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.

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.

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.

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.

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.

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.

Use of Leishmania major parasites expressing a recombinant Trypanosoma cruzi antigen as live vaccines against Chagas disease.

Introduction: Trypanosoma cruzi is the protozoan parasite causing Chagas disease, a Neglected Tropical Disease that affects 8 million people and causes 12,000 deaths per year, primarily because of cardiac pathology. Effective vaccination for T. cruzi remains an elusive goal. The use of a live vaccine vector, especially one that mimics the pathogen target, may be superior to the use of recombinant protein or DNA vaccine formulations. Methods: We generated recombinant Leishmania major, a related trypanosomatid parasite, as a vaccine vehicle to express the immunogenic T. cruzi trans-sialidase (TS) antigen. The induction of T cell and antibody responses, as well as T. cruzi protective immunity generated by these vaccines were assessed in vivo. Results: We demonstrate that mice inoculated with these recombinant TS-expressing L. major parasites mount T cell and antibody responses directed against TS and are protected against future T. cruzi infection. We also show that the partially attenuated dhfr-ts- CC1 L. major strain, previously found to induce protective immunity to virulent L. major infection without causing pathology, can also be engineered to express the TS antigen. This latter recombinant may represent a safe and effective option to explore for ultimate use in humans. Discussion: Altogether, these data indicate that L. major can stably express a T. cruzi antigen and induce T. cruzi-specific protective immunity, warranting further investigation of attenuated Leishmania parasites as vaccine.

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.

Glycan cross-feeding supports mutualism between Fusobacterium and the vaginal microbiota.

Women with bacterial vaginosis (BV), an imbalance of the vaginal microbiome, are more likely to be colonized by potential pathogens such as Fusobacterium nucleatum, a bacterium linked with intrauterine infection and preterm birth. However, the conditions and mechanisms supporting pathogen colonization during vaginal dysbiosis remain obscure. We demonstrate that sialidase activity, a diagnostic feature of BV, promoted F. nucleatum foraging and growth on mammalian sialoglycans, a nutrient resource that was otherwise inaccessible because of the lack of endogenous F. nucleatum sialidase. In mice with sialidase-producing vaginal microbiotas, mutant F. nucleatum unable to consume sialic acids was impaired in vaginal colonization. These experiments in mice also led to the discovery that F. nucleatum may also "give back" to the community by reinforcing sialidase activity, a biochemical feature of human dysbiosis. Using human vaginal bacterial communities, we show that F. nucleatum supported robust outgrowth of Gardnerella vaginalis, a major sialidase producer and one of the most abundant organisms in BV. These results illustrate that mutually beneficial relationships between vaginal bacteria support pathogen colonization and may help maintain features of dysbiosis. These findings challenge the simplistic dogma that the mere absence of "healthy" lactobacilli is the sole mechanism that creates a permissive environment for pathogens during vaginal dysbiosis. Given the ubiquity of F. nucleatum in the human mouth, these studies also suggest a possible mechanism underlying links between vaginal dysbiosis and oral sex.

Associations between the vaginal microbiome and Candida colonization in women of reproductive age.

