In vitro biofilm formation of Gardnerella vaginalis and Escherichia coli associated with bacterial vaginosis and aerobic vaginitis.

Objective: To determine the optimum biofilm formation ratio of Gardnerella vaginalis (G. vaginalis) in a mixed culture with Escherichia coli (E. coli). Methods: G. vaginalis ATCC14018, E. coli ATCC25922, as well as five strains of G. vaginalis were selected from the vaginal sources of patients whose biofilm forming capacity was determined by the Crystal Violet method. The biofilm forming capacity of E. coli in anaerobic and non-anaerobic environments were compared using the identical assay. The Crystal Violet method was also used to determine the biofilm forming capacity of a co-culture of G. vaginalis and E. coli in different ratios. After Live/Dead staining, biofilm thickness was measured using confocal laser scanning microscopy, and biofilm morphology was observed by scanning electron microscopy. Results: The biofilm forming capacity of E. coli under anaerobic environment was similar to that in a 5% CO2 environment. The biofilm forming capacity of G. vaginalis and E. coli was stronger at 106:105 CFU/mL than at other ratios (P<0.05). Their thicknesses were greater at 106:105 CFU/mL than at the other ratios, with the exception of 106:102 CFU/mL (P<0.05), under laser scanning microscopy. Scanning electron microscopy revealed increased biofilm formation at 106:105 CFU/mL and 106:102 CFU/mL, but no discernible E. coli was observed at 106:102 CFU/mL. Conclusion: G. vaginalis and E. coli showed the greatest biofilm forming capacity at a concentration of 106:105 CFU/mL at 48 hours and could be used to simulate a mixed infection of bacterial vaginosis and aerobic vaginitis in vitro.

Quantitative modeling predicts mechanistic links between pre-treatment microbiome composition and metronidazole efficacy in bacterial vaginosis.

Bacterial vaginosis is a condition associated with adverse reproductive outcomes and characterized by a shift from a Lactobacillus-dominant vaginal microbiota to a polymicrobial microbiota, consistently colonized by strains of Gardnerella vaginalis. Metronidazole is the first-line treatment; however, treatment failure and recurrence rates remain high. To understand complex interactions between Gardnerella vaginalis and Lactobacillus involved in efficacy, here we develop an ordinary differential equation model that predicts bacterial growth as a function of metronidazole uptake, sensitivity, and metabolism. The model shows that a critical factor in efficacy is Lactobacillus sequestration of metronidazole, and efficacy decreases when the relative abundance of Lactobacillus is higher pre-treatment. We validate results in Gardnerella and Lactobacillus co-cultures, and in two clinical cohorts, finding women with recurrence have significantly higher pre-treatment levels of Lactobacillus relative to bacterial vaginosis-associated bacteria. Overall results provide mechanistic insight into how personalized differences in microbial communities influence vaginal antibiotic efficacy.

Antibacterial Activity of Lactobacillus Strains Isolated from Mongolian Yogurt against Gardnerella vaginalis.

Worldwide interest in the use of functional foods containing probiotic bacteria such as Lactobacillus and Bifidobacterium for health promotion and disease prevention has increased significantly. Probiotics have demonstrated beneficial properties including strengthening the body's natural defense system, inhibiting the growth of pathogenic bacteria, and regulating mental activity, but their effects on the human vagina have not been fully elucidated. The primary purpose of our study was to isolate Lactobacillus strains from old yogurt, a traditional dairy product, and investigate their probiotic potential with respect to the human vaginal system. Four Lactobacillus plantarum (L. plantarum) strains, named ZX1, ZX2, ZX27, and ZX69, were isolated from the yogurt samples. Simultaneously, we used a commercial Lactobacillus strain (Lactobacillus delbrueckii DM8909) as a control strain. We tested the antimicrobial activity of Lactobacillus isolates against Escherichia coli and Gardnerella vaginalis by agar spot and well diffusion tests. Then, we tested the antibiotic susceptibility of the 5 strains by using the minimal inhibitory concentration method. We attempted to detect possible bacteriocin genes by PCR sequencing technique. Using a chemically defined medium simulating genital tract secretions, we found that the selected Lactobacillus strains could alter the expression of known virulence genes in Gardnerella vaginalis. Bacteriocins derived from these isolated strains had potent antibacterial activity against G. vaginalis and E. coli, with the most effective activity observed in the case of ZX27. In addition, all strains including the L. delbrueckii DM8909 were positive for the presence of the plantaricin cluster of genes described in L. plantarum C11. The tested stains possessed the pln gene indicating that one of the antibacterial agents was plantaricin. We assume that the production of antimicrobial substances such as bacteriocins induce G. vaginalis to upregulate antimicrobial resistance genes. The new isolated strains have bacteriocin-related genes and can change the antimicrobial resistance gene transcription of G. vaginalis.

