Inhibitory effect of Lactobacillus gasseri CCFM1201 on Gardnerella vaginalis in mice with bacterial vaginosis.

Gardnerella vaginalis is the core pathogen of bacterial vaginosis (BV), the most common vaginal infection in women. G. vaginalis exerts pathogenicity through various factors, such as biofilm formation and the local host immune response stimulation. Therefore, this study aimed to evaluate the inhibitory effect of Lactobacillus gasseri CCFM1201 on G. vaginalis using experimental BV models. We evaluated L. gasseri in vitro to inhibit pathogen biofilm formation and adhesion capacity in HeLa cells using crystal violet staining. Further in vivo studies were conducted to assess the inhibitory effects of L. gasseri CCFM1201 on BV induced by G. vaginalis. L. gasseri exhibited strain-specific adhesion and inhibition of pathogen biofilm formation in vitro. L. gasseri CCFM1201 significantly reduced G. vaginalis in mice (p < 0.05), inhibited sialidase activity, modulated tumor necrosis factor-α and interleukin-1ß expression, and reduced myeloperoxidase activity (p < 0.05). Histopathological examination indicated that L. gasseri CCFM1201 improved inflammatory cell infiltration of vaginal tissue and restored its structure. Vaginal epithelial cell exfoliation, the main clinical feature of BV, was significantly improved by L. gasseri CCFM1201 intervention (p < 0.05). Thus, L. gasseri CCFM1201 is a potential candidate for treating G. vaginalis-induced vaginal diseases.

Characterization of Gardnerella vaginalis membrane vesicles reveals a role in inducing cytotoxicity in vaginal epithelial cells.

Bacterial vaginosis (BV) is a common polymicrobial infection affecting women in the reproductive age and is associated with adverse obstetric and gynaecological outcomes. Gardnerella vaginalis is the most virulent anaerobic bacterial species predominantly associated with BV. However, a clear understanding of the mechanisms by which it contributes to the pathogenesis and persistence of BV is lacking. In this report, we demonstrate for the first time, the isolation of membrane vesicles (MVs) from G. vaginalis ATCC 14019. These MVs are approximately 120-260 nm in diameter. Proteomic characterization of the MVs by LC-MS/MS led to the identification of 417 proteins, including proteins involved in cellular metabolism as well as molecular chaperones and certain virulence factors. Immunoblot analysis of the MVs confirmed the presence of vaginolysin, the most well-characterized virulence factor of G. vaginalis. The exposure of the vaginal epithelial cells, VK2/E6E7 to the G. vaginalis MVs resulted in the internalization of the MVs. The MVs induced cytotoxicity and an increase in the levels of the pro-inflammatory cytokine, IL-8 in VK2 cells as well lysis of erythrocytes. The results of the study indicate that G. vaginalis MVs may be involved in the delivery of cytotoxic proteins and other virulence factors to the host cells and could thereby contribute towards enhancing the cellular damage associated with pathogenesis of BV.

Inhibition of sialidase activity and cellular invasion by the bacterial vaginosis pathogen Gardnerella vaginalis.

Bacterial vaginosis is a genital tract infection, thought to be caused by transformation of a lactobacillus-rich flora to a dysbiotic microbiota enriched in mixed anaerobes. The most prominent of these is Gardnerella vaginalis (GV), an anaerobic pathogen that produces sialidase enzyme to cleave terminal sialic acid residues from human glycans. Notably, high sialidase activity is associated with preterm birth and low birthweight. We explored the potential of the sialidase inhibitor Zanamavir against GV whole cell sialidase activity using methyl-umbelliferyl neuraminic acid (MU-NANA) cleavage assays, with Zanamavir causing a 30% reduction in whole cell GV sialidase activity (p < 0.05). Furthermore, cellular invasion assays using HeLa cervical epithelial cells, infected with GV, demonstrated that Zanamivir elicited a 50% reduction in cell association and invasion (p < 0.05). Our data thus highlight that pharmacological sialidase inhibitors are able to modify BV-associated sialidase activity and influence host-pathogen interactions and may represent novel therapeutic adjuncts.

Polybacterial stimulation suggests discrete IL-6/IL-6R signaling in human fetal membranes: Potential implications on IL-6 bioactivity.

