Vaginal Microbial Communities Research Articles

The Role of Hydrogen-Peroxide (H2O2) Produced by Vaginal Microbiota in Female Reproductive Health.

Female reproductive health is strongly associated with healthy vaginal microbiota, which is thought to be ensured by the dominance of certain Lactobacillus species. Lactobacilli control the vaginal microenvironment through several factors and mechanisms. One of them is their ability to produce hydrogen peroxide (H2O2). The role of Lactobacillus-derived H2O2 in the vaginal microbial community has been intensively investigated in several studies with many designs. However, results and data are controversial and challenging to interpret in vivo. Defining the underlying mechanisms responsible for a physiological vaginal ecosystem is crucial since it could directly affect probiotic treatment attempts. This review aims to summarize current knowledge on the topic, focusing on probiotic treatment possibilities.

Association between monoaromatic hydrocarbons exposure and vaginal microbiota disorder in recurrent implantation failure

Recurrent implantation failure (RIF) is a significant challenge facing population development, with the impact of monoaromatic hydrocarbons (MACHs) exposure on embryo implantation and reproductive hazards remaining largely unclear. As such, we conducted a prospective randomized controlled cohort study to explore the potential correlations between exposure to MACHs and vaginal microbiota, and their potential adverse effects on embryo implantation. Our findings suggest that exposure to Mono(2-ethylhexyl) phthalate (MEHP), Methyl N-methylanthranilate, and Benzene is closely linked with the disruption of the microbial community in the RIF group. The RIF group thus exhibited a fragile vaginal microbial community structure, with lower lactic acid bacterial abundance, and higher potential pathogenic bacterial abundance, such as Streptococcus anginosus and Peptoniphilus asaccharolyticus. This abnormal vaginal microbiota composition was associated with adverse pregnancy outcomes, with sphingolipid metabolism dysfunction being a key factor in abnormal microbiota leading to such outcomes. Additionally, we found that exposure to Methyl N-methylanthranilate and MEHP was closely associated with decreased abundance of Lactobacillus (including L.crispatus and L.jensenii) and diminished reproductive tract protective barrier function. Our study underscores the significance of acknowledging the impact of MACHs, a ubiquitous everyday exposure, on female fertility.

Characterization of Vaginal Microbial Community Dynamics in the Pathogenesis of Incident Bacterial Vaginosis, a Pilot Study.

Despite more than 60 years of research, the etiology of bacterial vaginosis (BV) remains controversial. In this pilot study, we used shotgun metagenomic sequencing to characterize vaginal microbial community changes before the development of incident BV (iBV). A cohort of African American women with a baseline healthy vaginal microbiome (no Amsel criteria, Nugent score 0-3 with no Gardnerella vaginalis morphotypes) were followed for 90 days with daily self-collected vaginal specimens for iBV (≥2 consecutive days of a Nugent score of 7-10). Shotgun metagenomic sequencing was performed on select vaginal specimens from 4 women, every other day for 12 days before iBV diagnosis. Sequencing data were analyzed through Kraken2 and bioBakery 3 workflows, and specimens were classified into community state types. Quantitative polymerase chain reaction was performed to compare the correlation of read counts with bacterial abundance. Common BV-associated bacteria such as G. vaginalis , Prevotella bivia , and Fannyhessea vaginae were increasingly identified in the participants before iBV. Linear modeling indicated significant increases in G. vaginalis and F . vaginae relative abundance before iBV, whereas the relative abundance of Lactobacillus species declined over time. The Lactobacillus species decline correlated with the presence of Lactobacillus phages. We observed enrichment in bacterial adhesion factor genes on days before iBV. There were also significant correlations between bacterial read counts and abundances measured by quantitative polymerase chain reaction. This pilot study characterizes vaginal community dynamics before iBV and identifies key bacterial taxa and mechanisms potentially involved in the pathogenesis of iBV.

Bacterial Species from Vaginal Microbiota Differently Affect the Production of the E6 and E7 Oncoproteins and of p53 and p-Rb Oncosuppressors in HPV16-Infected Cells.