Gardnerella vaginalis Enhances Atopobium vaginae Viability in an in vitro Model.

Bacterial vaginosis (BV) is the most common vaginal infection among women of reproductive age. A hallmark of BV is the presence of a highly structured polymicrobial biofilm on the vaginal epithelium, presumably initiated by facultative anaerobes of the genus Gardnerella, which then becomes a scaffold for other species to adhere to. One of the species often found incorporated in Gardnerella mediated biofilms is Atopobium vaginae. Interestingly, A. vaginae is very rarely found without the presence of Gardnerella. However, not much is known regarding the interactions between A. vaginae and Gardnerella species. This study assessed biological interactions between Gardnerella vaginalis and A. vaginae. In our in vitro model, by using specific Gardnerella and A. vaginae Peptide Nucleic Acid (PNA)-Fluorescence In Situ Hybridization (FISH) probes, we confirmed that A. vaginae was able to incorporate a pre-formed G. vaginalis biofilm, accounting for up to 20% of the total number of biofilm cells. However, our findings showed that almost 92% of A. vaginae cells lost viability after 48 h of mono-species planktonic growth, but were able to maintain viability when co-cultured with Gardnerella or after pre-conditioning with cell-free supernatant of Gardnerella cultures. While the in vitro conditions are very different from the in vivo microenvironment, this study contributes to a better understanding of why A. vaginae vaginal colonization rarely occurs in the absence of Gardnerella. Overall, this highlights the importance of microbial interactions between BV-associated bacteria and demands more studies focused on the polymicrobial bacterial communities found in BV.

An Updated Conceptual Model on the Pathogenesis of Bacterial Vaginosis.

Bacterial vaginosis (BV) is the most common cause of vaginal discharge. It is associated with an increased risk of preterm delivery, pelvic inflammatory disease, and an increased risk of acquisition of sexually transmitted infections including human immunodeficiency virus (HIV). The epidemiology of BV supports sexual transmission. However, its etiology remains unknown. At the center of the debate is whether BV is caused by a primary pathogen or a polymicrobial consortium of microorganisms that are sexually transmitted. We previously published a conceptual model hypothesizing that BV is initiated by sexual transmission of Gardnerella vaginalis. Critics of this model have iterated that G. vaginalis is found in virginal women and in sexually active women with a normal vaginal microbiota. In addition, colonization does not always lead to BV. However, recent advances in BV pathogenesis research have determined the existence of 13 different species within the genus Gardnerella. It may be that healthy women are colonized by nonpathogenic Gardnerella species, whereas virulent strains are involved in BV development. Based on our results from a recent prospective study, in addition to an extensive literature review, we present an updated conceptual model for the pathogenesis of BV that centers on the roles of virulent strains of G. vaginalis, as well as Prevotella bivia and Atopobium vaginae.

Selection of New Probiotics for Endometrial Health.

Microbiota is a crucial player in gynecologic health, in which bacteria can shift to a dysbiotic state triggering a pathogenic process. Based on an ecological understanding of the problem, the aim of this study is to select a potential probiotic strain to improve female reproductive tract based on its capacity to initially lower pH and to promote the reduction of pathogenic bacteria. Based on this rationale, strain Lactobacillus rhamnosus BPL005 was initially selected for its capacity to reduce in vitro pH levels and produce organic acids. Subsequently, strain L. rhamnosus BPL005 (CECT 8800) was demonstrated to have a protective role on endometrial infections in an in vitro model of bacterial colonization of primary endometrial epithelial cells with Atopobium vaginae, Gardnerella vaginalis, Propionibacterium acnes, and Streptococcus agalactiae. In this model, BPL005 when co-cultured with those pathogens was shown to lower pH and to produce organic acids, being lactic acid the most relevant. The co-cultivation of strain L. rhamnosus BPL005 with tested reference pathogens produced a significant reduction in P. acnes and St. agalactiae levels and a non-significant reduction in A. vaginae and G. vaginalis. The colonization of L. rhamnosus BPL005 in the culture decreased IL-6, IL-8, and MCP-1, heightened in the presence of pathogens, and increased IL-1RA and IL-1 beta. Finally, safety was evaluated showing no signs of cytotoxicity, irritation in vaginal tests, or allergic contact dermatitis potential through the Local Lymph Node Assay. Overall, these results show the potential of L. rhamnosus BPL005 strain as a probiotic in gynecological health.

