Gardnerella Vaginalis Alters Cervicovaginal Epithelial Cell Function Through Epithelial Cell-type Specific Immune Responses

Background: The cervicovaginal (CV) microbiome is highly associated with vaginal health and disease in both pregnant and non-pregnant individuals. An overabundance of Gardnerella vaginalis in the CV space is commonly associated with adverse reproductive outcomes including bacterial vaginosis (BV), sexually transmitted diseases and preterm birth while the presence of Lactobacillus spp is often associated with reproductive health. While host-microbial interactions are hypothesized to contribute to CV health and disease, the mechanisms by which these interactions regulate CV epithelial function remain largely unknown. Results: Using an in vitro co-culture model, we assessed the effects of Lactobacillus crispatus and G. vaginalis on the CV epithelial barrier, the immune mediators that could be contributing to decreased barrier integrity and the immune signaling pathways regulating the immune response. G. vaginalis, but not L. crispatus, significantly increased epithelial cell death and decreased epithelial barrier integrity in an epithelial cell-specific manner. A G. vaginalis-mediated epithelial immune response including NFkB activation and proinflammatory cytokine release was initiated partially through TLR2 dependent signaling pathways. Additionally, investigation of the cytokine immune profile in human CV fluid showed distinctive clustering of cytokines by G. vaginalis abundance and birth outcome. Conclusions: The results of this study show both microbe- and epithelial cell-type specific effects on CV epithelial function. Altered epithelial barrier function through cell death and immune mediated mechanisms by G. vaginalis, but not L. crispatus, indicates that host epithelial cells respond to bacteria-associated signals, resulting in altered epithelial function and ultimately CV disease. Additionally, distinct immune signatures associated with G. vaginalis or birth outcome provide further evidence that host-microbial interactions may contribute significantly to the biological mechanisms regulating reproductive outcomes.

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.

Agents causing genital infections in routine cytological tests: frequency and characteristics of Papanicolaou smears

Urinary tract infections are responsible for most human infections, these are caused by bacteria, fungi, protozoa and associated microorganisms. The goal of this study was to determine the rate of vaginal infection-causing agents in routine cytological exams and also to evaluate the characteristics of positive tested Pap smears. A retrospective documental with descriptive aspect research was performed in a Clinical Pathology laboratory from Maceió-AL. The results of the Pap smears exams for Trichomonas vaginalis, Gardnerella vaginalis, Candida spp and HPV were arranged in a database as well as other data such as bacterial, protozoan, fungal and viral coinfections. The sample was composed by 18.645 women who have undergone Pap smear exams from 2013 to 2017. Of these analyzed exams, 27.4% in 2013, 10.9% in 2014, 10.6% in 2015, 15.2% in 2016 and 13.67% in 2017 were within normal range, however more than half of these exams presented some infections caused by unspecific or microbiological agents. By analyzing all the reports, 4.073 (21.84%) presented inflammations caused by some species of infectious agent with the following rate order: G. vaginalis and T. vaginalis. Furthermore, it was possible to confirm high rates of coinfection by and Candida spp. The rate of genital infections in this study highlights that there is a public health matter that must be controlled, which points a greater need for monitoring, guidance and actions towards greater awareness in order to prevent these problems.

The Role of Genital Tract Microbiome in Fertility: A Systematic Review

The human microbiome plays a crucial role in determining the health status of every human being, and the microbiome of the genital tract can affect the fertility potential before and during assisted reproductive treatments (ARTs). This review aims to identify and appraise studies investigating the correlation of genital microbiome to infertility. Publications up to February 2021 were identified by searching the electronic databases PubMed/MEDLINE, Scopus and Embase and bibliographies. Only full-text original research articles written in English were considered eligible for analysis, whereas reviews, editorials, opinions or letters, case studies, conference papers, and abstracts were excluded. Twenty-six articles were identified. The oldest studies adopted the exclusive culture-based technique, while in recent years PCR and RNA sequencing based on 16S rRNA were the most used technique. Regardless of the anatomical site under investigation, the Lactobacillus-dominated flora seems to play a pivotal role in determining fertility, and in particular Lactobacillus crispatus showed a central role. Nonetheless, the presence of pathogens in the genital tract, such as Chlamydia trachomatis, Gardnerella vaginalis, Ureaplasma species, and Gram-negative stains microorganism, affected fertility also in case of asymptomatic bacterial vaginosis (BV). We failed to identify descriptive or comparative studies regarding tubal microbiome. The microbiome of the genital tract plays a pivotal role in fertility, also in case of ARTs. The standardization of the sampling methods and investigations approaches is warranted to stratify the fertility potential and its subsequent treatment. Prospective tubal microbiome studies are warranted.