The polybacterial invasion of the amniotic cavity and risk of preterm birth is often due to cervicovaginal bacteria such as genital mycoplasmas (Mycoplasma hominis and Ureaplasma urealyticum) and Gardnerella vaginalis. The most studied biomarker associated with preterm birth is interleukin-6 (IL-6), a pleiotropic cytokine that performs different functions based on classical or trans-signaling mechanisms. This study evaluated the changes in IL-6 and IL-6 function associated accessory molecules by human fetal membranes to determine the functional availability of IL-6 assessment in an in vitro model of polybacterial infection. Fetal membranes were treated with LPS or heat-inactivated genital mycoplasmas and G. vaginalis alone or in combination. IL-6 and its soluble receptors (sgp130, sIL-6R) were assessed in conditioned medium by immunoassays and membrane-bound receptors were evaluated in the tissue using immunohistochemistry and RT-PCR. Data from protein and gene expression were evaluated using linear mixed effects models. Data from immunohistochemistry were evaluated using one-way analysis of variance followed by the Tukey test. Genital mycoplasmas alone, or in combination, inhibited IL-6 trans-signaling with increased sgp130 production. G. vaginalis activated the classical IL-6 signaling pathway, as did LPS. Polybacterial treatment resulted in a balanced response with neither pathway being favored. The increase in IL-6 production by fetal membranes in response to infection is likely a non-specific innate response and not an indicator of a functional mediator of any labor-inducing pathways. This suggests that correlating the risk of adverse pregnancy outcomes and designing interventions based on IL-6 levels without considering soluble receptors may be an ineffective strategy.

Higher prevalence of colonization with Gardnerella vaginalis and gram-negative anaerobes in patients with recurrent miscarriage and elevated peripheral natural killer cells.

The role of vaginal infections in recurrent miscarriage (RM) is discussed controversially and screening is not recommended in international guidelines. Peripheral and uterine NK cells (pNK, uNK) play an important role in the establishment of a healthy pregnancy and are targets of immune diagnostics in RM patients. The aim of this study was to analyze the composition of the vaginal microbiota in RM patients and to correlate the findings to clinical characteristics as well as NK cell parameters. In total, n=243 RM patients with ≥3 consecutive miscarriages were recruited between 11/2011 and 03/2016. Vaginal swabs were analyzed by microbiological culture. Further, a cervical swab was taken in n=187 patients and the presence of Chlamydia trachomatis was evaluated by a molecular assay. Peripheral blood levels of CD45+CD3-CD56+CD16+ pNK (determined by four-color fluorescence flow cytometry) and CD56+ uNK (uterine biopsy, determined by immunohistochemistry) were analyzed. The prevalence of Gardnerella vaginalis colonization in RM patients was 19.0%, gram-negative anaerobes 20.5%, Candida species 7.9%, group B Streptococcus 11.0% and Enterobacteriaceae 14.8%. Commensal lactobacilli were absent in 14.5% of the women. Chlamydia trachomatis was detected in n=1 case (0.53%). The prevalence of Gardnerella vaginalis and gram-negative anaerobes in RM patients with elevated pNK (>280/µl, n=69) was significantly higher (p=0.012, p=0.04) compared to patients with normal pNK (n=174). In conclusion, RM patients with elevated pNK suffer more often from colonization by Gardnerella vaginalis and gram-negative anaerobes. This might indicate an association between the vaginal microbiota, local inflammation, changes in immune parameters and miscarriage.

Thymbra capitata essential oil as potential therapeutic agent against Gardnerella vaginalis biofilm-related infections.

AIM: To evaluate the antibacterial activity of Thymbra capitata essential oil and its main compound, carvacrol, against Gardnerella vaginalis grown planktonically and as biofilms, and its effect of vaginal lactobacilli. MATERIALS & METHODS: Minimal inhibitory concentration, minimal lethal concentration determination and flow cytometry analysis were used to assess the antibacterial effect against planktonic cells. Antibiofilm activity was measured through quantification of biomass and visualization of biofilm structure by confocal laser scanning microscopy. RESULTS: T. capitata essential oil and carvacrol exhibited a potent antibacterial activity against G. vaginalis cells. Antibiofilm activity was more evident with the essential oil than carvacrol. Furthermore, vaginal lactobacilli were significantly more tolerant to the essential oil. CONCLUSION: T. capitata essential oil stands up as a promising therapeutic agent against G. vaginalis biofilm-related infections.

