The role of bacterial genital infections in spontaneous preterm delivery: a case-control study.

Background: Spontaneous preterm delivery is defined as the beginning of the birth process before the 37th week of pregnancy. The presence of microorganisms in the fetal membranes is accompanied by an increase in the production of prostaglandin, one of the important factors associated with the prevalence of preterm birth. The invasion of microorganisms leads to the production of protease, coagulase, and elastase, which directly stimulate the onset of childbirth. We investigated the role of genital infections in women with preterm birth. Methods: The present case-control study was conducted in the west of Iran on 100 women with spontaneous preterm delivery (following 24 weeks of gestation and before 36 weeks and 6 days) as the case group and 100 women with normal delivery as controls. A questionnaire was applied to collect the data. Polymerase chain reaction and pathological examination of the placenta were performed. Results: The average age in women with normal delivery (30.92 ± 5.10) in women with spontaneous preterm delivery (30.27 ± 4.93). The prevalence of Chlamydia trachomatis, Neisseria gonorrhea, Listeria monocytogenes, and Mycoplasma genitalium infections was zero in both groups. The highest prevalence of Gardnerella vaginalis was 19 (19%) in the case group and Ureaplasma parvum 15 (15%) in the control group. Also, Placental inflammation was zero in controls and 7(7%) in the patient group. There was a significant relationship between Gardnerella vaginalis bacteria and spontaneous preterm delivery. Conclusion: The results of our study showed that except for Gardnerella vaginalis bacteria, there is no significant relationship between the above bacterial infections and spontaneous preterm birth. Moreover, despite the significant reduction in the prevalence of many sexually transmitted infections in this research, it is still suggested to increase the awareness of people, including pregnant women, about the ways it can be transmitted by gynecologists and health and treatment centers.

Molecular characterization of vaginal microbiota using a new 22-species qRT-PCR test to achieve a relative-abundance and species-based diagnosis of bacterial vaginosis.

Background: Numerous bacteria are involved in the etiology of bacterial vaginosis (BV). Yet, current tests only focus on a select few. We therefore designed a new test targeting 22 BV-relevant species. Methods: Using 946 stored vaginal samples, a new qPCR test that quantitatively identifies 22 bacterial species was designed. The distribution and relative abundance of each species, α- and ß-diversities, correlation, and species co-existence were determined per sample. A diagnostic index was modeled from the data, trained, and tested to classify samples into BV-positive, BV-negative, or transitional BV. Results: The qPCR test identified all 22 targeted species with 95 - 100% sensitivity and specificity within 8 hours (from sample reception). Across most samples, Lactobacillus iners, Lactobacillus crispatus, Lactobacillus jensenii, Gardnerella vaginalis, Fannyhessea (Atopobium) vaginae, Prevotella bivia, and Megasphaera sp. type 1 were relatively abundant. BVAB-1 was more abundant and distributed than BVAB-2 and BVAB-3. No Mycoplasma genitalium was found. The inter-sample similarity was very low, and correlations existed between key species, which were used to model, train, and test a diagnostic index: MDL-BV index. The MDL-BV index, using both species and relative abundance markers, classified samples into three vaginal microbiome states. Testing this index on our samples, 491 were BV-positive, 318 were BV-negative, and 137 were transitional BV. Although important differences in BV status were observed between different age groups, races, and pregnancy status, they were statistically insignificant. Conclusion: Using a diverse and large number of vaginal samples from different races and age groups, including pregnant women, the new qRT-PCR test and MDL-BV index efficiently diagnosed BV within 8 hours (from sample reception), using 22 BV-associated species.

Characterization of a Lactobacillus gasseri strain as a probiotic for female vaginitis.

