Harnessing the power within: engineering the microbiome for enhanced gynecologic health.

Abstract: Although numerous studies have demonstrated the impact of microbiome manipulation on human health, research on the microbiome's influence on female health remains relatively limited despite substantial disease burden. In light of this, we present a selected review of clinical trials and preclinical studies targeting both the vaginal and gut microbiomes for the prevention or treatment of various gynecologic conditions. Specifically, we explore studies that leverage microbiota transplants, probiotics, prebiotics, diet modifications, and engineered microbial strains. A healthy vaginal microbiome for females of reproductive age consists of lactic acid-producing bacteria predominantly of the Lactobacillus genus, which serves as a protective barrier against pathogens and maintains a balanced ecosystem. The gut microbiota's production of short-chain fatty acids, metabolism of primary bile acids, and modulation of sex steroid levels have significant implications for the interplay between host and microbes throughout the body, ultimately impacting reproductive health. By harnessing interventions that modulate both the vaginal and gut microbiomes, it becomes possible to not only maintain homeostasis but also mitigate pathological conditions. While the field is still working toward making broad clinical recommendations, the current studies demonstrate that manipulating the microbiome holds great potential for addressing diverse gynecologic conditions. Lay summary: Manipulating the microbiome has recently entered popular culture, with various diets thought to aid the microbes that live within us. These microbes live in different locations of our body and accordingly help us digest food, modulate our immune system, and influence reproductive health. The role of the microbes living in and influencing the female reproductive tract remains understudied despite known roles in common conditions such as vulvovaginal candidiasis (affecting 75% of females in their lifetime), bacterial vaginosis (25% of females in their lifetime), cervical HPV infection (80% of females in their lifetime), endometriosis (6-10% of females of reproductive age), and polycystic ovary syndrome (10-12% of females of reproductive age). Here, we review four different approaches used to manipulate the female reproductive tract and gastrointestinal system microbiomes: microbiota transplants, probiotics, prebiotics, and dietary interventions, and the use of engineered microbial strains. In doing so, we aim to stimulate discussion on new ways to understand and treat female reproductive health conditions.

Resident microbes shape the vaginal epithelial glycan landscape.

Epithelial cells are covered in carbohydrates (glycans). This glycan coat or "glycocalyx" interfaces directly with microbes, providing a protective barrier against potential pathogens. Bacterial vaginosis (BV) is a condition associated with adverse health outcomes in which bacteria reside in direct proximity to the vaginal epithelium. Some of these bacteria, including Gardnerella, produce glycosyl hydrolase enzymes. However, glycans of the human vaginal epithelial surface have not been studied in detail. Here, we elucidate key characteristics of the "normal" vaginal epithelial glycan landscape and analyze the impact of resident microbes on the surface glycocalyx. In human BV, glycocalyx staining was visibly diminished in electron micrographs compared to controls. Biochemical and mass spectrometric analysis showed that, compared to normal vaginal epithelial cells, BV cells were depleted of sialylated N- and O-glycans, with underlying galactose residues exposed on the surface. Treatment of primary epithelial cells from BV-negative women with recombinant Gardnerella sialidases generated BV-like glycan phenotypes. Exposure of cultured VK2 vaginal epithelial cells to recombinant Gardnerella sialidase led to desialylation of glycans and induction of pathways regulating cell death, differentiation, and inflammatory responses. These data provide evidence that vaginal epithelial cells exhibit an altered glycan landscape in BV and suggest that BV-associated glycosidic enzymes may lead to changes in epithelial gene transcription that promote cell turnover and regulate responses toward the resident microbiome.

Gardnerella Vaginolysin Potentiates Glycan Molecular Mimicry by Neisseria gonorrhoeae.