New Systems for Studying Intercellular Interactions in Bacterial Vaginosis.

Bacterial vaginosis (BV) affects almost a quarter of US women, making it a condition of major public health relevance. Key questions remain regarding the etiology of BV, mechanisms for its association with poor reproductive health outcomes, and reasons for high rates of treatment failure. New model systems are required to answer these remaining questions, elucidate the complex host-microbe and microbe-microbe interactions, and develop new, effective interventions. In this review, we cover the strengths and limitations of in vitro and in vivo model systems to study these complex intercellular interactions. Furthermore, we discuss advancements needed to maximize the translational utility of the model systems. As no single model can recapitulate all of the complex physiological and environmental conditions of the human vaginal microenvironment, we conclude that combinatorial approaches using in vitro and in vivo model systems will be required to address the remaining fundamental questions surrounding the enigma that is BV.

Gardnerella vaginalis outcompetes 29 other bacterial species isolated from patients with bacterial vaginosis, using in an in vitro biofilm formation model.

Despite the worldwide prevalence of bacterial vaginosis (BV), its etiology is still unknown. Although BV has been associated with the presence of biofilm, the ability of BV-associated bacteria to form biofilms is still largely unknown. Here, we isolated 30 BV-associated species and characterized their virulence, using an in vitro biofilm formation model. Our data suggests that Gardnerella vaginalis had the highest virulence potential, as defined by higher initial adhesion and cytotoxicity of epithelial cells, as well as the greater propensity to form a biofilm. Interestingly, we also demonstrated that most of the BV-associated bacteria had a tendency to grow as biofilms.

Reciprocal interference between Lactobacillus spp. and Gardnerella vaginalis on initial adherence to epithelial cells.

Bacterial vaginosis (BV) is the most common vaginal disorder in women of child-bearing age. It is widely accepted that the microbial switch from normal microflora to the flora commonly associated with BV is characterized by a decrease in vaginal colonization by specific Lactobacillus species together with an increase of G. vaginalis and other anaerobes. However, the order of events leading to the development of BV remains poorly characterized and it is unclear whether the decrease in lactobacilli is a cause or a consequence of the increase in the population density of anaerobes. Our goal was to characterize the interaction between two Gardnerella vaginalis strains, one of which was isolated from a healthy woman (strain 5-1) and the other from a woman diagnosed with BV (strain 101), and vaginal lactobacilli on the adherence to cervical epithelial cells. In order to simulate the transition from vaginal health to BV, the lactobacilli were cultured with the epithelial cells first, and then the G. vaginalis strain was introduced. We quantified the inhibition of G. vaginalis adherence by the lactobacilli and displacement of adherent lactobacilli by G. vaginalis. Our results confirmed that pathogenic G vaginalis 101 had a higher capacity for adhesion to the cervical epithelial cells than strain 5-1. Interestingly, strain 101 displaced L. crispatus but not L. iners whereas strain 5-1 had less of an effect and did not affect the two species differently. Furthermore, L. iners actually enhanced adhesion of strain 101 but not of strain 5-1. These results suggest that BV-causing G. vaginalis and L. iners do not interfere with one another, which may help to explain previous reports that women who are colonized with L. iners are more likely to develop BV.

Quantitative analysis of initial adhesion of bacterial vaginosis-associated anaerobes to ME-180 cells.

Bacterial vaginosis is the leading vaginal disorder but the transition from health to this dysbiotic condition remains poorly characterized. Our goal was to quantify the ability of BV-associated anaerobes to adhere to epithelial cells in the presence of lactobacilli. Gardnerella vaginalis outcompeted Lactobacillus crispatus and Lactobacillus iners actually enhanced its adherence.

Clinical features of bacterial vaginosis in a murine model of vaginal infection with Gardnerella vaginalis.