Vaginitis, a prevalent gynecological condition in women, is mainly caused by an imbalance in the vaginal micro-ecology. The two most common types of vaginitis are vaginal bacteriosis and vulvovaginal candidiasis, triggered by the virulent Gardnerella vaginalis and Candida albicans, respectively. In this study, a strain capable of inhibiting G. vaginalis and C. albicans was screened from vaginal secretions and identified as Lactobacillus gasseri based on 16S rRNA sequences. The strain, named L. gasseri VHProbi E09, could inhibit the growth of G. vaginalis and C. albicans under co-culture conditions by 99.07% ± 0.26% and 99.95% ± 0.01%, respectively. In addition, it could significantly inhibit the adhesion of these pathogens to vaginal epithelial cells. The strain further showed the ability to inhibit the enteropathogenic bacteria Escherichia coli and Salmonella enteritidis, to tolerate artificial gastric and intestinal fluids and to adhere to intestinal Caco-2 cells. These results suggest that L. gasseri VHProbi E09 holds promise for clinical trials and animal studies whether administered orally or directly into the vagina. Whole-genome analysis also revealed a genome consisting of 1752 genes for L. gasseri VHProbi E09, with subsequent analyses identifying seven genes related to adhesion and three genes related to bacteriocins. These adhesion- and bacteriocin-related genes provide a theoretical basis for understanding the mechanism of bacterial inhibition of the strain. The research conducted in this study suggests that L. gasseri VHProbi E09 may be considered as a potential probiotic, and further research can delve deeper into its efficacy as an agent which can restore a healthy vaginal ecosystem.

Vaginal flora in HPV infection: a cross­sectional analysis.

OBJECTIVE: The vaginal flora has been reported to be associated with human papillomavirus (HPV) infection. The purpose of this study was to investigate the characteristics of the cervical microbiota in patients with HPV infection and to analyse the changes in the vaginal flora and enzyme profiles in females with HPV infection. METHODS: We conducted a cross-sectional study involving 206 participants who underwent HPV genotyping, sexually transmitted diseases pathogen testing, cytology examination, and microbiome analysis. Additionally, we collected 115 HPV-negative samples and 48 HPV-positive samples for 16S rRNA amplicon sequencing. The vaginal microbial communities of both groups were analysed for diversity and differences to explore their association with HPV infection. RESULTS: The abundance of Lactobacillus was found to be reduced, while Gardnerella vaginalis was significantly more prevalent in the HPV + group. In terms of alpha diversity indices, the Shannon index (P = .0036) and Simpson index (P = .02) were higher in the HPV + group compared to the HPV - group, indicating greater community diversity in the HPV + group. Among the 10 sexually transmitted diseases pathogens analysed, Uup3 and Uup6 were significantly associated with HPV infection. Statistically significant differences were observed in Nugent scores and bacterial vaginosis between the two groups (P < .05). In functional analysis, 11 proteins and 13 enzymes were found to be significantly altered in the HPV + group. CONCLUSION: Our study demonstrates that disruptions in the vaginal flora are associated with HPV infection. Reduced levels of Lactobacillus, increased prevalence of Gardnerella, and abnormal enzyme profiles are closely linked to HPV infection.

The purpose of this study was to investigate the characteristics of the cervical microbiota in patients with human papillomavirus infection and to analyse the changes in the vaginal flora and enzyme profiles in females with human papillomavirus infection. We conducted a cross-sectional study involving 206 participants who underwent human papillomavirus genotyping, sexually transmitted diseases pathogen testing, cytology examination, and microbiome analysis. Additionally, we collected 115 HPV-negative samples and 48 HPV-positive samples for 16S rRNA amplicon sequencing. The abundance of Lactobacillus was found to be reduced, while Gardnerella vaginalis was significantly more prevalent in the HPV + group. In functional analysis, 11 proteins and 13 enzymes were found to be significantly altered in the HPV + group. Our study demonstrates that disruptions in the vaginal flora are associated with HPV infection. Reduced levels of Lactobacillus, increased prevalence of Gardnerella, and abnormal enzyme profiles are closely linked to HPV infection.