Vaginal dysbiosis is characterized by a decrease in the relative abundance of Lactobacillus species in favor of other species. This condition facilitates infections by sexually transmitted pathogens including high risk (HR)-human papilloma viruses (HPVs) involved in the development of cervical cancer. Some vaginal dysbiosis bacteria contribute to the neoplastic progression by inducing chronic inflammation and directly activating molecular pathways involved in carcinogenesis. In this study, SiHa cells, an HPV-16-transformed epithelial cell line, were exposed to different representative vaginal microbial communities. The expression of the HPV oncogenes E6 and E7 and the production of relative oncoproteins was evaluated. The results showed that Lactobacillus crispatus and Lactobacillus gasseri modulated the basal expression of the E6 and E7 genes of SiHa cells and the production of the E6 and E7 oncoproteins. Vaginal dysbiosis bacteria had contrasting effects on E6/E7 gene expression and protein production. The expression of the E6 and E7 genes and the production of the relative oncoproteins was increased by strains of Gardnerella vaginalis and, to a lesser extent, by Megasphaera micronuciformis. In contrast, Prevotella bivia decreased the expression of oncogenes and the production of the E7 protein. A decreased amount of p53 and pRb was found in the cultures of SiHa cells with M. micronuciformis, and accordingly, in the same cultures, a higher percentage of cells progressed to the S-phase of the cell cycle compared to the untreated or Lactobacillus-stimulated cultures. These data confirm that L. crispatus represents the most protective component of the vaginal microbiota against neoplastic progression of HR-HPV infected cells, while M. micronuciformis and, to a lesser extent, G. vaginalis may directly interfere in the oncogenic process, inducing or maintaining the production of viral oncoproteins.

Transport and Utilization of Glycogen Breakdown Products by Gardnerella spp. from the Human Vaginal Microbiome.

Multiple Gardnerella species frequently cooccur in vaginal microbiomes, and several factors, including competition for nutrients such as glycogen could determine their population structure. Although Gardnerella spp. can hydrolyze glycogen to produce glucose, maltose, maltotriose, and maltotetraose, how these sugars are transported and utilized for growth is unknown. We determined the distribution of genes encoding transporter proteins associated with the uptake of glucose, maltose, and malto-oligosaccharides and maltodextrins among Gardnerella species. A total of five different ABC transporters were identified in Gardnerella spp. of which MusEFGK2I and MalXFGK were conserved across all 15 Gardnerella isolates. RafEFGK and TMSP (trehalose, maltose, sucrose, and palatinose) operons were specific to G. vaginalis while the MalEFG transporter was identified in G. leopoldii only. Although no glucose specific sugar-symporters were identified, putative "glucose/galactose porters" and components of a phosphotransferase system were identified. In laboratory experiments, all Gardnerella isolates grew more in the presence of glucose, maltose, maltotriose, and maltotetraose compared to unsupplemented media. In addition, most isolates (10/15) showed significantly more growth on maltotetraose compared to glucose (Kruskal Wallis, P < 0.05) suggesting their preference for longer chain malto-oligosaccharides. Our findings show that although putative MusEFGK2I and MalXFGK transporters are found in all Gardnerella spp., some species-specific transporters are also present. Observed distribution of genes encoding transporter systems was consistent with laboratory observations that Gardnerella spp. grow better on longer chain malto-oligosaccharides. IMPORTANCE Increased abundance of Gardnerella spp. is a diagnostic characteristic of bacterial vaginosis, an imbalance in the human vaginal microbiome associated with troubling symptoms and negative reproductive health outcomes, including increased transmission of sexually transmitted infections and preterm birth. Competition for nutrients is likely an important factor in causing dramatic shifts in the vaginal microbial community. Gardnerella produces enzymes to digest glycogen, an important nutrient source for vaginal bacteria, but little is known about the mechanisms in Gardnerella for uptake of the products of this digestion, or whether Gardnerella use some or all of the products. Our results indicate that Gardnerella may have evolved to preferentially use a subset of the glycogen breakdown products, which would help them reduce direct competition with some other bacteria in the vagina.

Glycogen-Degrading Activities of Catalytic Domains of α-Amylase and α-Amylase-Pullulanase Enzymes Conserved in Gardnerella spp. from the Vaginal Microbiome.

Gardnerella spp. are associated with bacterial vaginosis in which normally dominant lactobacilli are replaced with facultative and anaerobic bacteria, including Gardnerella spp. Co-occurrence of multiple species of Gardnerella is common in the vagina, and competition for nutrients such as glycogen likely contributes to the differential abundances of Gardnerella spp. Glycogen must be digested into smaller components for uptake, a process that depends on the combined action of glycogen-degrading enzymes. In this study, the ability of culture supernatants of 15 isolates of Gardnerella spp. to produce glucose, maltose, maltotriose, and maltotetraose from glycogen was demonstrated. Carbohydrate-active enzymes (CAZymes) were identified bioinformatically in Gardnerella proteomes using dbCAN2. Identified proteins included a single-domain α-amylase (EC 3.2.1.1) (encoded by all 15 isolates) and an α-amylase-pullulanase (EC 3.2.1.41) containing amylase, carbohydrate binding modules, and pullulanase domains (14/15 isolates). To verify the sequence-based functional predictions, the amylase and pullulanase domains of the α-amylase-pullulanase and the single-domain α-amylase were each produced in Escherichia coli. The α-amylase domain from the α-amylase-pullulanase released maltose, maltotriose, and maltotetraose from glycogen, and the pullulanase domain released maltotriose from pullulan and maltose from glycogen, demonstrating that the Gardnerella α-amylase-pullulanase is capable of hydrolyzing α-1,4 and α-1,6 glycosidic bonds. Similarly, the single-domain α-amylase protein also produced maltose, maltotriose, and maltotetraose from glycogen. Our findings show that Gardnerella spp. produce extracellular amylase enzymes as "public goods" that can digest glycogen into maltose, maltotriose, and maltotetraose that can be used by the vaginal microbiota. IMPORTANCE Increased abundance of Gardnerella spp. is a diagnostic characteristic of bacterial vaginosis, an imbalance in the human vaginal microbiome associated with troubling symptoms, and negative reproductive health outcomes, including increased transmission of sexually transmitted infections and preterm birth. Competition for nutrients is likely an important factor in causing dramatic shifts in the vaginal microbial community, but little is known about the contribution of bacterial enzymes to the metabolism of glycogen, a major food source available to vaginal bacteria. The significance of our research is characterizing the activity of enzymes conserved in Gardnerella species that contribute to the ability of these bacteria to utilize glycogen.