Bacterial vaginosis (BV) is a dysbiosis of the vaginal flora characterized by a shift from a Lactobacillus-dominant environment to a polymicrobial mixture including Actinobacteria and gram-negative bacilli. BV is a common vaginal condition in women and is associated with increased risk of sexually transmitted infection and adverse pregnancy outcomes such as preterm birth. Gardnerella vaginalis is one of the most frequently isolated bacterial species in BV. However, there has been much debate in the literature concerning the contribution of G. vaginalis to the etiology of BV, since it is also present in a significant proportion of healthy women. Here we present a new murine vaginal infection model with a clinical isolate of G. vaginalis. Our data demonstrate that this model displays key features used clinically to diagnose BV, including the presence of sialidase activity and exfoliated epithelial cells with adherent bacteria (reminiscent of clue cells). G. vaginalis was capable of ascending uterine infection, which correlated with the degree of vaginal infection and level of vaginal sialidase activity. The host response to G. vaginalis infection was characterized by robust vaginal epithelial cell exfoliation in the absence of histological inflammation. Our analyses of clinical specimens from women with BV revealed a measureable epithelial exfoliation response compared to women with normal flora, a phenotype that, to our knowledge, is measured here for the first time. The results of this study demonstrate that G. vaginalis is sufficient to cause BV phenotypes and suggest that this organism may contribute to BV etiology and associated complications. This is the first time vaginal infection by a BV associated bacterium in an animal has been shown to parallel the human disease with regard to clinical diagnostic features. Future studies with this model should facilitate investigation of important questions regarding BV etiology, pathogenesis and associated complications.

Presence of a polymicrobial endometrial biofilm in patients with bacterial vaginosis.

OBJECTIVE: To assess whether the bacterial vaginosis biofilm extends into the upper female genital tract. STUDY DESIGN: Endometrial samples obtained during curettage and fallopian tube samples obtained during salpingectomy were collected. Endometrial and fallopian tube samples were analyzed for the presence of bacteria with fluorescence-in-situ-hybridisation (FISH) analysis with probes targeting bacterial vaginosis-associated and other bacteria. RESULTS: A structured polymicrobial Gardnerella vaginalis biofilm could be detected in part of the endometrial and fallopian tube specimens. Women with bacterial vaginosis had a 50.0% (95% CI 24.0-76.0) risk of presenting with an endometrial Gardnerella vaginalis biofilm. Pregnancy (AOR  = 41.5, 95% CI 5.0-341.9, p<0.001) and the presence of bacterial vaginosis (AOR  = 23.2, 95% CI 2.6-205.9, p<0.001) were highly predictive of the presence of uterine or fallopian bacterial colonisation when compared to non-pregnant women without bacterial vaginosis. CONCLUSION: Bacterial vaginosis is frequently associated with the presence of a structured polymicrobial Gardnerella vaginalis biofilm attached to the endometrium. This may have major implications for our understanding of the pathogenesis of adverse pregnancy outcome in association with bacterial vaginosis.

An adherent Gardnerella vaginalis biofilm persists on the vaginal epithelium after standard therapy with oral metronidazole.

OBJECTIVE: The purpose of this study was to determine the efficacy of standard treatment with oral metronidazole in the eradication of the bacterial vaginosis biofilm. STUDY DESIGN: We conducted an interventional follow-up study in which 18 patients with bacterial vaginosis were treated with oral metronidazole during 1 week and subsequently had a single random follow-up assessment at 1-week intervals, up to 5 weeks, with 3 patients representing each point in time. Follow-up assessment included conventional scoring of the vaginal microflora and determination of bacterial biofilm characteristics on a vaginal biopsy through bacterial 16/23S recombinant DNA-based fluorescence in-situ hybridization. RESULTS: Although all patients recovered, we consistently observed the resurgence with treatment cessation of a dense and active bacterial biofilm on the vaginal mucosa, primarily consisting of Gardnerella vaginalis and Atopobium vaginae. CONCLUSION: A large reservoir of the core bacteria to bacterial vaginosis persists as a biofilm after metronidazole treatment.

Estudio descriptivo sobre gardnerella vaginalis asociada a leucorrea en 112 pacientes

Se describe un estudio sobre leucorrea asociada a Gardnerella vaginalis demostrada en 112 pacientes que consultaron entre el 1 de Octubre de 1989 y el 31 de Marzo de 1993. La infección por Gardnerella se estableció según los siguientes criterios: Flujo homogéneo y adherente a la pared vaginal, pH mayor de 4.5, prueba de aminas positiva si se alcaliniza la secresión vaginal y presencia de "células guía" o "clave" en los extendidos citológicos. Se describen las principales manifestaciones clínicas encontradas en el presente estudio que pueden sugerir leucorrea asociada a G. vaginalis haciendo énfasis en la nila o escasa reacción inflamatoria y la ausencia de bacilos de Doderlein.