Bacterial vaginosis (BV) is a dysbiotic condition of the vaginal microbiome associated with higher risk of infection by Neisseria gonorrhoeae-the cause of gonorrhea. Here we test if one known facet of BV-the presence of bacterial cytolysins-leads to mobilization of intracellular contents that enhance gonococcal virulence. We cloned and expressed recombinant vaginolysin (VLY), a cytolysin produced by the BV-associated bacterium Gardnerella, verifying that it liberates contents of cervical epithelial (HeLa) cells, while vector control preparations did not. We tested if VLY mediates a well-known gonococcal virulence mechanism-the molecular mimicry of host glycans. To evade host immunity, N. gonorrhoeae caps its lipooligosaccharide (LOS) with α2-3-linked sialic acid. For this, gonococci must scavenge a metabolite made inside host cells. Flow cytometry-based lectin-binding assays showed that gonococci exposed to vaginolysin-liberated contents of HeLa cells displayed greater sialic acid capping of their LOS. This higher level of bacterial sialylation was accompanied by increased binding of the complement regulatory protein factor H, and greater resistance to complement attack. Together these results suggest that cytolytic activities present during BV may enhance the ability of N. gonorrhoeae to capture intracellular metabolites and evade host immunity via glycan molecular mimicry.

Mesh and layered electrospun fiber architectures as vehicles for Lactobacillus acidophilus and Lactobacillus crispatus intended for vaginal delivery.

Bacterial vaginosis (BV) is a recurrent condition that affects millions of women worldwide. The use of probiotics is a promising alternative or an adjunct to traditional antibiotics for BV prevention and treatment. However, current administration regimens often require daily administration, thus contributing to low user adherence and recurrence. Here, electrospun fibers were designed to separately incorporate and sustain two lactic acid producing model organisms, Lactobacillus crispatus (L. crispatus) and Lactobacillus acidophilus (L. acidophilus). Fibers were made of polyethylene oxide and polylactic-co-glycolic acid in two different architectures, one with distinct layers and the other with co-spun components. Degradation of mesh and layered fibers was evaluated via mass loss and scanning electron microscopy. The results show that after 48 h and 6 days, cultures of mesh and layered fibers yielded as much as 108 and 109 CFU probiotic/mg fiber in total, respectively, with corresponding daily recovery on the order of 108 CFU/(mg·day). In addition, cultures of the fibers yielded lactic acid and caused a significant reduction in pH, indicating a high level of metabolic activity. The formulations did not affect vaginal keratinocyte viability or cell membrane integrity in vitro. Finally, mesh and layered probiotic fiber dosage forms demonstrated inhibition of Gardnerella, one of the most prevalent and abundant bacteria associated with BV, respectively resulting in 8- and 6.5-log decreases in Gardnerella viability in vitro after 24 h. This study provides initial proof of concept that mesh and layered electrospun fiber architectures developed as dissolving films may offer a viable alternative to daily probiotic administration.

Rapid-dissolving electrospun nanofibers for intra-vaginal antibiotic or probiotic delivery.

The emergence of probiotics as an alternative and adjunct to antibiotic treatment for microbiological disturbances of the female genitourinary system requires innovative delivery platforms for vaginal applications. This study developed a new, rapid-dissolving form using electrospun polyethylene oxide (PEO) fibers for delivery of antibiotic metronidazole or probiotic Lactobacillus acidophilus, and performed evaluation in vitro and in vivo. Fibers did not generate overt pathophysiology or encourage Gardnerella growth in a mouse vaginal colonization model, inducing no alterations in vaginal mucosa at 24 hr post-administration. PEO-fibers incorporating metronidazole (100 µg MET/mg polymer) effectively prevented and treated Gardnerella infections (∼3- and 2.5-log reduction, respectively, 24 hr post treatment) when administered vaginally. Incorporation of live Lactobacillus acidophilus (107 CFU/mL) demonstrated viable probiotic delivery in vitro by PEO and polyvinyl alcohol (PVA) fibers to inhibit Gardnerella (108 CFU/mL) in bacterial co-cultures (9.9- and 7.0-log reduction, respectively, 24 hr post-inoculation), and in the presence of vaginal epithelial cells (6.9- and 8.0-log reduction, respectively, 16 hr post-inoculation). Administration of Lactobacillus acidophilus in PEO-fibers achieved vaginal colonization in mice similar to colonization observed with free Lactobacillus. acidophilus. These experiments provide proof-of-concept for rapid-dissolving electrospun fibers as a successful platform for intra-vaginal antibiotic or probiotic delivery.