Potential vaginal probiotics: safety, tolerability and preliminary effectiveness.

Vaginal discharge is one of the common reasons for gynaecologist consultation, as bacterial vaginosis and candidiasis are the main causes of discharge. These patients frequently experience numerous problems due to recurrent infections, side effects and drug resistance therefore alternative drugs are needed. Our primary aim was to evaluate safety and tolerability of the potentially probiotic Lactobacillus crispatus strains in volunteer women considering themselves healthy. We also monitored the effects of these strains on vaginal health parameters and lactobacilli counts in vagina and intestine. Forty women were recruited into trial. Absence of chronic diseases was confirmed by questionnaire and blood analysis at screening visit. In randomised double-blind placebo-controlled crossover study the eligible participants were randomly allocated to one of four groups and had to consume one of the two study products (Pro I or Pro II) - a capsule containing 3 strains, 109 cfu per strain, or placebo for 1 week. Treatment period was followed by 2-week washout period and continued with second treatment and washout period. Individuals receiving firstly probiotic, received later placebo and vice versa. Blood, vaginal and faecal samples were collected, and self-reported questionnaires were applied. Thirty subjects completed the trial. The probiotic capsules were well-tolerated. The Pro II intake resulted in a significant decrease in Nugent score (from median 3.0 to 2.0, mean 3.9 to 2.6, P=0.002) and reduction in Gardnerella vaginalis counts (log10 3.57 to 2.38; P=0.027). Reduction of total vaginal bacterial counts was revealed in Pro I group (log10 7.99 to 7.72; P=0.048). In conclusion, the selected vaginal L. crispatus strains are well tolerable and Pro II mixture is prospectively effective in reducing Nugent score and vaginal counts of G. vaginalis. Therefore, these strains seem to be promising candidates for development of novel evidence-based well-focused probiotics to target female urogenital tract disorders.

Antimicrobial and inflammatory properties of South African clinical Lactobacillus isolates and vaginal probiotics.

Bacterial vaginosis (BV) causes genital inflammation and increased HIV acquisition risk. The standard-of-care for BV, antibiotic therapy, is associated with high recurrence rates. Probiotics may improve treatment outcomes, although substantial heterogeneity in efficacy has been observed during clinical trials. To evaluate the potential to improve existing probiotics, we compared the inflammatory and antimicrobial (adhesion, H2O2, D-lactate and L-lactate production) characteristics of 23 vaginal Lactobacillus isolates from South African women, commercial vaginal probiotics (L. casei rhamnosus, L. acidophilus) and 4 reference strains. All lactobacilli induced inflammatory cytokine production by genital epithelial cells and produced D-lactate. Of six isolates assessed, five suppressed inflammatory responses to Gardnerella vaginalis. Although the L. acidophilus probiotic was the most adherent, many clinical isolates produced greater amounts of H2O2, D-lactate and L-lactate than the probiotics. The most L-lactate and H2O2 were produced by L. jensenii (adjusted p = 0.0091) and L. mucosae (adjusted p = 0.0308) species, respectively. According to the characteristics evaluated, the top 10 isolates included 4 L. jensenii, 2 L. crispatus, 1 L. mucosae, 1 L. vaginalis and the L. acidophilus probiotic. There is potential to develop an improved vaginal probiotic using clinical Lactobacillus isolates. Inflammatory profiles are critical to evaluate as some isolates induced substantial cytokine production.

Interaction of Gardnerella vaginalis and Vaginolysin with the Apical versus Basolateral Face of a Three-Dimensional Model of Vaginal Epithelium.

