased adherence capacity to epithelial cells in the presence of pathogenic G. vaginalis strains. Furthermore, the results showed that L. crispatus could be important for antagonizing the pathogenic strains of G. vaginalis. In turn, L. casei was the least adherent of the all lactobacilli used in this study. As a result, adherence studies help to provide insight into the clinical situation in which indigenous vaginal lactobacilli can interfere with G. vaginalis presence in vaginal microflora.Fundação para a Ciência e a Tecnologia (FCT) - Projeto FCOMP-01-0124-FEDER-008991 (PTDC/BIA-MIC/098228/2008).This work was supported by European Union funds (FEDER/COMPETE

Can cooperation within the vaginal microbiome lead to the development of bacterial vaginosis?

Our well-being and our microbes - Annual Meeting of the New Zealand Microbiological Society[Excerpt] Despite being a controversial topic over the past few decades, Gardnerella vaginalis has been attributed a central role in bacterial vaginosis (BV) development. An important milestone in BV research was the discovery that the different species involved in BV were associated in a structured polymicrobial biofilm, dominated by G. vaginalis. Subsequent studies demonstrated that G. vaginalis biofilms display a high resistance to the protective mechanisms of normal vaginal microflora, as well as an increased tolerance to antibiotics. Despite the increased evidence of the pivotal role of G. vaginalis in BV biofilm development, the importance of the other BV-associated anaerobes should not be neglected. There are some observational studies that correlate bacterial co-colonization between G. vaginalis and some other specific BV-associated bacteria during BV. Furthermore, synergistic interactions can occur between BV-associated species and G. vaginalis, leading to increased biofilm formation in dual-species biofilms. We hypothesized that differential bacterial interactions can occur during BV development. To test our hypothesis, we examined the ecological interactions between G. vaginalis and other 15 BV-associated anaerobes that we had previously shown to enhance biofilm formation by G. vaginalis, using a dual-species biofilm model. [...]info:eu-repo/semantics/publishedVersio

Genetic heterogeneity and taxonomic diversity among Gardnerella species

Gardnerella spp. are the most frequent organisms found in bacterial vaginosis (BV). However, colonization by Gardnerella spp. does not always lead to BV. Developments in molecular genetics have shed new light on the diversity of the genus Gardnerella, leading to an emended description of Gardnerella vaginalis and, for the first time, the inclusion of more species of Gardnerella, namely, Gardnerella leopoldii, Gardnerella piotii, and Gardnerella swidsinskii. While no association had been made between the novel Gardnerella species and virulence potential, there is genomic evidence of a higher prevalence of some genotypes in BV cases, as compared with colonization in women in whom BV has been clinically ruled out. Gardnerella vaginalis has been considered a pivotal player in the progression of bacterial vaginosis (BV), a condition associated with serious health complications. However, G. vaginalis is also commonly found in asymptomatic or BV-negative women. This has generated interest in the question of whether genetic differences among isolates might distinguish pathogenic from commensal isolates. G. vaginalis was the only recognized species in its genus for four decades, but recently an emended description of G. vaginalis and descriptions of three new species Gardnerella leopoldii, Gardnerella piotii, and Gardnerella swidsinskii have been proposed. This review provides background on the heterogeneity and diversity within the genus Gardnerella, highlighting the main features that distinguish species and clades, and how these features may impact BV development.J.C. and N.C. acknowledge the projects PTDC/BIA-MIC/28271/2017 (POCI-01-0145-FEDER-028271), and the strategic funding of unit UID/BIO/04469/2019, supported by the Portuguese Foundation for Science and Technology.info:eu-repo/semantics/publishedVersio

Evaluation of different culture media to support in vitro growth and biofilm formation of bacterial vaginosis-associated anaerobes

Background Bacterial vaginosis (BV) is one of the most common vaginal infections worldwide. It is associated with the presence of a dense polymicrobial biofilm on the vaginal epithelium, formed mainly by Gardnerella species. The biofilm also contains other anaerobic species, but little is known about their role in BV development. Aim To evaluate the influence of different culture media on the planktonic and biofilm growth of six cultivable anaerobes frequently associated with BV, namely Gardnerella sp., Atopobium vaginae, Lactobacillus iners, Mobiluncus curtisii, Peptostreptococcus anaerobius and Prevotella bivia. Methods A total of nine different culture media compositions, including commercially available and chemically defined media simulating genital tract secretions, were tested in this study. Planktonic cultures and biofilms were grown under anaerobic conditions (10% carbon dioxide, 10% helium and 80% nitrogen). Planktonic growth was assessed by optical density measurements, and biofilm formation was quantified by crystal violet staining. Results Significant planktonic growth was observed for Gardnerella sp., A. vaginae and L. iners in New York City III broth, with or without ascorbic acid supplementation. Biofilm quantification showed high in vitro biofilm growth for Gardnerella sp., P. anaerobius and P. bivia in almost all culture media excluding Brucella broth. Contrary, only New York City III broth was able to promote biofilm formation for A. vaginae, L. iners and M. curtisii. Conclusions Our data demonstrate that New York City III broth relative to the other tested media is the most conducive for future studies addressing polymicrobial biofilms development as this culture medium allowed the formation of significant levels of single-species biofilms.This work was supported by the research project [PTDC/BIA-MIC/28271/2017] under the scope of COMPETE 2020 [POCI-01-0145-FEDER-028271], supported by the Portuguese Foundation for Science and Technology (FCT), and by the strategic funding of unit [UIDB/04469/2020]. Aliona S. Rosca received financial support from individual Grant [PD/BD/128037/2016]. Nuno Cerca received support from the National Institute of Allergy and Infectious Diseases (R01AI146065-01A1, granted to Christina A. Muzny, MD, MSPH). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.info:eu-repo/semantics/publishedVersio