Synergistic anti-virulence efficacy of citral and carvacrol against mixed vaginitis causing Candida albicans and Gardnerella vaginalis: An in vitro and in vivo study.

Mixed vaginitis due to bacterial vaginosis (BV) and vulvovaginal candidiasis (VVC) is the most prevalent form and presents a significant therapeutic challenge globally. Since, the administration of monotherapy leads to subsequent recurrent infections, synergistic therapy that completely eradicates both pathogens is of dire need to manage mixed vaginities scenario and to prevent its recurrence. The current investigation was focused on exploring the synergistic inhibitory efficacy of phytochemicals against the virulence traits of individual and mixed species of C. albicans and G. vaginalis in vitro and in vivo (Galleria mellonella). Out of five phytochemicals (carvacrol, thymol, cinnamaldehyde, eugenol, and borneol) screened for synergism with citral [(Ct) as the prime molecule owing to its myriad therapeutic potential], carvacrol (Ca) in combination with citral exhibited promising synergistic effect. Time-kill kinetics and one-minute contact-killing assays demonstrated the phenomenal microbicidal effect of Ct-Ca combination against both mono and dual-species within 30 min and one-minute time intervals, respectively. Furthermore, the sub-CMICs (synergistic combinatorial MIC) of Ct-Ca have significantly eradicated the mature biofilms and remarkably reduced the virulence attributes of both C. albicans and G. vaginalis (viz., yeast to hyphae transition, filamentation, protease production, and hydrophobicity index), in single and dual species states. The non-toxic nature of Ct-Ca combination was authenticated using in vitro (human erythrocyte cells) and in vivo (Galleria mellonella) models. In addition, the in vivo efficacy evaluation and subsequent histopathological investigation was done using the invertebrate model system G. mellonella, which further ascertained the effectiveness of Ct-Ca combination in fighting off the infection caused by individual and mixed species of C. albicans and G. vaginalis. Concomitantly, the current work is the first of its kind to delineate the in vitro interaction of C. albicans and G. vaginalis mixed species at their growth and biofilm states, together emphasizes the promising therapeutic potential of acclaimed phytochemicals as combinatorial synergistic therapy against mixed vaginitis.

Phenylacetic acid, an anti-vaginitis metabolite produced by the vaginal symbiotic bacterium Chryseobacterium gleum.

The human microbiome contains genetic information that regulates metabolic processes in response to host health and disease. While acidic vaginal pH is maintained in normal conditions, the pH level increases in infectious vaginitis. We propose that this change in the vaginal environment triggers the biosynthesis of anti-vaginitis metabolites. Gene expression levels of Chryseobacterium gleum, a vaginal symbiotic bacterium, were found to be affected by pH changes. The distinctive difference in the metabolic profiles between two C. gleum cultures incubated under acidic and neutral pH conditions was suggested to be an anti-vaginitis molecule, which was identified as phenylacetic acid (PAA) by spectroscopic data analysis. The antimicrobial activity of PAA was evaluated in vitro, showing greater toxicity toward Gardnerella vaginalis and Candida albicans, two major vaginal pathogens, relative to commensal Lactobacillus spp. The activation of myeloperoxidase, prostaglandin E2, and nuclear factor-κB, and the expression of cyclooxygenase-2 were reduced by an intravaginal administration of PAA in the vaginitis mouse model. In addition, PAA displayed the downregulation of mast cell activation. Therefore, PAA was suggested to be a messenger molecule that mediates interactions between the human microbiome and vaginal health.

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.

The microbiota of sexual intercourse and its effect on prostatitis.