Studies have implicated Gardnerella vaginalis as an important etiological agent in bacterial vaginosis (BV). It produces a cholesterol-dependent cytolysin, vaginolysin (VLY). In this study, we sought to characterize the interaction between vaginal epithelium, G. vaginalis, and VLY using EpiVaginal tissues from MatTek. These tissues are three-dimensional and have distinct apical and basolateral sides, enabling comparison of the effects of G. vaginalis and VLY following exposure to either side. We measured cytotoxicity, cytokine production, and bacterial growth, following apical versus basolateral exposure. G. vaginalis exhibited more-rapid growth in coculture with the tissue model when it was exposed to the apical side. VLY permeabilized cells on the basolateral side of the tissues but failed to permeabilize apical epithelial cells. Cytokine secretion in response to VLY and G. vaginalis also depended on the polarity of exposure. VLY did not cause significant changes in cytokine levels when exposed apically. Apical tissue challenge by G. vaginalis appeared to dampen the inflammatory response, as decreases in granulocyte-macrophage colony-stimulating factor (GM-CSF) (6.6-fold), RANTES (14.8-fold), and interferon gamma inducible protein 10 kDa (IP-10) (53-fold) and an increase in interleukin-1 receptor antagonist (IL-1ra) (5-fold) were observed. In vivo, G. vaginalis normally colonizes the apical face of the vaginal epithelium. Results from this study suggest that while G. vaginalis may grow on the apical face of the vaginal epithelium, its VLY toxin does not target these cells in this model. This phenomenon could have important implications regarding colonization of the vagina by G. vaginalis and may suggest an explanation for the lack of an overt immune response to this organism.

Significant increase in cultivation of Gardnerella vaginalis, Alloscardovia omnicolens, Actinotignum schaalii, and Actinomyces spp. in urine samples with total laboratory automation.

While total laboratory automation (TLA) is well established in laboratory medicine, only a few microbiological laboratories are using TLA systems. Especially in terms of speed and accuracy, working with TLA is expected to be superior to conventional microbiology. We compared in total 35,564 microbiological urine cultures with and without incubation and processing with BD Kiestra TLA for a 6-month period each retrospectively. Sixteen thousand three hundred thirty-eight urine samples were analyzed in the pre-TLA period and 19,226 with TLA. Sixty-two percent (n = 10,101/16338) of the cultures processed without TLA and 68% (n = 13,102/19226) of the cultures processed with TLA showed growth. There were significantly more samples with two or more species per sample and with low numbers of colony forming units (CFU) after incubation with TLA. Regarding the type of bacteria, there were comparable amounts of Enterobacteriaceae in the samples, slightly less non-fermenting Gram-negative bacteria, but significantly more Gram-positive cocci, and Gram-positive rods. Especially Alloscardivia omnicolens, Gardnerella vaginalis, Actinomyces spp., and Actinotignum schaalii were significantly more abundant in the samples incubated and processed with TLA. The time to report was significantly lower in the TLA processed samples by 1.5 h. We provide the first report in Europe of a large number of urine samples processed with TLA. TLA showed enhanced growth of non-classical and rarely cultured bacteria from urine samples. Our findings suggest that previously underestimated bacteria may be relevant pathogens for urinary tract infections. Further studies are needed to confirm our findings.

Anti-inflammatory effect of two Lactobacillus strains during infection with Gardnerella vaginalis and Candida albicans in a HeLa cell culture model.

Lactobacilli are the dominant bacteria of the vaginal tract of healthy women and they play a major role in the maintenance of mucosal homeostasis, preventing genital infections, such as bacterial vaginosis (BV) and vulvovaginal candidiasis (VVC). It is now known that one mechanism of this protection is the influence that lactobacilli can exert on host immune responses. In this context, we evaluated two Lactobacillus strains (L. plantarum 59 and L. fermentum 137) for their immunomodulatory properties in response to Gardnerella vaginalis (BV) or Candida albicans (VVC) infections in a HeLa cell infection model. G. vaginalis and C. albicans triggered the secretion of pro-inflammatory cytokines (TNF-α, IL-1ß, IL-6 and IL-8) and the activation of NF-κB in HeLa cells, in contrast to L. plantarum 59 and L. fermentum 137. Treatments with the Lactobacillus strains or their cell-free supernatants before (pre-treatment) or after (post-treatment) the challenge with the pathogens resulted in decreased secretion of pro-inflammatory cytokines and decreased activation of NF-κB. The treatments with Lactobacillus strains not only decreased the secretion of IL-8, but also its expression, as confirmed by gene reporter luciferase assay, suggesting transcription-level control by lactobacilli. In conclusion, L. plantarum 59 and L. fermentum 137 were confirmed to have an anti-inflammatory effect against G. vaginalis and C. albicans and they were able to influence signalling in NF-κB pathway, making them interesting candidates as probiotics for the prevention or treatment of BV and VVC.