Oropharyngeal, proximal colonic, and vaginal microbiomes of healthy Korean native black pig gilts

BackgroundExploring the microbiome in multiple body sites of a livestock species informs approaches to promote its health and performance through efficient and sustainable modulation of these microbial ecosystems. Here, we employed 16S rRNA gene sequencing to describe the microbiome in the oropharyngeal cavity, proximal colon, and vaginal tract of Jeju Black pigs (JBP), which are native to the Korean peninsula.ResultsWe sampled nine 7-month-old JBP gilts raised under controlled conditions. The most abundant phyla that we found within the oropharyngeal microbiota were Proteobacteria, Bacteroidetes, Fusobacteria and Firmicutes, collectively providing core features from twenty-five of their genera. We also found a proximal colonic microbial core composed of features from twenty of the genera of the two predominant phyla, Firmicutes, and Bacteroidetes. Remarkably, within the JBP vaginal microbiota, Bacteroidetes dominated at phylum level, contrary to previous reports regarding other pig breeds. Features of the JBP core vaginal microbiota, came from seventeen genera of the major phyla Bacteroidetes, Firmicutes, Proteobacteria, and Fusobacteria. Although these communities were distinct, we found some commonalities amongst them. Features from the genera Streptococcus, Prevotella, Bacillus and an unclassified genus of the family Ruminococcaceae were ubiquitous across the three body sites. Comparing oropharyngeal and proximal colonic communities, we found additional shared features from the genus Anaerorhabdus. Between oropharyngeal and vaginal ecosystems, we found other shared features from the genus Campylobacter, as well as unclassified genera from the families Fusobacteriaceae and Flavobacteriaceae. Proximal colonic and vaginal microbiota also shared features from the genera Clostridium, Lactobacillus, and an unclassified genus of Clostridiales.ConclusionsOur results delineate unique and ubiquitous features within and across the oropharyngeal, proximal colonic and vaginal microbial communities in this Korean native breed of pigs. These findings provide a reference for future microbiome-focused studies and suggest a potential for modulating these communities, utilizing ubiquitous features, to enhance health and performance of the JBP.

Causes and Consequences of Preterm Birth, A Systematic Review

To systematically review the potential causes and possible effects of preterm birth [&lt;37 weeks gestational duration]. We searched PubMed, google scholar, clinicaltrials.gov and science direct for English language articles published from 2004 to march 2021. All kinds of study schemes were considered acceptable, comprising case –control, cohort studies, experimental and cross- sectional studies. Significant evidences indicate that social stress, elevated cadmium exposure, genomic variations, vitamin D deficiency, pre-conception hepatitis B infection, declined vaginal microbial community, intrauterine infection, reduction in cervical consistency index, strong exposure of creatinine corrected thallium, systemic autoimmune diseases, ozone, primary traffic air pollutants, road traffic noise, potential exposure of arsenic, HIV exposure, maternal thyroid dysfunction, maternal plasma protein level and COVID-19 exposure in pregnant females are the major risk factors for PTBs. Results of earlier investigations indicated prominent risk of insulin resistance, hypertension, neurological defects, heart failure, Chronic kidney disease, Lung function impairment, lower birth weight, thalamocortical system defects, cancer, altered cardiac phenotype and cardio metabolic diseases in survivors of preterm births. This review will help clinicians to isolate the fundamental etiology and to proactively identify, cope and improve outcomes of at-risk pregnancies.