It is estimated that microorganisms colonize 90% of the body surface. In some tracts, such as the genitourinary tract, the microbiota varies throughout life, influenced by hormonal stimulation and sexual practices. This study evaluated the semen differences and presence of Lactobacillus crispatus, Lactobacillus iners, Gardnerella vaginalis and Atopobium vaginae in semen samples from patients with symptoms of chronic prostatitis and men asymptomatic for urogenital infections. Fifty-three semen samples were included: 22 samples from men with symptoms of chronic prostatitis and 31 asymptomatic men (control group). In addition to the presence of L. crispatus, L. iners, G. vaginalis and A. vaginae, semen parameters, total antioxidant capacity of seminal plasma, prostatic antigen and some proinflammatory cytokines were evaluated in each semen sample. Volunteers with symptoms of chronic prostatitis presented a lower percentage of sperm morphology (4.3% vs. control group 6.0%, p = 0.004); in the semen samples of volunteers in the group asymptomatic for urogenital infections, microorganisms associated with the vaginal microbiota were detected more frequently. The presence of bacteria in the vaginal microbiota can also benefit male reproductive health, which undergoes various modifications related to lifestyle habits that are susceptible to modification. Microorganisms associated with the vaginal microbiota, such as L. crispatus, L. iners, G. vaginalis and A. vaginae, may have a protective role against the development of male genitourinary diseases such as prostatitis.

Mucus production, host-microbiome interactions, hormone sensitivity, and innate immune responses modeled in human cervix chips.

Modulation of the cervix by steroid hormones and commensal microbiome play a central role in the health of the female reproductive tract. Here we describe organ-on-a-chip (Organ Chip) models that recreate the human cervical epithelial-stromal interface with a functional epithelial barrier and production of mucus with biochemical and hormone-responsive properties similar to living cervix. When Cervix Chips are populated with optimal healthy versus dysbiotic microbial communities (dominated by Lactobacillus crispatus and Gardnerella vaginalis, respectively), significant differences in tissue innate immune responses, barrier function, cell viability, proteome, and mucus composition are observed that are similar to those seen in vivo. Thus, human Cervix Organ Chips represent physiologically relevant in vitro models to study cervix physiology and host-microbiome interactions, and hence may be used as a preclinical testbed for development of therapeutic interventions to enhance women's health.

Extracellular vesicles from vaginal Gardnerella vaginalis and Mobiluncus mulieris contain distinct proteomic cargo and induce inflammatory pathways.

Colonization of the vaginal space with bacteria such as Gardnerella vaginalis and Mobiluncus mulieris is associated with increased risk for STIs, bacterial vaginosis, and preterm birth, while Lactobacillus crispatus is associated with optimal reproductive health. Although host-microbe interactions are hypothesized to contribute to reproductive health and disease, the bacterial mediators that are critical to this response remain unclear. Bacterial extracellular vesicles (bEVs) are proposed to participate in host-microbe communication by providing protection of bacterial cargo, delivery to intracellular targets, and ultimately induction of immune responses from the host. We evaluated the proteome of bEVs produced in vitro from G. vaginalis, M. mulieris, and L. crispatus, identifying specific proteins of immunologic interest. We found that bEVs from each bacterial species internalize within cervical and vaginal epithelial cells, and that epithelial and immune cells express a multi-cytokine response when exposed to bEVs from G. vaginalis and M. mulieris but not L. crispatus. Further, we demonstrate that the inflammatory response induced by G. vaginalis and M. mulieris bEVs is TLR2-specific. Our results provide evidence that vaginal bacteria communicate with host cells through secreted bEVs, revealing a mechanism by which bacteria lead to adverse reproductive outcomes associated with inflammation. Elucidating host-microbe interactions in the cervicovaginal space will provide further insight into the mechanisms contributing to microbiome-mediated adverse outcomes and may reveal new therapeutic targets.

Establishment and Validation of a Mouse Bacterial Vaginosis Model.