Release Kinetics of Metronidazole from 3D Printed Silicone Scaffolds for Sustained Application to the Female Reproductive Tract.

Sustained vaginal administration of antibiotics or probiotics has been proposed to improve treatment efficacy for bacterial vaginosis. 3D printing has shown promise for development of systems for local agent delivery. In contrast to oral ingestion, agent release kinetics can be fine-tuned by the 3D printing of specialized scaffold designs tailored for particular treatments while enhancing dosage effectiveness via localized sustained release. It has been challenging to establish scaffold properties as a function of fabrication parameters to obtain sustained release. In particular, the relationships between scaffold curing conditions, compressive strength, and drug release kinetics remain poorly understood. This study evaluates 3D printed scaffold formulation and feasibility to sustain the release of metronidazole, a commonly used antibiotic for BV. Cylindrical silicone scaffolds were printed and cured using three different conditions relevant to potential future incorporation of temperature-sensitive labile biologics. Compressive strength and drug release were monitored for 14d in simulated vaginal fluid to assess long-term effects of fabrication conditions on mechanical integrity and release kinetics. Scaffolds were mechanically evaluated to determine compressive and tensile strength, and elastic modulus. Release profiles were fitted to previous kinetic models to differentiate potential release mechanisms. The Higuchi, Korsmeyer-Peppas, and Peppas-Sahlin models best described the release, indicating similarity to release from insoluble or polymeric matrices. This study shows the feasibility of 3D printed silicone scaffolds to provide sustained metronidazole release over 14d, with compressive strength and drug release kinetics tuned by the fabrication parameters.

Formulation and characterization of pressure-assisted microsyringe 3D-printed scaffolds for controlled intravaginal antibiotic release.

Bacterial vaginosis (BV) is a highly recurrent vaginal condition linked with many health complications. Topical antibiotic treatments for BV are challenged with drug solubility in vaginal fluid, lack of convenience and user adherence to daily treatment protocols, among other factors. 3D-printed scaffolds can provide sustained antibiotic delivery to the female reproductive tract (FRT). Silicone vehicles have been shown to provide structural stability, flexibility, and biocompatibility, with favorable drug release kinetics. This study formulates and characterizes novel metronidazole-containing 3D-printed silicone scaffolds for eventual application to the FRT. Scaffolds were evaluated for degradation, swelling, compression, and metronidazole release in simulated vaginal fluid (SVF). Scaffolds retained high structural integrity and sustained release. Minimal mass loss (<6%) and swelling (<2%) were observed after 14 days in SVF, relative to initial post-cure measurements. Scaffolds cured for 24 hr (50 °C) demonstrated elastic behavior under 20% compression and 4.0 N load. Scaffolds cured for 4 hr (50 °C), followed by 72 hr (4 °C), demonstrated the highest, sustained, metronidazole release (4.0 and 27.0 µg/mg) after 24 hr and 14 days, respectively. Based upon daily release profiles, it was observed that the 24 hr timepoint had the greatest metronidazole release of 4.08 µg/mg for scaffolds cured at 4 hr at 50 °C followed by 72 hr at 4 °C. For all curing conditions, release of metronidazole after 1 and 7 days showed > 4.0-log reduction in Gardnerella concentration. Negligible cytotoxicity was observed in treated keratinocytes comparable to untreated cells, This study shows that pressure-assisted microsyringe 3D-printed silicone scaffolds may provide a versatile vehicle for sustained metronidazole delivery to the FRT.

Fabrication and characterization of bioprints with Lactobacillus crispatus for vaginal application.