Amniotic fluid and vaginal microbiota in pregnant women with gestational diabetes mellitus by metagenomics

AimsTo compared the amniotic fluid and vaginal microbiota of pregnant women, who developed gestational diabetes mellitus to those who did not, and explored if any differences exist in the composition and functional genes of their amniotic fluid and vaginal microbiota. MethodsWe compared the amniotic fluid and vaginal microbiota of five GDM patients and five non-GDM pregnant women in the third trimester of pregnancy by using metagenomics, and analyzed the characteristics, functions, and differences between two groups. ResultsThe analysis of the amniotic fluid showed significant differences in genus and species. In contrast, there was no significant difference in vaginal microbial community structure between two groups. The alpha diversity in the GDM group was higher than the control, but the difference was not statistically significant. There was a statistically significant difference in the species-level beta-diversity measured by Bray–Curtis distance of the amniotic fluid communities between two groups, while no significant differences were observed in the vaginal microbiota. Regarding the functions, we observed that the microbial communities of the amniotic fluid and vaginal secretions were involved in the regulation of a variety of host metabolic pathways, including carbohydrate metabolism, membrane transport, energy, amino acid metabolism, nucleotide metabolism, etc. No significant differences were observed between two groups, but different sites exist. ConclusionThe amniotic fluid of this study was discovered to have a heterogeneous, albeit small in number, community of microorganisms.Our research suggests that gestational diabetes has a very limited impact on the amniotic fluid and vaginal microbiome. The composition and the functions of the microbiota at different sites are different.

HPV-related cervical diseases: Alteration of vaginal microbiotas and promising potential for diagnosis.

Vaginal microbiota is closely associated with women's health, however, the correlation between HPV-related cervical disease (HRCD) and vaginal microbiota is still obscure. In this study, patients with HRCD (n = 98) and healthy controls (n = 58) in Hangzhou were recruited, and their vaginal microbiota were collected and analyzed. The composition of the vaginal microbial community was explored, and a disease classification model was developed by random forest algorithm. The results suggested that the diversity of vaginal microbiota was significantly higher in HRCDs than that in healthy controls (p < 0.05). Firmicutes is the dominant phylum in vaginal microbiota, and Lactobacillus was identified as the most altered genus between two groups (p < 0.01). Kyoto Encyclopedia of Genes and Genomesanalysis suggested the difference in vaginal microbial community functions between two groups. Furthermore, we identified 10 biomarkers as the optimal marker sets for the random forest model and found a higher probability of disease values in HRCD group in discovery cohort (p < 0.0001), with an area under the receiver operating characteristiccurve reaching 89.7% (95% confidence interval: 78.3%-100%). We further validated the model in both validation and independent diagnosis cohorts, confirming its accuracy in the prediction of HRCD. In conclusion, this study revealed the community composition of vaginal microbiota in HRCDs and successfully constructed a diagnostic model for HRCD.

Comparison of Metagenomic Sequencing and the NanoString nCounter Analysis System for the Characterization of Bacterial and Viral Communities in Vaginal Samples.

ABSTRACTDNA sequencing assays have been used to characterize the vaginal microbiome and to identify associations with clinical outcomes. The purpose of this study was to evaluate the utility of the NanoString nCounter platform, a more efficient assay compared to sequencing, for the characterization of vaginal microbial communities. A panel of NanoString nCounter probes was designed to detect common vaginal bacteria and viruses with relevance to reproductive health. A defined synthetic community of microbes and 43 clinical samples were interrogated with NanoString nCounter assays and compared to known compositions or metagenomic shotgun sequencing (MSS) results. The NanoString nCounter platform and MSS were able to distinguish closely related microbes. In clinical samples, the relative abundance of bacterial species was similar between the two assays. The assays sometimes disagreed when targets were present at low abundance. More viruses were detected by MSS than by nCounter. However, the nCounter assays are able to provide results in about 30 h with minimal hands-on time, whereas MSS requires at least 138 to 178 h with extensive hands-on time. The reagent cost for the two assays was similar, but the overall cost of the nCounter was lower due to the minimal hands-on time. MSS can be used to inform the design of a targeted multiplex panel for the assessment of vaginal microbial communities, thereby allowing for more cost-effective and rapid screening of patient samples for research studies. The sensitivity for low abundance microbes could be improved, possibly by adding additional target amplification cycles before nCounter assessment. This approach has potential as an assay with both research and clinical applications.IMPORTANCE Metagenomic shotgun sequencing can inform the design of a targeted multiplex panel by which the NanoString nCounter platform can assess vaginal microbial communities, thereby allowing for more cost-effective and rapid screening of patient samples.

Development of a New Biomarker Model for Predicting Preterm Birth in Cervicovaginal Fluid.