BACKGROUND: Bacterial vaginosis (BV) is a common vaginal infection without a reliable animal model. To establish a novel mouse BV model, we evaluated multiple parameters of various identified bacteria-infected mice, including Staphylococcus aureus (SA), Escherichia coli (EC), Streptococcus agalactiae, ß-Hemolytic streptococcus, and Gardnerella vaginalis (GV). METHODS: Mature female KM mice were randomly allocated to a vehicle group (group A, without any treatment) and experimental groups. After vaginal secretions were harvested, experimental groups were divided into phosphate buffer solution group (PBS, group B), control group including SA, and EC with a 1:1 ratio (group C), SA, EC, and Streptococcus agalactiae with a 1:2:1 ratio group (group D), SA, EC, and ß-Hemolytic streptococcus with a 1:2:1 ratio group (group E), and GV group (group F). The vaginal secretions of experimental mice were collected by flushing with 100 mL sterile PBS on days 2, 4, and 6. Vaginal secretions were examined by Gram staining, sialidase assay, ammonia test, and pH value measurement. IL-6 and IL-10 levels in mouse serum were detected by enzyme-linked immunosorbent assay. Hematoxylineosin staining and mouse cervicovaginal tissue histopathological scores were observed. The diagnostic test results were analyzed by logistic regression analysis and receiver operating characteristic curves. The Shapiro-Wilk analysis of variance, or rank-sum test, was used for normal distribution analysis. Pearson's correlation and chi-squared test determined the correlation and comparison data expressed as a percentage or frequency. RESULTS: There was less severe vaginal morphology in GV-infected mice compared to other bacteria-infected mice. The sialidase assay, the ammonia test, and the pH values of vaginal secretions showed significant differences between GV-infected and uninfected mice. Serum IL-6 and IL-10 levels and vaginal histological scoring increased in other bacteria-infected mice, but GV-infected mice showed only a mildly increasing trend of IL-10 levels and vaginal histological scoring compared to control mice. CONCLUSIONS: GV-infected mice showed clinical features similar to human BV infection, including vaginal anatomical and pathological indices, and biochemical and immune parameters. Serum IL-10 level has potential for use in BV diagnosis.

Gardnerella Vaginalis Bacteremia in a Pregnant Woman with Vaginal Myomectomy.

BACKGROUND: This case involves a 28-year-old pregnant woman (39w+2) who was admitted to obstetrics due to abdominal tightness and bacteremia with Gardnerella vaginalis which developed after caesarean section and vaginal myomectomy. METHODS: A blood culture was performed, and the bacteria were identified through mass spectrometry. RESULTS: Mass spectrometry data indicated that the infection bacteria were Gardnerella vaginalis. The patient's temperature returned to normal after oral ampicillin in combination with clindamycin. CONCLUSIONS: Gardnerella vaginalis bacteremia is very rare in clinical practice, and the combination of ampicillin and clindamycin has a good therapeutic effect. This study may provide a reference for the diagnosis and treatment of Gardnerella vaginalis bacteremia.

The role of sialidases in the pathogenesis of bacterial vaginosis and their use as a promising pharmacological target in bacterial vaginosis.

Bacterial vaginosis (BV) is an infection of the genital tract characterized by disturbance of the normally Lactobacilli-dominated vaginal flora due to the overgrowth of Gardnerella and other anaerobic bacteria. Gardnerella vaginalis, an anaerobic pathogen and the major pathogen of BV, produces sialidases that cleave terminal sialic acid residues off of human glycans. By desialylation, sialidases not only alter the function of sialic acid-containing glycoconjugates but also play a vital role in the attachment, colonization and spread of many other vaginal pathogens. With known pathogenic effects, excellent performance of sialidase-based diagnostic tests, and promising therapeutic potentials of sialidase inhibitors, sialidases could be used as a biomarker of BV. This review explores the sources of sialidases and their role in vaginal dysbiosis, in aims to better understand their participation in the pathogenesis of BV and their value in the diagnosis and treatment of BV.

Optimized bacterial absolute quantification method by qPCR using an exogenous bacterial culture as a normalization strategy in triple-species BV-like biofilms.