Development of an in vitro vaginal exudate adhesion model for Bacterial Vaginosis

Microbiotec'17 - Congress of Microbiology and Biotechnology 2017Background: Bacterial vaginosis (BV) is the worldwide leading vaginal disorder commonly recognized between menarche and menopause in women of all ethnicities. It is associated with serious health problems relating to both fertility and pregnancy. This dysbiosis is characterized by a reduction in lactic acid-producing bacteria, mainly Lactobacillus spp., accompanied by an overgrowth of strict or facultative anaerobic bacteria, predominantly Gardnerella vaginalis. However, G. vaginalis is also present in healthy women and its vaginal colonization does not always lead to BV. To better understand the complex interactions that occur between host and microorganisms, and as well as between microorganisms in the vaginal microenvironment, development of in vitro models that can simulate the in vivo conditions is required, since no adequate animal model exists.This work was supported by national funds (FCT) by the strategic project of UID/BIO/04469/2013 and by BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by European Regional Development Fund under the scope of Norte2020. AR had an individual FCT fellowship (PD/BD/128037/2016). NC is an Investigator FCT.info:eu-repo/semantics/publishedVersio

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.This work was supported by the Portuguese Foundation for Science and Technology. AR and NC are funded by the individual Grants [PD/BD/128037/2016] and the strategic funding of unit [UID/BIO/04469/2019], respectively. JC and LS are funded by the research project [PTDC/BIA-MIC/28271/2017], under the scope of COMPETE 2020 [POCI-01-0145-FEDER-028271].info:eu-repo/semantics/publishedVersio

Atopobium vaginae and Prevotella bivia are able to incorporate and influence gene expression in a pre-formed Gardnerella vaginalis biofilm

Bacterial vaginosis (BV) is associated with a highly structured polymicrobial biofilm on the vaginal epithelium where Gardnerella species presumably play a pivotal role. Gardnerella vaginalis, Atopobium vaginae, and Prevotella bivia are vaginal pathogens detected during the early stages of incident BV. Herein, we aimed to analyze the impact of A. vaginae and P. bivia on a pre-established G. vaginalis biofilm using a novel in vitro triple-species biofilm model. Total biofilm biomass was determined by the crystal violet method. We also discriminated the bacterial populations in the biofilm and in its planktonic fraction by using PNA FISH. We further analyzed the influence of A. vaginae and P. bivia on the expression of key virulence genes of G. vaginalis by quantitative PCR. In our tested conditions, A. vaginae and P. bivia were able to incorporate into pre-established G. vaginalis biofilms but did not induce an increase in total biofilm biomass, when compared with 48-h G. vaginalis biofilms. However, they were able to significantly influence the expression of HMPREF0424_0821, a gene suggested to be associated with biofilm maintenance in G. vaginalis. This study suggests that microbial relationships between co-infecting bacteria can deeply affect the G. vaginalis biofilm, a crucial marker of BV.This research was partially funded by the National Institute of Allergy and Infectious Diseases (R01AI146065-01A1). It was also partially funded by the Portuguese Foundation for Science and Technology (FCT), by the research project (PTDC/BIA-MIC/28271/2017), under the scope of COMPETE 2020 (POCI-01-0145-FEDER-028271), and by the strategic funding of unit (UIDB/04469/2020).info:eu-repo/semantics/publishedVersio

Antimicrobial activity of Thymbra capitata and its major components agasint Gardnerella spp.

[Excerpt] Introduction: Bacterial vaginosis (BV) is a common vaginal disorder that affects women in reproductive age. This condition is characterized by a shift in the vaginal microflora from the dominant beneficial Lactobacillus to an overgrowth of strictly and anaerobic bacteria. It is noteworthy that BV involves the presence of multispecies biofilm, mainly composed by Gardnerella spp.. A major issue related to BV is the recurrent failure of conventionally antibiotic therapies. Therefore, essential oils (EOs) have been considered as a possible treatment option. Thymbra capitata EO has already demonstrated a good potential to eradicate bacteria. [...]info:eu-repo/semantics/publishedVersio