Preterm birth (PTB) is a social problem that adversely affects not only the survival rate of the fetus, but also the premature babies and families, so there is an urgent need to find accurate biomarkers. We noted that among causes, eubiosis of the vaginal microbial community to dysbiosis leads to changes in metabolite composition. In this study, short chain fatty acids (SCFAs) representing dysbiosis were derivatized using (N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide, MTBSTFA) and targeted analysis was conducted in extracted organic phases of cervicovaginal fluid (CVF). In residual aqueous CVF, polar metabolites produced biochemistry process were derivatized using methoxyamine and N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA), and non-targeted analysis were conducted. Nine SCFAs were quantified, and 58 polar metabolites were detected in 90 clinical samples using gas chromatography/mass spectrometry (GC/MS). The criteria of statistical analysis and detection rate of clinical sample for development of PTB biomarkers were presented, and 19 biomarkers were selected based on it, consisting of 1 SCFA, 2 organic acids, 4 amine compounds, and 12 amino acids. In addition, the model was evaluated as a suitable indicator for predicting PTB without distinction between sample collection time. We hope that the developed biomarkers based on microbiota-derived metabolites could provide useful diagnostic biomarkers for actual patients and pre-pregnancy.

Predicting preterm birth through vaginal microbiota, cervical length, and WBC using a machine learning model.

An association between the vaginal microbiome and preterm birth has been reported. However, in practice, it is difficult to predict premature birth using the microbiome because the vaginal microbial community varies highly among samples depending on the individual, and the prediction rate is very low. The purpose of this study was to select markers that improve predictive power through machine learning among various vaginal microbiota and develop a prediction algorithm with better predictive power that combines clinical information. As a multicenter case–control study with 150 Korean pregnant women with 54 preterm delivery group and 96 full-term delivery group, cervicovaginal fluid was collected from pregnant women during mid-pregnancy. Their demographic profiles (age, BMI, education level, and PTB history), white blood cell count, and cervical length were recorded, and the microbiome profiles of the cervicovaginal fluid were analyzed. The subjects were randomly divided into a training (n = 101) and a test set (n = 49) in a two-to-one ratio. When training ML models using selected markers, five-fold cross-validation was performed on the training set. A univariate analysis was performed to select markers using seven statistical tests, including the Wilcoxon rank-sum test. Using the selected markers, including Lactobacillus spp., Gardnerella vaginalis, Ureaplasma parvum, Atopobium vaginae, Prevotella timonensis, and Peptoniphilus grossensis, machine learning models (logistic regression, random forest, extreme gradient boosting, support vector machine, and GUIDE) were used to build prediction models. The test area under the curve of the logistic regression model was 0.72 when it was trained with the 17 selected markers. When analyzed by combining white blood cell count and cervical length with the seven vaginal microbiome markers, the random forest model showed the highest test area under the curve of 0.84. The GUIDE, the single tree model, provided a more reasonable biological interpretation, using the 10 selected markers (A. vaginae, G. vaginalis, Lactobacillus crispatus, Lactobacillus fornicalis, Lactobacillus gasseri, Lactobacillus iners, Lactobacillus jensenii, Peptoniphilus grossensis, P. timonensis, and U. parvum), and the covariates produced a tree with a test area under the curve of 0.77. It was confirmed that the association with preterm birth increased when P. timonensis and U. parvum increased (AUC = 0.77), which could also be explained by the fact that as the number of Peptoniphilus lacrimalis increased, the association with preterm birth was high (AUC = 0.77). Our study demonstrates that several candidate bacteria could be used as potential predictors for preterm birth, and that the predictive rate can be increased through a machine learning model employing a combination of cervical length and white blood cell count information.

The association between the pre-pregnancy vaginal microbiome and time-to-pregnancy: a Chinese pregnancy-planning cohort study

BackgroundAlthough sexually transmitted infections are regarded as the main cause of tubal infertility, the association between the common vaginal microbiome and female fecundability has yet to be determined. The objective of this study was to find convincing evidence relating to the impact of the vaginal bacterial structure on the fecundability of women planning pregnancy.MethodsWe recruited women who took part in the Free Pre-pregnancy Health Examination Project from 13 June 2018 to 31 October 2018 (n = 89, phase I) and from 1 November 2018 to 30 May 2020 (n = 389, phase II). We collected pre-pregnancy vaginal swabs from each subject; then, we followed up each subject to acquire the pregnancy-planning outcome in 1 year. In phase I, 16S rRNA gene sequencing was performed to investigate the vaginal bacterial content between the pregnancy and non-pregnancy groups. These findings were verified in phase II by applying a quantitative real-time polymerase chain reaction for the measurement of the absolute abundance of specific species. Cox models were used to estimate fecundability ratios (FR) for each vaginal microbiome type.ResultsIn phase I, 59.6% (53/89) of women became pregnant within 1 year. The principal coordinate analysis showed that the pre-pregnancy vaginal microbial community structures of the pregnant and non-pregnant groups were significantly different (PERMANOVA test, R2 = 0.025, P = 0.049). The abundance of the genus Lactobacillus in the pregnancy group was higher than that of the non-pregnant group (linear discriminant analysis effect size (LDA) > 4.0). The abundance of the genus Gardnerella in the non-pregnant group was higher than those in the pregnant group (LDA > 4.0). In phase II, female fecundability increased with higher absolute loads of Lactobacillus gasseri (quartile Q4 vs Q1, FR = 1.71, 95%CI 1.02–2.87) but decreased with higher absolute loads of Fannyhessea vaginae (Q4 vs Q1, FR = 0.62, 95%CI 0.38–1.00). Clustering analysis showed that the vaginal microbiome of type D (characterized by a higher abundance of Lactobacillus iners, a lower abundance of Lactobacillus crispatus and Lactobacillus gassri) was associated with a 55% reduction of fecundability (FR = 0.45, 95%CI 0.26–0.76) compared with type A (featuring three Lactobacillus species, low Gardnerella vaginalis and Fannyhessea vaginae abundance).ConclusionsThis cohort study demonstrated an association between the pre-pregnancy vaginal microbiome and female fecundability. A vaginal microbiome characterized by a higher abundance of L. iners and lower abundances of L. crispatus and L. gasseri appeared to be associated with a lower fecundability. Further research now needs to confirm whether manipulation of the vaginal microenvironment might improve human fecundability.