Bacterial vaginosis (BV) is characterized by low levels of lactobacilli and overgrowth of potential pathogens in the female genital tract. Current antibiotic treatments often fail to treat BV in a sustained manner, and > 50% of women experience recurrence within 6 months post-treatment. Recently, lactobacilli have shown promise for acting as probiotics by offering health benefits in BV. However, as with other active agents, probiotics often require intensive administration schedules incurring difficult user adherence. Three-dimensional (3D)-bioprinting enables fabrication of well-defined architectures with tunable release of active agents, including live mammalian cells, offering the potential for long-acting probiotic delivery. One promising bioink, gelatin alginate has been previously shown to provide structural stability, host compatibility, viable probiotic incorporation, and cellular nutrient diffusion. This study formulates and characterizes 3D-bioprinted Lactobacillus crispatus-containing gelatin alginate scaffolds for gynecologic applications. Different weight to volume (w/v) ratios of gelatin alginate were bioprinted to determine formulations with highest printing resolution, and different crosslinking reagents were evaluated for effect on scaffold integrity via mass loss and swelling measurements. Post-print viability, sustained-release, and vaginal keratinocyte cytotoxicity assays were conducted. A 10:2 (w/v) gelatin alginate formulation was selected based on line continuity and resolution, while degradation and swelling experiments demonstrated greatest structural stability with dual genipin and calcium crosslinking, showing minimal mass loss and swelling over 28 days. 3D-bioprinted L. crispatus-containing scaffolds demonstrated sustained release and proliferation of live bacteria over 28 days, without impacting viability of vaginal epithelial cells. This study provides in vitro evidence for 3D-bioprinted scaffolds as a novel strategy to sustain probiotic delivery with the ultimate goal of restoring vaginal lactobacilli following microbiological disturbances.

Lactobacillus crispatus-loaded electrospun fibers yield viable and metabolically active bacteria that kill Gardnerella in vitro.

Bacterial vaginosis (BV) is a common condition that affects one-third of women worldwide. BV is characterized by low levels of healthy lactobacilli and an overgrowth of common anaerobes such as Gardnerella. Antibiotics for BV are administered orally or vaginally; however, approximately half of those treated will experience recurrence within 6 months. Lactobacillus crispatus present at high levels has been associated with positive health outcomes. To address the high recurrence rates following BV treatment, beneficial bacteria have been considered as an alternative or adjunct modality. This study aimed to establish proof-of-concept for a new long-acting delivery vehicle for L. crispatus. Here, it is shown that polyethylene oxide (PEO) fibers loaded with L. crispatus can be electrospun with poly(lactic-co-glycolic acid) (PLGA) fibers (ratio 1:1), and that this construct later releases L. crispatus as metabolically viable bacteria capable of lactic acid production and anti-Gardnerella activity. Probiotic-containing fibers were serially cultured in MRS (deMan, Rogosa, Sharpe) broth with daily media replacement and found to yield viable L. crispatus for at least 7 days. Lactic acid levels and corresponding pH values generally corresponded with levels of L. crispatus cultured from the fibers and strongly support the conclusion that fibers yield viable L. crispatus that is metabolically active. Cultures of L. crispatus-loaded fibers limited the growth of Gardnerella in a dilution-dependent manner during in vitro assays in the presence of cultured vaginal epithelial cells, demonstrating bactericidal potential. Exposure of VK2/E6E7 cells to L. crispatus-loaded fibers resulted in minimal loss of viability relative to untreated cells. Altogether, these data provide proof-of-concept for electrospun fibers as a candidate delivery vehicle for application of vaginal probiotics in a long-acting form.

Cataloging natural sialic acids and other nonulosonic acids (NulOs), and their representation using the Symbol Nomenclature for Glycans.