The role of microbiota in pathogenesis and development of viral infections

Introduction and purpose The microbiota plays an important role in human metabolism, immune system, and development of many chronic diseases, cancers, and infectious diseases. The aim of the study is to present the role of gut microbiota in viral infections, including HBV, Herpesviridae, HIV, and SARS-CoV-2. The newest publications from the last 5 years available on the PubMed database were taken into account. A brief description of the state of knowledge The mechanism by which bacterias have an impact on viral infection is based on a synthesis of specific short-chain fatty acids (SCFAs) and modulation of cytokine release and immune system function by bacterias. Domination of Gardnerella vaginalis and lack of Lactobacillus in the vaginal microbiome increased the risk of HSV-2 infection in women. Inflammation of the genital tract can influence susceptibility to HIV infection, but probiotics via enhancement of the gut barrier integrity, change TH17/Treg ratio, can restore microbiome composition. LPS - component of the structure of Gram-negative bacteria can be a marker of HBV infection. In the airway microbiome of patients with COVID-19 opportunistic microorganisms were identified. Conclusions Diet, intake of probiotics, fecal microbiota transplantation (FMT) are interventions that might be efficient methods in prophylaxis and treatment of viral diseases. Further studies are needed to evaluate the mechanism of action of microbiome in pathogenesis of infectious diseases.

Age and sex trends of Gardnerella vaginalis infection in patients with sexually transmitted infections in Korea

Background and Objectives: Gardnerella vaginalis and Candida albicans are the most common causative agents of bac- terial vaginosis, and infections with these pathogens lead to inflammation, endometritis, and pruritus. The aim of this retro- spective study was to determine the trends of G. vaginalis infections based on real-time PCR data according to age and sex in patients with sexually transmitted diseases. Materials and Methods: A total of 59,381 specimens isolated at a clinical laboratory from September 2018 to December 2020 were subjected to real-time PCR for the detection of G. vaginalis DNA. Sample types included catheter, pus, tissue, swab, and urine samples. Results: Among 59,381 samples, 20,718 (34.8%) were positive for G. vaginalis. Of the positive samples, 13,186 (63.7%) were from male patients and 7,532 (36.3%) were from female patients. Average patient age was 39.1 years (the average age of male and female patients was 38.34 and 40.43 years, respectively). Female patients younger than 19 years exhibited the highest incidence of G. vaginalis, at 71.57%, followed by 68.46% incidence in those aged 20-29 years; the lowest incidence was in women aged 40-49 years. Further, among specimen types, the highest number of G. vaginalis-positive specimens was obtained by the swab sampling method. Conclusion: From 2018 to 2020 in Korea, the number of tests conducted for bacterial vaginosis has increased, while the incidence of G. vaginalis infections appears to have decreased. the finding that female adolescents have a high tendency to carry the pathogen is important. and for effective surveillance of BV, sampling by cotton swabs and detection by multiplex PCR might be a good approach.

Gardnerella Vaginalis in Recurrent Urinary Tract Infection is Associated with Dysbiosis of the Bladder Microbiome