Genetic Elements Orchestrating Lactobacillus crispatus Glycogen Metabolism in the Vagina.

Glycogen in the female lower reproductive tract is a major carbon source for colonization and acidification by common vaginal Lactobacillus species, such as Lactobacillus crispatus. Previously, we identified the amylopullulanase encoding gene pulA of Lactobacillus crispatus to correlate with the ability to autonomously utilize glycogen for growth. Here, we further characterize genetic variation and differential regulation of pulA affecting the presence of its gene product on the outer surface layer. We show that alpha-glucan degrading activity dissipates when Lactobacillus crispatus is grown on glucose, maltose and maltotriose, in agreement with carbon catabolite repression elements flanking the pulA gene. Proteome analysis of the S-layer confirmed that the amylopullulanase protein is highly abundant in an S-layer enriched fraction, but not in a strain with a defective amylopullulanase variant or in an amylopullulanase-sufficient strain grown on glucose. In addition, we provide evidence that Lactobacillus crispatus pulA mutants are relevant in vivo, as they are commonly observed in metagenome datasets of human vaginal microbial communities. Analysis of the largest publicly available dataset of 1507 human vaginal metagenomes indicates that among the 270 samples that contain a Lactobacillus crispatus pulA gene, 62 samples (23%) had a defective variant of this gene. Taken together, these results demonstrate that both environmental, as well as genetic factors explain the variation of Lactobacillus crispatus alpha-glucosidases in the vaginal environment.

Prenatal Stress and the Microbiome in the Time of COVID- A Prospective Cohort Study