Nonulosonic acids or non-2-ulosonic acids (NulOs) are an ancient family of 2-ketoaldonic acids (α-ketoaldonic acids) with a 9-carbon backbone. In nature, these monosaccharides occur either in a 3-deoxy form (referred to as "sialic acids") or in a 3,9-dideoxy "sialic-acid-like" form. The former sialic acids are most common in the deuterostome lineage, including vertebrates, and mimicked by some of their pathogens. The latter sialic-acid-like molecules are found in bacteria and archaea. NulOs are often prominently positioned at the outermost tips of cell surface glycans, and have many key roles in evolution, biology and disease. The diversity of stereochemistry and structural modifications among the NulOs contributes to more than 90 sialic acid forms and 50 sialic-acid-like variants described thus far in nature. This paper reports the curation of these diverse naturally occurring NulOs at the NCBI sialic acid page (https://www.ncbi.nlm.nih.gov/glycans/sialic.html) as part of the NCBI-Glycans initiative. This includes external links to relevant Carbohydrate Structure Databases. As the amino and hydroxyl groups of these monosaccharides are extensively derivatized by various substituents in nature, the Symbol Nomenclature For Glycans (SNFG) rules have been expanded to represent this natural diversity. These developments help illustrate the natural diversity of sialic acids and related NulOs, and enable their systematic representation in publications and online resources.

Metabolic Network Models of the Gardnerella Pangenome Identify Key Interactions with the Vaginal Environment.

Gardnerella is the primary pathogenic bacterial genus present in the polymicrobial condition known as bacterial vaginosis (BV). Despite BV's high prevalence and associated chronic and acute women's health impacts, the Gardnerella pangenome is largely uncharacterized at both the genetic and functional metabolic levels. Here, we used genome-scale metabolic models to characterize in silico the Gardnerella pangenome metabolic content. We also assessed the metabolic functional capacity in a BV-positive cervicovaginal fluid context. The metabolic capacity varied widely across the pangenome, with 38.15% of all reactions being core to the genus, compared to 49.60% of reactions identified as being unique to a smaller subset of species. We identified 57 essential genes across the pangenome via in silico gene essentiality screens within two simulated vaginal metabolic environments. Four genes, gpsA, fas, suhB, and psd, were identified as core essential genes critical for the metabolic function of all analyzed bacterial species of the Gardnerella genus. Further understanding these core essential metabolic functions could inform novel therapeutic strategies to treat BV. Machine learning applied to simulated metabolic network flux distributions showed limited clustering based on the sample isolation source, which further supports the presence of extensive core metabolic functionality across this genus. These data represent the first metabolic modeling of the Gardnerella pangenome and illustrate strain-specific interactions with the vaginal metabolic environment across the pangenome. IMPORTANCE Bacterial vaginosis (BV) is the most common vaginal infection among reproductive-age women. Despite its prevalence and associated chronic and acute women's health impacts, the diverse bacteria involved in BV infection remain poorly characterized. Gardnerella is the genus of bacteria most commonly and most abundantly represented during BV. In this paper, we use metabolic models, which are a computational representation of the possible functional metabolism of an organism, to investigate metabolic conservation, gene essentiality, and pathway utilization across 110 Gardnerella strains. These models allow us to investigate in silico how strains may differ with respect to their metabolic interactions with the vaginal-host environment.

Gardnerella Exposures Alter Bladder Gene Expression and Augment Uropathogenic Escherichia coli Urinary Tract Infection in Mice.

The anaerobic actinobacterium Gardnerella was first isolated from the bladder by suprapubic aspiration more than 50 years ago. Since then, Gardnerella has been increasingly recognized as a common and often abundant member of the female urinary microbiome (urobiome). Some studies even suggest that the presence of Gardnerella is associated with urological disorders in women. We recently reported that inoculation of Gardnerella into the bladders of mice results in urothelial exfoliation. Here, we performed whole bladder RNA-seq in our mouse model to identify additional host pathways involved in the response to Gardnerella bladder exposure. The transcriptional response to Gardnerella reflected the urothelial turnover that is a consequence of exfoliation while also illustrating the activation of pathways involved in inflammation and immunity. Additional timed exposure experiments in mice provided further evidence of a potentially clinically relevant consequence of bladder exposure to Gardnerella-increased susceptibility to subsequent UTI caused by uropathogenic Escherichia coli. Together, these data provide a broader picture of the bladder's response to Gardnerella and lay the groundwork for future studies examining the impact of Gardnerella on bladder health.