Colonization of the cervicovaginal space with Gardnerella vaginalis leads to local inflammation and cervical remodeling in pregnant mice.

The role of the cervicovaginal (CV) microbiome in regulating cervical function during pregnancy is poorly understood. Gardnerella vaginalis (G. vaginalis) is the most common bacteria associated with the diagnosis of bacterial vaginosis (BV). While BV has been associated with preterm birth (PTB), clinical trials targeting BV do not decrease PTB rates. It remains unknown if G. vaginalis is capable of triggering molecular, biomechanical and cellular events that could lead to PTB. The objective of this study was to determine if cervicovaginal colonization with G. vaginalis, in pregnant mice, induced cervical remodeling and modified cervical function. CD-1 timed-pregnant mice received a 5X108 CFU/mL intravaginal inoculation of G. vaginalis or control on embryonic day 12 (E12) and E13. On E15, the mice were sacrificed and cervicovaginal fluid (CVF), amniotic fluid (AF), cervix, uterus, placentas and fetal membranes (FM) were collected. Genomic DNA was isolated from the CVF, placenta, uterus and FM and QPCR was performed to confirm colonization. IL-6 was measured in the CVF and AF and soluble e-cadherin (seCAD) was assessed in the CVF by ELISA. RNA was extracted from the cervices to evaluate IL-10, IL-8, IL-1ß, TNF-α, Tff-1, SPINK-5, HAS-1 and LOX expression via QPCR. Mucicarmine and trichrome staining was used to assess cervical mucin and collagen. Biomechanical properties of the cervix were studied using quasi-static tensile load-to-failure biomechanical tests. G. vaginalis successfully colonized the CV space. This colonization induced immune responses (increased IL-6 levels in CVF and AF, increased mRNA expression of cervical cytokines), altered the epithelial barrier (increased seCAD in the CVF), induced cervical remodeling (increased mucin production, altered collagen) and altered cervical biomechanical properties (a decrease in biomechanical modulus and an increase in maximum strain). The ability of G. vaginalis to induce these molecular, immune, cellular and biomechanical changes suggests that this bacterium may play a pathogenic role in premature cervical remodeling leading to PTB.

Innate immune components affect growth and virulence traits of bacterial-vaginosis-associated and non-bacterial-vaginosis-associated Gardnerella vaginalis strains similarly.

Mucosal surfaces of the female reproductive tract contain a variety of antimicrobial components that provide the first line of defense against bacteria involved in the development of bacterial vaginosis (BV). Microbiological analysis of BV has shown Gardnerella vaginalis to be a prominent species in BV development. However, G. vaginalis colonization does not always lead to BV. Over the last decade, phenotypic and genotypic studies have demonstrated the existence of strain variants. Therefore, this study aimed to investigate if the major components of the vaginal immune response, specifically lysozyme, lactoferrin and ß-defensin 2, differently affected virulence traits of G. vaginalis strains isolated from healthy women or from women with BV. Gardnerella vaginalis strains were first genotyped by the clade classification system and then phenotypically characterized. Our results revealed that key differences in initial adhesion existed among the isolates but that these differences could not be predicted using the clade-genotyping approach. Importantly, we found that growth, initial adhesion and biofilm formation were strongly affected by lysozymes, but at similar levels in both groups, suggesting that the response to host immune components is not a distinguishing characteristic of isolates from women with BV versus those from healthy women.

Method of Selection of Bacteria Antibiotic Resistance Genes Based on Clustering of Similar Nucleotide Sequences.

A new method for selection of bacterium antibiotic resistance genes is proposed and tested for solving the problems related to selection of primers for PCR assay. The method implies clustering of similar nucleotide sequences and selection of group primers for all genes of each cluster. Clustering of resistance genes for six groups of antibiotics (aminoglycosides, ß-lactams, fluoroquinolones, glycopeptides, macrolides and lincosamides, and fusidic acid) was performed. The method was tested for 81 strains of bacteria of different genera isolated from patients (K. pneumoniae, Staphylococcus spp., S. agalactiae, E. faecalis, E. coli, and G. vaginalis). The results obtained by us are comparable to those in the selection of individual genes; this allows reducing the number of primers necessary for maximum coverage of the known antibiotic resistance genes during PCR analysis.