BackgroundGiven the emerging importance of the role of the gut microbiota-brain-axis in mediating prenatal stress-induced neurodevelopmental outcomes, a prospective cohort study was conducted. The COVID-19 Pandemic occurred halfway through study recruitment (n=35). The study aims to a) evaluate perceived stress across gestation, b) determine whether maternal microbiome composition changes with stress, and c) discern the influence of the COVID-19 pandemic on maternal stress, psychometric scores, and alterations in the microbiome.MethodsThis longitudinal study design includes five time points across pregnancy and the post-partum period, at which biological samples were collected and psychometrics administered. Samples include maternal rectal and vaginal swabs. Psychometrics include measures of perceived stress, anxiety, depression, sleep, diet, and childhood adversity. Study participants identify as 62.9% White and 31.4% Black or African American. Finally, PacBio full-length 16S rRNA sequencing using SMRT Cell technology is used to identify the maternal rectal and vaginal microbial communities.ResultsParticipants delivering during the pandemic reporting greater perceived stress (p≤0.05). Of note, there were no significant differences in anxiety or depressive symptoms across gestation in the pre-pandemic participants as compared to participants during the pandemic. During the second trimester, increased depression associated with increased rectal alpha diversity, and increased perceived stress was associated with increased levels of Prevotella, Sneathia, and Gardnerella in the rectal samples. In contrast, participants with increased depressive symptoms during the third trimester had reduced vaginal alpha diversity measures at delivery.ConclusionsFindings suggests maternal perceived stress and depressive symptoms are associated with alterations in maternal microbiotaKeywordsGut Microbiome, Prenatal Maternal Stress, Gut-Brain Axis BackgroundGiven the emerging importance of the role of the gut microbiota-brain-axis in mediating prenatal stress-induced neurodevelopmental outcomes, a prospective cohort study was conducted. The COVID-19 Pandemic occurred halfway through study recruitment (n=35). The study aims to a) evaluate perceived stress across gestation, b) determine whether maternal microbiome composition changes with stress, and c) discern the influence of the COVID-19 pandemic on maternal stress, psychometric scores, and alterations in the microbiome. Given the emerging importance of the role of the gut microbiota-brain-axis in mediating prenatal stress-induced neurodevelopmental outcomes, a prospective cohort study was conducted. The COVID-19 Pandemic occurred halfway through study recruitment (n=35). The study aims to a) evaluate perceived stress across gestation, b) determine whether maternal microbiome composition changes with stress, and c) discern the influence of the COVID-19 pandemic on maternal stress, psychometric scores, and alterations in the microbiome. MethodsThis longitudinal study design includes five time points across pregnancy and the post-partum period, at which biological samples were collected and psychometrics administered. Samples include maternal rectal and vaginal swabs. Psychometrics include measures of perceived stress, anxiety, depression, sleep, diet, and childhood adversity. Study participants identify as 62.9% White and 31.4% Black or African American. Finally, PacBio full-length 16S rRNA sequencing using SMRT Cell technology is used to identify the maternal rectal and vaginal microbial communities. This longitudinal study design includes five time points across pregnancy and the post-partum period, at which biological samples were collected and psychometrics administered. Samples include maternal rectal and vaginal swabs. Psychometrics include measures of perceived stress, anxiety, depression, sleep, diet, and childhood adversity. Study participants identify as 62.9% White and 31.4% Black or African American. Finally, PacBio full-length 16S rRNA sequencing using SMRT Cell technology is used to identify the maternal rectal and vaginal microbial communities. ResultsParticipants delivering during the pandemic reporting greater perceived stress (p≤0.05). Of note, there were no significant differences in anxiety or depressive symptoms across gestation in the pre-pandemic participants as compared to participants during the pandemic. During the second trimester, increased depression associated with increased rectal alpha diversity, and increased perceived stress was associated with increased levels of Prevotella, Sneathia, and Gardnerella in the rectal samples. In contrast, participants with increased depressive symptoms during the third trimester had reduced vaginal alpha diversity measures at delivery. Participants delivering during the pandemic reporting greater perceived stress (p≤0.05). Of note, there were no significant differences in anxiety or depressive symptoms across gestation in the pre-pandemic participants as compared to participants during the pandemic. During the second trimester, increased depression associated with increased rectal alpha diversity, and increased perceived stress was associated with increased levels of Prevotella, Sneathia, and Gardnerella in the rectal samples. In contrast, participants with increased depressive symptoms during the third trimester had reduced vaginal alpha diversity measures at delivery. ConclusionsFindings suggests maternal perceived stress and depressive symptoms are associated with alterations in maternal microbiota Findings suggests maternal perceived stress and depressive symptoms are associated with alterations in maternal microbiota

181 Evaluation of the Fecal Microbiota in Commercial Sows with Variable Risk for Pelvic Organ Prolapse During Late Gestation

Abstract Sow mortality has increased during the last decade with a large portion due to pelvic organ prolapse (POP) manifesting during late gestation and early lactation. Approximately 21% of sow mortality is attributed to POP, creating a significant economic and animal welfare concern. The study objective was to identify differences in sow fecal microbiota associated with POP risk and to determine if fecal and vaginal microbial communities are correlated. Using an established perineal scoring (PS) system, 2,864 sows were assigned PS1 (low), PS2 (moderate), or PS3 (high) based on phenotypic observations and presumed POP risk during gestation week 15. Of those sows scored, 1.0%, 2.7%, and 23.4% of PS1, PS2, and PS3 assigned sows, respectively, subsequently experienced POP. At the time of scoring, fecal swabs were collected from sows (n = 213) and DNA was extracted for 16S rRNA gene Illumina MiSeq sequencing, and analyzed using mothur, phyloseq and SAS. Additionally, co-occurrence networks were constructed between fecal and vaginal microbial communities using CoNet (V1.1.1), to identify correlations in taxa abundance. Differences in fecal community composition (PERMANOVA; P &amp;lt; 0.05), structure (alpha diversity measurements; P &amp;lt; 0.05), and 13 individual operational taxonomic units (OTU; Q &amp;lt; 0.05) were detected between PS1 and PS3 sows. The abundance of several taxa were correlated across sample collection sites, suggesting the fecal and vaginal microbial communities may influence one another. Collectively, fewer differences exist in fecal microbiota in sows differing risk for POP than previously observed in vaginal microbiota, suggesting the vaginal microbial communities may be a better indicator of POP risk. Understanding correlations between the fecal and vaginal microbial communities may provide strategies or targets for combatting POP. This project was supported by the National Pork Board and the Foundation for Food and Agriculture Research.

Community Structure and Ecological Network's Changes of Vaginal Microbiome in Women Right After Delivery.