Models of Murine Vaginal Colonization by Anaerobically Grown Bacteria.

The mammalian vagina can be colonized by many bacterial taxa. The human vaginal microbiome is often dominated by Lactobacillus species, but one-in-four women experience bacterial vaginosis, in which a low level of lactobacilli is accompanied by an overgrowth of diverse anaerobic bacteria. This condition has been associated with many health complications, including risks to reproductive and sexual health. While there is growing evidence showing the complex nature of microbial interactions in human vaginal health, the individual roles of these different anaerobic bacteria are not fully understood. This is complicated by the lack of adequate models to study anaerobically grown vaginal bacteria. Mouse models allow us to investigate the biology and virulence of these organisms in vivo. Other mouse models of vaginal bacterial inoculation have previously been described. Here, we describe methods for the inoculation of anaerobically grown bacteria and their viable recovery in conventionally raised C57Bl/6 mice. A new, less stressful procedural method for vaginal inoculation and washing is also described. Inoculation and viable recovery of Gardnerella are outlined in detail, and strategies for additional anaerobes such as Prevotella bivia and Fusobacterium nucleatum are discussed.

Use of the International Consultation on Incontinence Questionnaires Bladder Diary in Men Seeking therapy for Lower Urinary Tract Symptoms.

BACKGROUND: Completion rates and correspondence to other measures need to be established for the International Consultation on Incontinence Questionnaire (ICIQ) bladder diary (ICIQ-BD) in the assessment of male lower urinary tract symptoms (LUTS). OBJECTIVE: To evaluate ICIQ-BD completion rates, frequency, volume, and sensation reporting for men. DESIGN, SETTING, AND PARTICIPANTS: Baseline data from the Urodynamics for Prostate Surgery Trial; Randomised Evaluation of Assessment Methods (UPSTREAM) randomised controlled trial evaluating 820 men at 26 UK hospitals, looking at the ICIQ-BD, uroflowmetry, International Prostate Symptom Score, and ICIQ symptom score for male LUTS (ICIQ-MLUTS), were assessed. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The ICIQ-BD, IPSS, ICIQ-MLUTS, and uroflowmetry data at baseline obtained from UPSTREAM were assessed. Correlations were analysed by Pearson's correlation coefficient, and comparison between groups were performed using paired or unpaired t tests or Tukey's test. All statistical tests were two sided and the strength of evidence was presented using p values. RESULTS AND LIMITATIONS: Of the participants, 25.0% (205/820) provided complete voiding and bedtime information for 3 d, 41.2% (338/820) omitted bedtime information, and the remainder omitted some or all voiding information. Median values (minimum - maximum) of 24-h, daytime, and night-time frequencies were 9.7 (3.3-24.0), 7.7 (3.3-22.7), and 1.7 (0.0-5.7), respectively. The mean voided volume per micturition for day and night times were 175.8 ± 74.2 and 264.4 ± 150.7 ml (p < 0.001), respectively. For fully completed diaries, day- and night-time frequency showed a weak-to-moderate correlation with symptom score questionnaires. More severe nocturia was generally reported in symptom scores than in the ICIQ-BD. In patients with high bother for increased daytime frequency (symptom), the mean daytime frequency (ICIQ-BD) was 9.6 ± 3.2 versus 7.6 ± 2.2 for low bother (p < 0.001). High bother for nocturia showed night-time frequency of 2.3 ± 1.2 versus 1.5 ± 1.1 for low bother (p < 0.001). For fully and partially completed diaries, ICIQ-BD sensation scores correlated weakly with symptom scores. Voided volumes from the bladder diary and uroflowmetry correlated weakly. CONCLUSIONS: Two-thirds of men (543/820) fully completed voiding information in the ICIQ-BD, but many omitted bedtime information, limiting the ability to quantify nocturia and diagnose nocturnal polyuria. PATIENT SUMMARY: Most men with urinary symptoms complete a bladder diary fully but may fail to indicate bedtimes. Extra information from a diary helps support symptom questionnaires to explain a patient's urinary habits.