Characterization and complete genome sequences of L. rhamnosus DSM 14870 and L. gasseri DSM 14869 contained in the EcoVag® probiotic vaginal capsules.

Lactobacillus rhamnosus DSM 14870 and Lactobacillus gasseri DSM 14869 were previously isolated from the vaginal epithelial cells (VEC) of healthy women and selected for the development of the vaginal EcoVag® probiotic capsules. EcoVag® was subsequently shown to provide long-term cure and reduce relapse of bacterial vaginosis (BV) as an adjunct to antibiotic therapy. To identify genes potentially involved in probiotic activity, we performed genome sequencing and characterization of the two strains. The complete genome analysis of both strains revealed the presence of genes encoding functions related to adhesion, exopolysaccharide (EPS) biosynthesis, antimicrobial activity, and CRISPR adaptive immunity but absence of antibiotic resistance genes. Interesting features of L. rhamnosus DSM 14870 genome include the presence of the spaCBA-srtC gene encoding spaCBA pili and interruption of the gene cluster encoding long galactose-rich EPS by integrases. Unique to L. gasseri DSM 14869 genome was the presence of a gene encoding a putative (1456 amino acid) new adhesin containing two rib/alpha-like repeats. L. rhamnosus DSM 14870 and L. gasseri DSM 14869 showed acidification of the culture medium (to pH 3.8) and a strong adhesion capability to the Caco-2 cell line and VEC. L. gasseri DSM 14869 could produce a thick (40nm) EPS layer and hydrogen peroxide. L. rhamnosus DSM 14870 was shown to produce SpaCBA pili and a 20nm EPS layer, and could inhibit the growth of Gardnerella vaginalis, a bacterium commonly associated with BV. The genome sequences provide a basis for further elucidation of the molecular basis for their probiotic functions.

Evaluating the utility of syndromic case management for three sexually transmitted infections in women visiting hospitals in Delhi, India.

Utility of syndromic case management (SCM) in women visiting obstetrics & gynecology department needs to be evaluated as it is subjective and imperfect. Consequently, antibiotic resistance has accelerated along with increased risk of infection to the partners. To understand the effectiveness and/or inadequacies of SCM, 11000 women were recruited and examined by clinicians for infection by Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), Trichomonas vaginalis (TV), Bacterial vaginosis (BV) and others. Amongst these patients, 1797 (16.3%) reported vaginal discharge (VD). Other symptoms included: vaginitis (97%), cervicitis (75%), genital ulcers (60%), abnormal vaginal discharge (55%) and lower abdominal pain (48%). The patients were treated for single or co-infections using pre-packed National Aids Control Program III STI/RTI Kits. However, based on PCR diagnostics, 1453/1797 (81%) subjects were uninfected for NG/TV/CT. Amongst 344 (19%) infected patients, 257 (75%) carried infection with single pathogen (TV/NG/CT) while 87/344 (25%) were co-infected with multiple pathogens. Prevalence of TV, NG & CT was 4%, 7% and 8% respectively. Co-infection with CT + NG was highest, 51% (44/87), whereas, co-infection with CT + TV was 21% and NG + TV was 18% while co-infection with all three pathogens was 1.3%. We conclude that SCM is imprecise and successful intervention requires accurate and confirmatory diagnostic approach.

Gardnerella vaginalis population dynamics in bacterial vaginosis.

Bacterial vaginosis (BV) is the leading cause of vaginal discharge and is associated with the facultative Gram-variable bacterium Gardnerella vaginalis, whose population structure consists of four clades. Our goal was to determine if these clades differ with regard to abundance during BV. We performed a short-term longitudinal study of BV. Patients were evaluated according to the Amsel criteria and Nugent scoring at initial diagnosis, immediately after treatment and at a 40- to 45-day follow-up visit. G. vaginalis clade abundance was determined by quantitative real-time polymerase chain reactions (qPCRs). Among all specimens, the abundance of clades 1 and 4 were higher than that of clades 2 and 3 (P < 0.001). In general, the abundance of each clade increased with the degree of vaginal dysbiosis, as determined by the Nugent score and was greater in women with Amsel 4 compared with those with Amsel 0. Only clade 1 abundance was greater when Amsel 0 or 1 specimens were compared with Amsel 2 or 3 specimens (P < 0.01). Following antimicrobial treatment, abundance of clades 1 (P < 0.001) and 4 (P < 0.05) decreased regardless of the clinical and microbiological outcome, whereas clade 2 only decreased in women who had a sustained treatment response for 40-45 days (P < 0.01). Recurrent BV was characterized by post-treatment increases of clade 1 and 2 (P < 0.01). Clades 1 and 4 predominate in vaginal specimens. Clade abundance differs with regard to the Nugent score, the Amsel criteria, and response to therapy and BV recurrence.

