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Vaginal microbiota composition affects many facets of reproductive health. Lactobacillus iners-dominated microbial communities are associated with poorer outcomes, including higher risk of bacterial vaginosis (BV), compared with vaginal microbiota rich in L. crispatus. Unfortunately, standard-of-care metronidazole therapy for BV typically results in dominance of L. iners, probably contributing to post-treatment relapse. Here we generate an L. iners isolate collection comprising 34 previously unreported isolates from 14 South African women with and without BV and 4 previously unreported isolates from 3 US women. We also report an associated genome catalogue comprising 1,218 vaginal Lactobacillus isolate genomes and metagenome-assembled genomes from300 women across 4 continents. We show that, unlike L. crispatus, L. iners growth is dependent on L-cysteine in vitro and we trace this phenotype to the absence of canonical cysteine biosynthesis pathways and a restricted repertoire of cysteine-related transport mechanisms. We further show that cysteine concentrations in cervicovaginal lavage samples correlate with Lactobacillus abundance in vivo and that cystine uptake inhibitors selectively inhibit L. iners growth in vitro. Combining an inhibitor with metronidazole promotes L. crispatus dominance of defined BV-like communities in vitro by suppressing L. iners growth. Our findings enable a better understanding of L. iners biology and suggest candidate treatments to modulate the vaginal microbiota to improve reproductive health for women globally.

We compared the effect of commercial vaginal douching products on Lactobacillus crispatus, L. jensenii, L. gasseri, L. iners, E. coli, and immortalized vaginal epithelial cells (VK2). All studied douching products (vinegar, iodine and baking soda based) induced epithelial cell death, and all inhibited growth of E. coli. Co-culture of vaginal epithelial cells with any of the lactobacilli immediately following exposure to douching products resulted in a trend to less human cell death. However, co-culture of epithelial cells with L. iners was associated with higher production of IL6 and IL8, and lower IL1RA regardless of presence or type of douching solution. Co-culture with L. crispatus or L. jensenii decreased IL6 production in the absence of douches, but increased IL6 production after exposure to vinegar. Douching products may be associated with epithelial disruption and inflammation, and may reduce the anti-inflammatory effects of beneficial lactobacilli.

Vaginal microbiota composition affects several important reproductive health outcomes. Lactobacillus crispatus-dominant bacterial communities have favorable associations whereas anaerobe-dominant communities deficient of lactobacilli are linked to poor outcomes, including bacterial vaginosis (BV). Lactobacillus iners, the most abundant vaginal species worldwide, has adverse associations compared to L. crispatus, but standard metronidazole treatment for BV promotes L. iners-dominance, likely contributing to post-treatment relapse. L. iners is under-studied because it fails to grow in standard Lactobacillus media in vitro. Here we trace this in vitro phenotype to a species-specific cysteine requirement associated with limitations in cysteine-related transport mechanisms and show that vaginal cysteine concentrations correlate with Lactobacillus abundance in vivo. We demonstrate that cystine uptake inhibitors selectively impede L. iners growth and that combining an inhibitor with metronidazole thus promotes L. crispatus dominance of defined BV-like communities. These findings identify a novel target for therapeutic vaginal microbiota modulation to improve reproductive health.

Background We compared vaginal microbial communities in postmenopausal black and white women. Methods Shotgun sequencing of vaginal swabs from postmenopausal women self-identified as black or white was compared using MiRKAT. Results Vaginal community dominance by Lactobacillus crispatus or Lactobacillusgasseri was more common in 44 postmenopausal black women (n = 12, 27%) than among 44 matched white women (n = 2, 5%; P = .01). No individual taxa were significantly more abundant in either group. Conclusions We identified small overall differences in vaginal microbial communities of black and white postmenopausal women. L. crispatus dominance was more common in black women. Clinical Trials Registration NCT02516202 (MsFLASH05) and NCT01418209 (MsFLASH03).