Prostate Surgery for Men with Lower Urinary Tract Symptoms: Do We Need Urodynamics to Find the Right Candidates? Exploratory Findings from the UPSTREAM Trial.

BACKGROUND: Identifying men whose lower urinary tract symptoms (LUTS) may benefit from surgery is challenging. OBJECTIVE: To identify routine diagnostic and urodynamic measures associated with treatment decision-making, and outcome, in exploratory analyses of the UPSTREAM trial. DESIGN, SETTING, AND PARTICIPANTS: A randomised controlled trial was conducted including 820 men, considering surgery for LUTS, across 26 hospitals in England (ISCTRN56164274). INTERVENTION: Men were randomised to a routine care (RC) diagnostic pathway (n = 393) or a pathway that included urodynamics (UDS) in addition to RC (n = 427). OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Men underwent uroflowmetry and completed symptom questionnaires, at baseline and 18 mo after randomisation. Regression models identified baseline clinical and symptom measures that predicted recommendation for surgery and/or surgical outcome (measured by the International Prostate Symptom Score [IPSS]). We explored the association between UDS and surgical outcome in subgroups defined by routine measures. RESULTS AND LIMITATIONS: The recommendation for surgery could be predicted successfully in the RC and UDS groups (area under the receiver operating characteristic curve 0.78), with maximum flow rate (Qmax) and age predictors in both groups. Surgery was more beneficial in those with higher symptom scores (eg, IPSS >16), age <74 yr, Qmax <9.8 ml/s, bladder outlet obstruction index >47.6, and bladder contractility index >123.0. In the UDS group, urodynamic measures were more strongly predictive of surgical outcome for those with Qmax >15, although patient-reported outcomes were also more predictive in this subgroup. CONCLUSIONS: Treatment decisions were informed with UDS, when available, but without evidence of change in the decisions reached. Despite the small group sizes, exploratory analyses suggest that selective use of UDS could detect obstructive pathology, missed by routine measures, in certain subgroups. PATIENT SUMMARY: Baseline clinical and symptom measurements were able to predict treatment decisions. The addition of urodynamic test results, while useful, did not generally lead to better surgical decisions and outcomes over routine tests alone.

Bladder Exposure to Gardnerella Activates Host Pathways Necessary for Escherichia coli Recurrent UTI.

Recurrent urinary tract infections (rUTI) are a costly clinical problem affecting millions of women worldwide each year. The majority of rUTI cases are caused by uropathogenic Escherichia coli (UPEC). Data from humans and mouse models indicate that some instances of rUTI are caused by UPEC emerging from latent reservoirs in the bladder. Women with vaginal dysbiosis, typically characterized by high levels of Gardnerella and other anaerobes, are at increased risk of UTI. Multiple studies have detected Gardnerella in urine collected by transurethral catheterization (to limit vaginal contamination), suggesting that some women experience routine urinary tract exposures. We recently reported that inoculation of Gardnerella into the bladder triggers rUTI from UPEC bladder reservoirs in a mouse model. Here we performed whole bladder RNA-seq to identify host pathways involved in Gardnerella-induced rUTI. We identified a variety host pathways differentially expressed in whole bladders following Gardnerella exposure, such as pathways involved in inflammation/immunity and epithelial turnover. At the gene level, we identified upregulation of Immediate Early (IE) genes, which are induced in various cell types shortly following stimuli like infection and inflammation. One such upregulated IE gene was the orphan nuclear receptor Nur77 (aka Nr4a1). Pilot experiments in Nur77-/- mice suggest that Nur77 is necessary for Gardnerella exposure to trigger rUTI from UPEC reservoirs. These findings demonstrate that bladder gene expression can be impacted by short-lived exposures to urogenital bacteria and warrant future examination of responses in distinct cell types, such as with single cell transcriptomic technologies. The biological validation studies in Nur77-/- mice lay the groundwork for future studies investigating Nur77 and the Immediate Early response in rUTI.