Quantitative Polymerase Chain Reaction (qPCR) is a widely used method in molecular biology to quantify target DNA sequences. Despite its accuracy, there are important experimental controls that should be considered to avoid biased results. One of them is gDNA loss during extraction, which is higher among samples with lower bacterial concentrations. Improvement in qPCR quantification procedures is mandatory to obtain reproducible and accurate results. Herein, we report an improved qPCR method for bacterial quantification of Gardnerella vaginalis, Prevotella bivia, and Fannyhessea vaginae, three key-bacterial vaginosis (BV)-associated bacteria (BVAB) thought to play important roles in the pathogenesis of this common vaginal infection. The formation of a mature biofilm on vaginal epithelial cells is an unique feature of BV and, despite over 60 years of research, the exact etiology of BV remains unknown. Here, we optimized a qPCR method that accurately quantified triple-species biofilms containing these key BVAB, after the addition of an exogenous bacterial control containing a fixed concentration of Escherichia coli, prior to gDNA extraction. This improved method minimized and normalized the inherent losses associated with bacterial centrifugation, which allows better sensitivity at lower bacterial concentrations.

Lactobacillus crispatus CCFM1339 Inhibits Vaginal Epithelial Barrier Injury Induced by Gardnerella vaginalis in Mice.

The vaginal epithelial barrier, which integrates mechanical, immune, chemical, and microbial defenses, is pivotal in safeguarding against external pathogens and upholding the vaginal microecological equilibrium. Although the widely used metronidazole effectively curtails Gardnerella vaginalis, a key pathogen in bacterial vaginosis, it falls short in restoring the vaginal barrier or reducing recurrence rates. Our prior research highlighted Lactobacillus crispatus CCFM1339, a vaginally derived Lactobacillus strain, for its capacity to modulate the vaginal epithelial barrier. In cellular models, L. crispatus CCFM1339 fortified the integrity of the cellular monolayer, augmented cellular migration, and facilitated repair. Remarkably, in animal models, L. crispatus CCFM1339 substantially abated the secretion of the barrier disruption biomarker E-cadherin (from 101.45 to 82.90 pg/mL) and increased the anti-inflammatory cytokine IL-10 (35.18% vs. the model), consequently mitigating vaginal inflammation in mice. Immunological assays in vaginal tissues elucidated increased secretory IgA levels (from 405.56 to 740.62 ng/mL) and curtailed IL-17 gene expression. Moreover, L. crispatus CCFM1339 enhanced Lactobacilli abundance and attenuated Enterobacterium and Enterococcus within the vaginal microbiome, underscoring its potential in probiotic applications for vaginal barrier regulation.

Gardnerella vaginalis ventilatory acquired pneumonia among patients with trauma.

Gardnerella vaginalis (G. vaginalis) is a bacterium rarely responsible for systemic infections and is exceptionally isolated from bronchopulmonary samples. Here, we report here two patients with trauma who were diagnosed with a G. vaginalis ventilatory acquired pneumonia (VAP) via mini bronchoalveolar lavage (mini-BAL). According to our observations, G. vaginalis was the only microorganism with a significant threshold and the identification was obtained by a reliable mean. There is no recommendation for antibiotic treatment for invasive G. vaginalis infection. We treated these infections with Cefotaxim and Metronidazole which clinically improved the infection. To determine whether the two patients were infected by the same strain, we used a random amplified polymorphic DNA (RAPD) technique. The two G. vaginalis organisms had distinct RAPD profiles, suggesting the absence of cross-transmission. These two cases of trauma and G. vaginalis VAP suggest that this infection cannot be ruled out and should alert the clinician to treat it.

Sexual transmission of urogenital bacteria: whole metagenome sequencing evidence from a sexual network study.