Background Up to 30% of women with vaginal symptoms are not assigned a diagnosis after standard diagnostic assessment. Methods We compared premenopausal women with idiopathic vaginitis (IV) or vulvodynia (VVD) to healthy controls. Microbiota were characterized using rRNA sequencing. Cytokines/chemokines (IL-10, IL-1α, IL-1β, IL-6, IL-8, IL-2, IL-18, IL-4, IL-9, and IL-13) were measured in vaginal lavage fluid using the Meso Scale Discovery platform or ELISA (IL-1ra). Immunoglobulins were measured in vaginal lavage fluid using a bead-based immunoassay (Millipore). Cases and controls were compared using Kruskal-Wallis, analysis of variance, and linear regression or (for microbiome composition) the Bray-Curtis dissimilarity statistic. Results We compared 20 women with IV, 30 with VVD, and 52 controls. Most (80%) had greater than 90% 16S rRNA gene sequences from Lactobacillus crispatus, L. jensenii, L. gasseri, or L. iners. In analyses adjusted for age and hormonal contraception (HC), Gardnerella vaginalis was less prevalent and abundant in women with VVD (2/30, 7%) versus controls (16/52, 31%) or IV (5/20, 25%) (P = 0.030). Bray-Curtis dissimilarity was not significantly different between IV and controls or VVD. Fungal sequences were only detected in 5 participants: 2 control, 1 IV, 2 VVD. In univariate analysis, cytokines were not associated with diagnosis. Median vaginal concentration of IgE (but not other immunoglobulins) was lower in women with VVD (P = 0.006). Conclusions Minimal differences in vaginal microbiota and inflammatory markers between women with IV, VVD or controls suggest no striking association between vaginal bacteria, fungi or inflammation and diagnosis in these women.

Our goal was to identify evidenced-based strategies to prevent and treat patients with neurogenic bladder who suffer from recurrent urinary tract infections. We focused on therapy beyond standard antibiotics to address a multitude of factors implicated in these complex infections. Anatomic and functional components specific to neurogenic bladder contribute to the risk of infection and require close clinical monitoring. The host-pathogen interaction is one that allows for colonization of bacteria in the bladder. We recognize that bacteriuria in the neurogenic bladder population does not equate with infection. Local antibiotic treatment and oral supplements are often not adequate to eliminate infection nor prevent recurrent infection due to biofilms. However, novel medical therapies, such as photodynamic therapy, bacterial interference, and infrared laser therapy to augment local immune cells, are promising options to prevent and treat symptomatic infection. A combination approach including management of anatomic and functional factors with medical intervention can significantly improve frequency of urinary infection. Further study of non-antibiotic therapeutic strategies is much needed as we recognize the complexity of the urinary biomes and the limitations of antibiotic therapies.

Half of all postmenopausal women report symptoms of vulvar, vaginal, or urinary discomfort with substantial impact on sexual function and quality of life; underlying mechanisms leading to symptoms are poorly understood.To examine the possibility that the vaginal microbiota and/or mucosal immune response contributes to the severity of bothersome vaginal symptoms, we conducted a substudy of samples from a randomized trial of vaginal treatment for genitourinary syndrome of menopause to compare these features between women whose symptoms improved and women whose symptoms did not improve.This is a secondary analysis of samples collected in a 12-week randomized trial of treatment with vaginal estradiol or moisturizer vs placebo for moderate-severe postmenopausal symptoms of vaginal discomfort. We randomly selected 20 women in each arm with ≥2-point decrease in most bothersome symptom severity (responders) and 20 matched controls with ≤1-point decrease (nonresponders). At 0, 4, and 12 weeks, we characterized vaginal microbiota (16S ribosomal RNA gene sequencing), vaginal fluid metabolites (broad-based metabolomic profiling), vaginal fluid-soluble immune markers (Meso Scale Discovery), pH, and vaginal maturation index. We compared responders with nonresponders at baseline and across all visits using linear mixed models to evaluate associations with microbiota, metabolites, and immune markers, incorporating visit and participant-specific random effects while controlling for treatment arm.Here, the mean age of women was 61 years (n=120), and most women (92%) were White. At enrollment, no significant differences were observed between responders and nonresponders in age, most bothersome symptom type or severity, microbiota composition or diversity, Lactobacillus dominance, metabolome, or immune markers. There was a significant decrease in diversity of the vaginal microbiota in both responders and nonresponders (P.001) over 12 weeks. Although this change did not differ by responder status, diversity was associated with treatment arm: more women in the estradiol arm (63%) had Lactobacillus-dominant, lower diversity bacterial communities than women in the moisturizer (35%) or dual placebo (23%) arms (P=.001) at 12 weeks. The metabolome, vaginal maturation index, and measured immune markers were not associated with responder status over the 12 weeks but varied by treatment arm.Postmenopausal vaginal symptom severity was not significantly associated with vaginal microbiota or mucosal inflammatory markers in this small study. Women receiving vaginal estradiol experienced greater abundance of lactobacilli and lower vaginal pH at end of treatment.