A mouse model displays host and bacterial strain differences in Aerococcus urinae urinary tract infection.

In recent years, the clinical significance of Aerococcus urinae has been increasingly recognized. A. urinae has been implicated in cases of urinary tract infection (UTI; acute cystitis and pyelonephritis) in both male and female patients, ranging from children to older adults. Aerococcus urinae can also be invasive, causing urosepsis, endocarditis, and musculoskeletal infections. Mechanisms of pathogenesis in A. urinae infections are poorly understood, largely due to the lack of an animal model system. In response to this gap, we developed a model of A. urinae urinary tract infection in mice. We compared A. urinae UTI in female C3H/HeN and C57BL/6 mice and compared four clinical isolates of A. urinae isolated from patients with UTI, urgency urinary incontinence, and overactive bladder. Our data demonstrate that host genetic background modulates A. urinae UTI. Female C57BL/6 female mice rapidly cleared the infection. Female C3H/HeN mice, which have inherent vesicoureteral reflux that flushes urine from the bladder up into the kidneys, were susceptible to prolonged bacteriuria. This result is consistent with the fact that A. urinae infections most frequently occur in patients with underlying urinary tract abnormalities or disorders that make them susceptible to bacterial infection. Unlike uropathogens such as E. coli, which cause infection and inflammation both of the bladder and kidneys in C3H/HeN mice, A. urinae displayed tropism for the kidney, persisting in kidney tissue even after clearance of bacteria from the bladder. Aerococcus urinae strains from different genetic clades displayed varying propensities to cause persistent kidney infection. Aerococcus urinae infected kidneys displayed histological inflammation, neutrophil recruitment and increased pro-inflammatory cytokines. These results set the stage for future research that interrogates host-pathogen interactions between A. urinae and the urinary tract.

Vaginal sialoglycan foraging by Gardnerella vaginalis: mucus barriers as a meal for unwelcome guests?

Bacterial vaginosis (BV) is a condition of the vaginal microbiome in which there are few lactobacilli and abundant anaerobic bacteria. Members of the genus Gardnerella are often one of the most abundant bacteria in BV. BV is associated with a wide variety of poor health outcomes for women. It has been recognized since the 1980s that women with BV have detectable and sometimes markedly elevated levels of sialidase activity in vaginal fluids and that bacteria associated with this condition produce this activity in culture. Mounting evidence collected using diverse methodologies points to the conclusion that BV is associated with a reduction in intact sialoglycans in cervicovaginal secretions. Here we review evidence for the contributions of vaginal bacteria, especially Gardnerella, in the processes of mucosal sialoglycan degradation, uptake, metabolism and depletion. Our understanding of the impacts of vaginal sialoglycan degradation is still limited. However, the potential implications of sialic acid depletion are discussed in light of our current understanding of the roles played by sialoglycans in vaginal physiology.

The structure and diversity of strain-level variation in vaginal bacteria.

The vaginal microbiome plays an important role in human health and species of vaginal bacteria have been associated with reproductive disease. Strain-level variation is also thought to be important, but the diversity, structure and evolutionary history of vaginal strains is not as well characterized. We developed and validated an approach to measure strain variation from metagenomic data based on SNPs within the core genomes for six species of vaginal bacteria: Gardnerella vaginalis, Lactobacillus crispatus, Lactobacillus iners, Lactobacillus jensenii, Lactobacillus gasseri and Atopobium vaginae. Despite inhabiting the same environment, strain diversity and structure varies across species. All species except L. iners are characterized by multiple distinct groups of strains. Even so, strain diversity is lower in the Lactobacillus species, consistent with a more recent colonization of the human vaginal microbiome. Both strain diversity and the frequency of multi-strain samples is related to species-level diversity of the microbiome in which they occur, suggesting similar ecological factors influencing diversity within the vaginal niche. We conclude that the structure of strain-level variation provides both the motivation and means of testing whether strain-level differences contribute to the function and health consequences of the vaginal microbiome.