Sexual transmission of the urogenital microbiota may contribute to adverse sexual and reproductive health outcomes. The extent of sexual transmission of the urogenital microbiota is unclear as prior studies largely investigated specific pathogens. We used epidemiologic data and whole metagenome sequencing to characterize urogenital microbiota strain concordance between participants of a sexual network study. Individuals who screened positive for genital Chlamydia trachomatis were enrolled and referred their sexual contacts from the prior 60-180 days. Snowball recruitment of sexual contacts continued for up to four waves. Vaginal swabs and penile urethral swabs were collected for whole metagenome sequencing. We evaluated bacterial strain concordance using inStrain and network analysis. We defined concordance as ≥99.99% average nucleotide identity over ≥50% shared coverage; we defined putative sexual transmission as concordance between sexual contacts with <5 single-nucleotide polymorphisms per megabase. Of 138 participants, 74 (54%) were female; 120 (87%) had genital chlamydia; and 43 (31%) were recruited contacts. We identified 115 strain-concordance events among 54 participants representing 25 bacterial species. Seven events (6%) were between sexual contacts including putative heterosexual transmission of Fannyhessea vaginae, Gardnerella leopoldii, Prevotella amnii, Sneathia sanguinegens, and Sneathia vaginalis (one strain each), and putative sexual transmission of Lactobacillus iners between female contacts. Most concordance events (108, 94%) were between non-contacts, including eight female participants connected through 18 Lactobacillus crispatus and 3 Lactobacillus jensenii concordant strains, and 14 female and 2 male participants densely interconnected through 52 Gardnerella swidsinskii concordance events.IMPORTANCEEpidemiologic evidence consistently indicates bacterial vaginosis (BV) is sexually associated and may be sexually transmitted, though sexual transmission remains subject to debate. This study is not capable of demonstrating BV sexual transmission; however, we do provide strain-level metagenomic evidence that strongly supports heterosexual transmission of BV-associated species. These findings strengthen the evidence base that supports ongoing investigations of concurrent male partner treatment for reducing BV recurrence. Our data suggest that measuring the impact of male partner treatment on F. vaginae, G. leopoldii, P. amnii, S. sanguinegens, and S. vaginalis may provide insight into why a regimen does or does not perform well. We also observed a high degree of strain concordance between non-sexual-contact female participants. We posit that this may reflect limited dispersal capacity of vaginal bacteria coupled with individuals' comembership in regional transmission networks where transmission may occur between parent and child at birth, cohabiting individuals, or sexual contacts.

Interpretation of vaginal metagenomic characteristics in different types of vaginitis.

Although vaginitis is closely related to vaginal microecology in females, the precise composition and functional potential of different types of vaginitis remain unclear. Here, metagenomic sequencing was applied to analyze the vaginal flora in patients with various forms of vaginitis, including cases with a clue cell proportion ranging from 1% to 20% (Clue1_20), bacterial vaginitis (BV), vulvovaginal candidiasis (VVC), and BV combined with VVC (VVC_BV). Our results identified Prevotella as an important biomarker between BV and Clue1_20. Moreover, a gradual decrease was observed in the relative abundance of shikimic acid metabolism associated with bacteria producing indole as well as a decline in the abundance of Gardnerella vaginalis in patients with BV, Clue1_20, and healthy women. Interestingly, the vaginal flora of patients in the VVC_BV group exhibited structural similarities to that of the VVC group, and its potentially functional characteristics resembled those of the BV and VVC groups. Finally, Lactobacillus crispatus was found in high abundance in healthy samples, greatly contributing to the stability of the vaginal environment. For the further study of L. crispatus, we isolated five strains of L. crispatus from healthy samples and evaluated their capacity to inhibit G. vaginalis biofilms and produce lactic acid in vitro to select the potential probiotic candidate for improving vaginitis in future clinical studies. Overall, we successfully identified bacterial biomarkers of different vaginitis and characterized the dynamic shifts in vaginal flora between patients with BV and healthy females. This research advances our understanding and holds great promise in enhancing clinical approaches for the treatment of vaginitis. IMPORTANCE: Vaginitis is one of the most common gynecological diseases, mostly caused by infections of pathogens such as Candida albicans and Gardnerella vaginalis. In recent years, it has been found that the stability of the vaginal flora plays an important role in vaginitis. Furthermore, the abundant Lactobacillus-producing rich lactic acid in the vagina provides a healthy acidic environment such as Lactobacillus crispatus. The metabolites of Lactobacillus can inhibit the colonization of pathogens. Here, we collected the vaginal samples of patients with bacterial vaginitis (BV), vulvovaginal candidiasis (VVC), and BV combined with VVC to discover the differences and relationships among the different kinds of vaginitis by metagenomic sequencing. Furthermore, because of the importance of L. crispatus in promoting vaginal health, we isolated multiple strains from vaginal samples of healthy females and chose the most promising strain with potential probiotic benefits to provide clinical implications for treatment strategies.