Phosphorylation and activation of the arachidonate-mobilizing phospholipase A2 in macrophages in response to bacteria.

The role of potential target enzymes in the protein-kinase-C-independent eicosanoid response triggered by certain bacteria in murine peritoneal macrophages [Svensson, U., Holst, E. & Sundler, R. (1991) Eur. J. Biochem. 202, 699-705] has been investigated. The eicosanoid response was found to be due to an increase in the mobilization of arachidonate rather than to inhibition of arachidonate esterification or activation of the cyclooxygenase pathway and to be accompanied by a persistent increase in the activity of the arachidonate-mobilizing phospholipase A2 (PLA2-85). Also, down-regulation of protein-kinase C by prolonged treatment with 4 beta-phorbol 12-myristate 13-acetate did not reduce the bacterial activation of PLA2-85. The increase in activity of PLA2-85, like the increase in eicosanoid formation, showed a lag period of approximately 10 min. Furthermore, exposure of 32P-labeled macrophages to either bacteria (Gardnerella vaginalis) or the protein-phosphatase inhibitor okadaic acid caused an increase in the phosphorylation of PLA2-85. Okadaic acid (0.5 microM), which itself caused arachidonate mobilization and activation of PLA2-85 after a lag period of approximately 45 min, greatly promoted the response to bacteria even at earlier time points. This study provides strong evidence that the eicosanoid response to bacteria in macrophages occurs via a protein-kinase-C-independent activation of PLA2-85 and that this activation is due to an increase in the phosphorylation of the enzyme.

[Clinical and colposcopic aspects of bacterial vaginosis]. / Aspects cliniques et colposcopiques des vaginoses bactériennes.

Bacterial vaginosis is characterized by an uniform, malodorous, white-grey discharge. The presenting symptom is generally the unpleasant smell of the vaginal discharge, particularly following the menses or intercourse. Other functional signs, such as pruritus, dysuria and dyspareunia are rare. Inflammatory signs are frequent, and can be revealed by colposcopy with the Lugol test: this shows punctuate colpitis with small regular points corresponding histologically to an inflammatory focus in the connective tissue. The term "vaginitis" is avoided because of the absence of polymorphonuclear cells in the vaginal discharge, despite the presence of inflammation. Bacterial vaginosis has been held responsible for prematurity, small birthweight and post-partum infection. Nonetheless, Gardnerella vaginalis and Mobiluncus spp can be recovered from the vaginal flora of women with no signs of inflammation.

Protein-kinase-C-independent activation of arachidonate release and prostaglandin E2 formation in macrophages interacting with certain bacteria.

Certain bacterial species, of which we selected Fusobacterium nucleatum, Gardnerella vaginalis, Peptostreptococcus anaerobius and Propionibacterium acnes, were found to induce release of arachidonic acid in a dose- and time-dependent manner in mouse macrophages. The release of arachidonic acid showed a characteristic lag period of approximately 10 min and was accompanied by selective transformation into prostaglandin E2. Bacteria killed by various methods caused a similar response, indicating that bacterial surface structures rather than secreted products were involved. Down-regulation of protein kinase C by treatment of macrophages with 4 beta-phorbol 12-myristate 13-acetate hardly affected the response to bacteria at all, except for a partial inhibition in the case of P. acnes. Furthermore, the generation of prostaglandin E2 was synergistically enhanced when macrophages were exposed to both bacteria and phorbol ester. It is also unlikely that bacterial activation was mediated exclusively via a rise in cytosolic [Ca2+], since bacteria stimulated the release of arachidonic acid also when [Ca2+] was clamped at various levels and since the response to bacteria was enhanced in an additive to synergistic manner when combined with calcium ionophore. Changes in protein phosphorylation in macrophages exposed to F. nucleatum (Gram-negative) were virtually identical to those seen with bacterial lipopolysaccharide, while P. anaerobius (Gram-positive) induced enhanced labeling of a single detectable phosphoprotein. In both cases, the changes in protein phosphorylation showed a time lag of 4-8 min and occurred independently of protein kinase C, consistent with a possible role in signal transduction. These results demonstrate that certain bacteria cause activation of arachidonic acid release and prostaglandin E2 formation in mouse macrophages; that the response is independent of protein kinase C and that it is not wholly mediated via a rise in cytosolic [Ca2+].