Background Cervicovaginal microbiota domination by Lactobacillus crispatus is associated with beneficial health outcomes, whereas L. iners dominance has more adverse associations. However bacterial vaginosis (BV) treatment with metronidazole (MTZ) typically leads to domination by L. iners rather than L. crispatus. L. iners differs from other lactobacilli by its inability to grow in MRS media. We hypothesized that exploring this growth difference would identify targets for selective L. iners inhibition. Methods Bacteria were grown anaerobically. Nutrient uptake and metabolism were assessed using UPLC-MS/MS and isotopically labeled substrates. Bacterial genome annotation employed Prodigal, Roary, and EggNOG. Competition experiments with mock mixed communities were analyzed by 16S rRNA gene sequencing. We confirmed result generalizability using a diverse collection of South African and North American strains and genomes. Results Supplementing MRS broth with L-cysteine (Cys) or L-cystine permitted robust L. iners growth, while L. crispatus grew without Cys supplementation. Despite their different growth requirements, neither species could synthesize Cys via canonical pathways. Adding the cystine uptake inhibitors S-methyl-L-cysteine (SMC, Fig 1) or seleno-DL-cystine (SDLC) blocked growth of L. iners but not other lactobacilli, suggesting L. iners lacks mechanisms other lactobacilli use to exploit complex exogenous Cys sources. Notably, cydABCD, an operon with Cys/glutathione transport and redox homeostasis activities, is absent from L. iners but present in non-iners Lactobacillus species. Consistent with possible roles for cydABCD in explaining the observed phenotypes, (1) L. iners failed to take up exogenous glutathione and (2) supplementing MRS with reducing agents permitted L. iners growth, which could be blocked by SMC or SDLC. In growth competitions testing L. iners and L. crispatus within mock BV-like communities, SMC plus MTZ outperformed MTZ alone in promoting L. crispatus dominance (Figs 2&3). Figure 1: S-methyl-L-cysteine (SMC) selectively blocks growth of L. iners but not other cervicovaginal Lactobacillus species in cysteine-supplemented MRS broth. Growth was measured by optical density and inhibition calculated relative to Cys-supplemented no-inhibitor control during exponential growth. Values displayed are median (+/- maximum/minimum) for 3 replicates from a single experiment. In all panels, representative data are shown from 1 of >=2 independent experiments for each bacterial strain and media condition. Results are representative of multiple strains for L. iners (n = 16), L. crispatus (n = 7), and L. jensenii (n = 2). Figure 2: Relative abundance of L. crispatus, L. iners, or various BV-associated bacteria in mock bacterial communities grown in rich, non-selective media with or without metronidazole (MTZ) and/or SMC. Relative abundance was determined by bacterial 16S rRNA gene sequencing. Data are shown for three representative mock communities with 5 replicates per media condition. Figure 3: Ratio of L. crispatus to other species in the mock bacterial communities depicted in Figure 2. Statistical significance determined via 1-way ANOVA of log10-transformed ratios with post-hoc Tukey test; selected pairwise comparisons are shown (***, p < 0.001). Conclusion L. iners has unique requirements for exogenous cysteine/cystine or a reduced environment for growth. Targeting cystine uptake to inhibit L. iners is a potential strategy for shifting cervicovaginal microbiota towards L. crispatus-dominant communities. Disclosures Douglas S. Kwon, MD, PhD, Day Zero Diagnostics (Consultant, Shareholder, Other Financial or Material Support, co-founder)

To evaluate magnetic resonance imaging findings that differentiate among periurethral bulking agents (primarily collagen), urethral diverticulum, and periurethral cyst. We searched our radiologic database retrospectively from 2001 to 2017 for periurethral cystic lesions, identifying a total of 50 patients with 68 lesions. Final diagnoses in 68 lesions were bulking agents (27), urethral diverticula (29), and periurethral cysts (12). Two abdominal radiologists, blinded to clinical history, independently evaluated T1, T2, and post-contrast images. The readers assessed number, morphological features, location, connection to urethra and mass effect, signal intensity, and enhancement for each lesion. Fisher exact test and logistic regression analysis were performed for each univariate significant feature. The operative and pathologic reports were the reference standard. Magnetic resonance imaging features found more often in bulking agents versus urethral diverticulum were multiple lesions (P = 0.011), upper or upper-mid-urethral location (P ≤ 0.0001), lack of internal fluid/fluid level (P = 0.002), no urethral connection (P = 0.005), T1 isointensity, and T2 mild hyperintensity compared to muscles but lower T2 signal than urine (P