Background: Recent studies on the urine microbiome have highlighted the importance of the gut-vagina-bladder axis in recurrent cystitis (RC). In particular, the role of Gardnerella as a covert pathogen that activates E. coli in animal experiments has been reported. Herein, we conducted a human bladder microbiome study to investigate the effect of Gardnerella on RC. Methods: Urine 16S ribosomal RNA gene sequencing via transurethral catheterization was conducted in the normal control group (NC) (n=18) and RC group (n=78). Results: The positive detection rate of Gardnerella species did not differ between the NC and RC groups (22.2% vs. 18.0%, p = 0.677). In addition, the Gardnerella-positive NC and Gardnerella-positive RC groups showed similar levels of microbiome diversity. The Gardnerella-positive group was categorized into three subgroups: Escherichia-dominant group, Gardnerella-dominant group, and Lactobacillus-dominant group, respectively. All of the Escherichia-dominant groups were associated with RC. Gardnerella-dominant or Lactobacillus-dominant groups expressed RC with symptoms when risk factors such as degree of Gardnerella proliferation or causative agents of bacterial vaginosis were present.Conclusion: Gardnerella can act as a covert pathogen of RC depending on other risk factors, and Gardnerella infection should be considered in patients with RC. New guideline recommendations regarding antibiotics selection based on a novel method to detect the cause of RC may be required to reduce antibiotics resistance.

Antimicrobial Activity of a Vaginal Gel Formulation: Considerations Related to Vaginal Infection and Dysbiosis

Many non-prescription preparations intended to treat or alleviate symptoms of vaginal infection are available in American and European markets, but many have scant preclinical or clinical research underpinning. Respecta®Balance Gel (RBG) is marketed as an adjunct to probiotic treatment and its relevant antimicrobial properties were studied. Key findings with the manufacturer-supplied gel showed reduced turbidity in broth-dilution tests by 50% against Candida albicans and Candida glabrata at RBG concentrations 0.2–0.4% of neat product, respectively. A 50% reduction in turbidity of Escherichia coli, Streptococcus agalactiae, Enterococcus faecalis ranged from 1.6–2.2% and Gardnerella vaginalis was shown by flow cytometry counts to undergo a 50% reduction at 0.3% RBG. Propidium iodide staining indicated a rapid reduction of cell integrity of G. vaginalis almost immediately while after 4 h 45% of E. coli cells were stained. The lactic acid in BHI inhibited bacteria and yeast at concentrations ranging from 0.2–1.8% but inhibition was not solely due to pH since a 1:4 dilution of RBG resulted in a pH near neutral (6.75). Other findings showed biofilm accumulation assessed after 10-days exposure of Candida spp. to RBG and was reduced by an average of one-third (community strains) to one-half (drug-resistant strains). One excipient of the RBG, disodium EDTA, inhibited the growth of bacteria and yeast at concentrations below those present in RBG and may accentuate the activity of the host defense factor, lactoferrin. We conclude that RBG is a potent inhibitor of vaginal microorganisms relevant to vaginitis or intrapartum infections and contains excipients that may contribute to its antimicrobial activity.

Tilapia Piscidin 4 (TP4) Reprograms M1 Macrophages to M2 Phenotypes in Cell Models of Gardnerella vaginalis-Induced Vaginosis

Gardnerella vaginalis is associated with bacterial vaginosis (BV). The virulence factors produced by G. vaginalis are known to stimulate vaginal mucosal immune response, which is largely driven by activated macrophages. While Tilapia piscidin 4 (TP4), an antimicrobial peptide isolated from Nile tilapia, is known to display a broad range of antibacterial functions, it is unclear whether TP4 can affect macrophage polarization in the context of BV. In this study, we used the culture supernatants from G. vaginalis to stimulate differentiation of THP-1 and RAW264.7 cells to an M1 phenotype. The treatment activated the NF-κB/STAT1 signaling pathway, induced reactive nitrogen and oxygen species, and upregulated inflammatory mediators. We then treated the induced M1 macrophages directly with a non-toxic dose of TP4 or co-cultured the M1 macrophages with TP4-treated vaginal epithelial VK2 cells. The results showed that TP4 could not only decrease pro-inflammatory mediators in the M1 macrophages, but it also enriched markers of M2 macrophages. Further, we found that direct treatment with TP4 switched M1 macrophages toward a resolving M2c phenotype via the MAPK/ERK pathway and IL-10-STAT3 signaling. Conversely, tissue repair M2a macrophages were induced by TP4-treated VK2 cells; TP4 upregulated TSG-6 in VK2 cells, which subsequently activated STAT6 and M2a-related gene expression in the macrophages. In conclusion, our results imply that TP4 may be able to attenuate the virulence of G. vaginalis by inducing resolving M2c and tissue repair M2a macrophage polarizations, suggesting a novel strategy for BV therapy.