In Vitro Activity of Lactobacillus fermentum LF5 Against Different Candida Species and Gardnerella vaginalis: A New Perspective to Approach Mixed Vaginal Infections?

GOALS: This study was undertaken to demonstrate the ability of Lactobacillus fermentum LF5 (DSM 32277) to inhibit in vitro different Candida species and Gardnerella vaginalis to weigh its potential effectiveness even in mixed vaginal infections. BACKGROUND: A wide female population is suffering from various vulvovaginal infections. These diseases are often associated with a decrease in the concentration of Lactobacilli in the vagina. Mixed vaginal infections represent >20% of women with vulvovaginal infection. STUDY: LF5 strain was cocultured in De Man, Rogosa and Sharpe with Candida according to a 1:100 ratio in favor of the yeast. Each culture was sampled after 24 hours of incubation for the selective enumeration of the yeasts performed on yeast extract glucose chloramphenicol agar medium.The growth of Gardnerella alone (positive control) and in the presence of different concentrations of neutralized supernatants of L. fermentum LF5 ranging from 5% to 20% was quantified by means of optical density at 600 nm (OD600). RESULTS: L. fermentum LF5 demonstrated the ability to inhibit significantly the growth of the 5 species of Candida by at least 4 logarithms.Furthermore, L. fermentum LF5 showed a significant activity after both 24 and 48 hours (46% and 82% with 20% of neutralized supernatant, respectively). A significant dose-dependent growth inhibition was recorded in particular after 48 hours of incubation, even achieving a 80% inhibition of G. vaginalis growth. CONCLUSIONS: The biotherapeutic LF5 could be the only documented strain effective in mixed forms. For this purpose, a human clinical trial is in progress.

Slight Pro-Inflammatory Immunomodulation Properties of Dendritic Cells by Gardnerella vaginalis: The "Invisible Man" of Bacterial Vaginosis?

Bacterial vaginosis (BV), the most common genital infection in reproductive-aged women, is associated with increased risk of sexually transmitted infections. Its etiology remains unclear, especially the role of Gardnerella (G.) vaginalis, an anaerobic bacterium characteristic of the BV-alteration of the vaginal ecosystem. In the genital mucosa, dendritic cells (DCs) sense bacteria of the microenvironment via receptors and then orchestrate the immune response by induction of different T cell subtypes. We investigated the interactions between G. vaginalis and human monocyte-derived DCs using a wide range of bacterial concentrations (multiplicity of infection from 0.01 to 100), and the effects of this pathogen on PHA-induced lymphocyte proliferation. As observed by electron microscopy and cytometry, G. vaginalis reduced the internalization ability of DCs by forming extracellular clusters and induced neither DC maturation, nor DC secretion of cytokines, except at the highest dose with a very early DC maturation state. The same profile was observed on lymphocytes with significant increases of proliferation and cytokine secretion only at the highest bacterial concentration. Our findings indicate that G. vaginalis possesses slight immune-stimulating activities against DCs and T cells, reflecting thus a defective inflammatory response and giving rise to the atypical, non- or low-grade, inflammatory clinical disease profile.

Escherichia coli and Enterococcus faecalis are able to incorporate and enhance a pre-formed Gardnerella vaginalis biofilm.

Gardnerella vaginalis is the most frequent microorganism found in bacterial vaginosis (BV), while Escherichia coli and Enterococcus faecalis are amongst the most frequent pathogens found in urinary tract infections (UTIs). This study aimed to evaluate possible interactions between UTIs pathogens and G. vaginalis using an in vitro dual-species biofilm model. Our results showed that dual-species biofilms reached significantly higher bacterial concentration than monospecies biofilms. Moreover, visualization of dual-populations species in the biofilms, using the epifluorescence microscopy, revealed that all of the urogenital pathogens coexisted with G. vaginalis. In conclusion, our work demonstrates that uropathogens can incorporate into mature BV biofilms.