Insertive vaginal sex is associated with altered penile immunology and enrichment of Gardnerella vaginalis in uncircumcised Ugandan men.

PROBLEM: HIV susceptibility is linked to the penile immune milieu (particularly IL-8 levels) and microbiome. The effects of insertive vaginal sex itself on penile immunology and microbiota are not well described. METHOD OF STUDY: We compared the immune milieu and microbiology of the coronal sulcus (CS) and distal urethra in 47 uncircumcised Ugandan men reporting ever (n = 42) or never (n = 5) having had vaginal intercourse. Soluble immune factors were assayed by multiplex ELISA, and penile bacteria abundance by 16S rRNA qPCR and sequencing. Co-primary endpoints were penile levels of IL-8 and soluble E-cadherin. RESULTS: Independent of classical STIs, men reporting prior vaginal sex demonstrated elevated IL-8 levels in both the coronal sulcus (1.78 vs. 0.81 log10 pg/mL, p = .021) and urethra (2.93 vs. 2.30 log10 pg/mL; p = .003), with a strong inverse relationship between urethral IL-8 levels and the time from last vaginal sex (r = -0.436; p = .004). Vaginal sex was also associated with elevated penile IL-1α/ß and soluble E-cadherin (sEcad), a marker of epithelial disruption. Gardnerella vaginalis (Gv) was only present in the penile microbiome of men reporting prior vaginal sex, and urethral Gv absolute abundance was strongly associated with urethral inflammation (r = 0.556; p < .001); corynebacteria were enriched in the CS of men reporting no prior vaginal sex and were associated with reduced CS inflammation. CONCLUSIONS: Sexual intercourse was associated with sustained changes in penile immunology, potentially mediated through microbial alterations, in particular the urethral abundance of G. vaginalis. Future studies should further characterize the effects of sexual debut on penile bacteria and immunology.

Deciphering Intermediate Nugent scores: Utility of Real-time PCR for Bacterial Vaginosis diagnosis.

PURPOSE: To optimize real-time PCR assays for diagnosis of Bacterial Vaginosis (BV) and determine cut-off loads by ROC analysis for Gardnerella vaginalis, Atopobium vaginae and Lactobacillus spp. as compared to Nugent scoring (Gold standard) in clinical samples. RESULTS: Out of 125 women, 34 were positive, 26 intermediate and 65 negative for BV by Nugent scoring. All three real-time PCR assays were found to be highly sensitive & specific and AUC suggested excellent diagnostic accuracy. An optimal cut-off was >9.45 × 103 copies/ ml, >3.34 × 103 copies/ ml & ≤ 18.63 × 103 copies/ ml for G. vaginalis, A. vaginae and Lactobacillus spp. respectively, in BV positives. Gram staining and qPCR were discordant only in patients with intermediate scores (n = 26) where qPCR identified 15 (57.69%) as positive and 11 (42.3%) as negative. CONCLUSION: PCR-based molecular BV diagnosis is more accurate and can be used for deciphering intermediate Nugent scores.