ObjectivesVaginal microbiota is not only an important source of bacterial colonization for neonates, but also plays a crucial role in maternal and neonatal health. This study aimed to investigate the vaginal microbial community structure right after delivery and its impact on the neonatal oral microbiome.MethodsIn this study, 27 women were recruited from Bao'an Maternal and Child Health Hospital. Bacterial compositions of vaginal samples before and right after delivery and neonatal oral samples right after birth were investigated using 16S rRNA sequencing of V3–V4 hyperregions.ResultsVaginal microbiome before delivery was dominated by the genus Lactobacillus. After delivery, the vaginal microbial community was altered, with significantly decreased proportion of Lactobacillus, increased alpha-diversity, and a more diverse ecological network. A large number subjects dominated by Lactobacillus species before delivery shifted to CST (community state type) IV after delivery. In addition, similar changes were observed in the neonatal oral microbiome, and its community profile was closer to vaginal samples after delivery than before delivery with principal coordinates analysis and microbial source tracking analysis.ConclusionThe vaginal microbiome was altered right after delivery and impacted the colonization of the neonatal oral microbiome in China. Further, it is vital to understand the longitudinal influence on maternal and neonatal health of vaginal microbiome community changes after delivery.

Effect of β-Estradiol on Mono- and Mixed-Species Biofilms of Human Commensal Bacteria Lactobacillus paracasei AK508 and Micrococcus luteus C01 on Different Model Surfaces

The impact of steroid hormones, and particularly estradiol, on human microbiota could be recognized as a substantial part of human-microbiota interactions. However, an area that remains poorly investigated is that of the skin and vaginal microbial communities and biofilms, which contain non-pathogenic bacteria of phyla Firmicutes and Actinobacteria, especially probiotic bacteria of the genus Lactobacillus and the widespread, safe skin genus, Micrococcus. Experiments with Lactobacillus paracasei AK508 and Micrococcus luteus C01 biofilms on PTFE cubes showed dose-dependent effects of estradiol at concentrations of 0.22 nM and 22 nM. The hormone mostly inhibits L. paracasei growth and stimulates M. luteus. The presented studies of colony-forming unit (CFU) amountsand cell aggregation in biofilms on glass fiber filters showed the same general tendencies. Estradiol generally increased the aggregation of cells in monospecies communities and potentially changed the synthesis of antibacterial metabolites in L. paracasei. The balance between two bacteria in mixed-species biofilms depended on the initial adhesion stage, and when this stage was reduced, micrococci were more resistant to the antagonistic action of L. paracasei. Moreover, in mixed-species biofilms, the effect of estradiol on lactobacilli altered from inhibition to stimulation, potentially due to the presence of M. luteus. At the same time, ethanol as a solvent for estradiol at the concentration 0.6% acted mostly as an antagonist of the hormone and had an opposite effect on bacteria; nevertheless, the overlapping of ethanol and estradiol effects was shown to be minimal. The data obtained prove the complexity of microbial interactions and the regulatory effect of estradiol on commensal bacteria biofilms.

Network-Based Differences in the Vaginal and Bladder Microbial Communities Between Women With and Without Urgency Urinary Incontinence.

BackgroundLittle is known about the relationship of proximal urogenital microbiomes in the bladder and the vagina and how this contributes to bladder health. In this study, we use a microbial ecology and network framework to understand the dynamics of interactions/co-occurrences of bacteria in the bladder and vagina in women with and without urgency urinary incontinence (UUI).MethodsWe collected vaginal swabs and catheterized urine specimens from 20 women with UUI (cases) and 30 women without UUI (controls). We sequenced the V4 region of the bacterial 16S rRNA gene and evaluated using alpha and beta diversity metrics. We used microbial network analysis to detect interactions in the microbiome and the betweenness centrality measure to identify central bacteria in the microbial network. Bacteria exhibiting maximum betweenness centrality are considered central to the microbe-wide networks and likely maintain the overall microbial network structure.ResultsThere were no significant differences in the vaginal or bladder microbiomes between cases and controls using alpha and beta diversity. Silhouette metric analysis identified two distinct microbiome clusters in both the bladder and vagina. One cluster was dominated by Lactobacillus genus while the other was more diverse. Network-based analyses demonstrated that vaginal and bladder microbial networks were different between cases and controls. In the vagina, there were similar numbers of genera and subgroup clusters in each network for cases and controls. However, cases tend to have more unique bacterial co-occurrences. While Bacteroides and Lactobacillus were the central bacteria with the highest betweenness centrality in controls, Aerococcus had the highest centrality in cases and correlated with bacteria commonly associated with bacterial vaginosis. In the bladder, cases have less than half as many network clusters compared to controls. Lactobacillus was the central bacteria in both groups but associated with several known uropathogens in cases. The number of shared bacterial genera between the bladder and the vagina differed between cases and controls, with cases having larger overlap (43%) compared to controls (29%).ConclusionOur study shows overlaps in microbial communities of bladder and vagina, with higher overlap in cases. We also identified differences in the bacteria that are central to the overall community structure.