BACKGROUND: The composition of bacteria within the vaginal microbiome has garnered a lot of recent attention and has been associated with reproductive health and disease. Despite the common occurrence of yeast (primarily Candida) within the vaginal microbiome, there is still an incomplete picture of relationships between yeast and bacteria (especially lactobacilli), as well as how such associations are governed. Such relationships could be important to a more holistic understanding of the vaginal microbiome and its connection to reproductive health. OBJECTIVE: The objective of the study was to perform molecular characterization of clinical specimens to define associations between vaginal bacteria (especially Lactobacillus species) and Candida colonization. In vitro studies were conducted to test the 2 most common dominant Lactobacillus species (Lactobacillus crispatus and Lactobacillus iners) in their ability to inhibit Candida growth and to examine the basis for such inhibition. STUDY DESIGN: A nested cross-sectional study of reproductive-age women from the Contraceptive CHOICE Project was conducted. Vaginal swabs from 299 women were selected to balance race and bacterial vaginosis status, resulting in a similar representation of black and white women in each of the 3 Nugent score categories (normal [0-3], intermediate [4-6], and bacterial vaginosis [7-10]). Sequencing of the 16S ribosomal gene (V4 region) was used to determine the dominant Lactobacillus species present (primarily Lactobacillus iners and Lactobacillus crispatus), defined as >50% of the community. Subjects without dominance by a single Lactobacillus species were classified as Diverse. A Candida-specific quantitative polymerase chain reaction targeting the internally transcribed spacer 1 was validated using vaginal samples collected from a second cohort of women and used to assess Candida colonization. Two hundred fifty-five nonpregnant women with sufficient bacterial biomass for analysis were included in the final analysis. Generalized linear models were used to evaluate associations between Lactobacillus dominance, sociodemographic and risk characteristics, and vaginal Candida colonization. In separate in vitro studies, the potential of cell-free supernatants from Lactobacillus crispatus and Lactobacillus iners cultures to inhibit Candida growth was evaluated. RESULTS: Forty-two women (16%) were vaginally colonized with Candida. Microbiomes characterized as Diverse (38%), Lactobacillus iners-dominant (39%), and Lactobacillus crispatus-dominant (20%) were the most common. The microbiome, race, and Candida colonization co-varied with a higher prevalence of Candida among black women and Lactobacillus iners-dominant communities compared with white women and Lactobacillus crispatus-dominant communities. Lactobacillus iners-dominant communities were more likely to harbor Candida than Lactobacillus crispatus-dominant communities (odds ratio, 2.85, 95% confidence interval, 1.03-7.21; Fisher exact test, P = .048). In vitro, Lactobacillus crispatus produced greater concentrations of lactic acid and exhibited significantly more pH-dependent growth inhibition of Candida albicans, suggesting a potential mechanism for the clinical observations. CONCLUSION: In nonpregnant women, Lactobacillus iners-dominant communities were significantly more likely to harbor Candida than Lactobacillus crispatus-dominant communities, suggesting that Lactobacillus species have different relationships with Candida. In vitro experiments indicate that Lactobacillus crispatus may impede Candida colonization more effectively than Lactobacillus iners through a greater production of lactic acid.

Identification and characterization of NanH2 and NanH3, enzymes responsible for sialidase activity in the vaginal bacterium Gardnerella vaginalis.

Gardnerella vaginalis is abundant in bacterial vaginosis (BV), a condition associated with adverse reproductive health. Sialidase activity is a diagnostic feature of BV and is produced by a subset of G. vaginalis strains. Although its genetic basis has not been formally identified, sialidase activity is presumed to derive from the sialidase A gene, named here nanH1 In this study, BLAST searches predicted two additional G. vaginalis sialidases, NanH2 and NanH3. When expressed in Escherichia coli, NanH2 and NanH3 both displayed broad abilities to cleave sialic acids from α2-3- and α2-6-linked N- and O-linked sialoglycans, including relevant mucosal substrates. In contrast, recombinant NanH1 had limited activity against synthetic and mucosal substrates under the conditions tested. Recombinant NanH2 was much more effective than NanH3 in cleaving sialic acids bearing a 9-O-acetyl ester. Similarly, G. vaginalis strains encoding NanH2 cleaved and foraged significantly more Neu5,9Ac2 than strains encoding only NanH3. Among a collection of 34 G. vaginalis isolates, nanH2, nanH3, or both were present in all 15 sialidase-positive strains but absent from all 19 sialidase-negative isolates, including 16 strains that were nanH1-positive. We conclude that NanH2 and NanH3 are the primary sources of sialidase activity in G. vaginalis and that these two enzymes can account for the previously described substrate breadth cleaved by sialidases in human vaginal specimens of women with BV. Finally, PCRs of nanH2 or nanH3 from human vaginal specimens had 81% sensitivity and 78% specificity in distinguishing between Lactobacillus dominance and BV, as determined by Nugent scoring.

Gardnerella vaginalis and Prevotella bivia Trigger Distinct and Overlapping Phenotypes in a Mouse Model of Bacterial Vaginosis.

BACKGROUND: Bacterial vaginosis (BV) is a common imbalance of the vaginal microbiota characterized by overgrowth of diverse Actinobacteria, Firmicutes, and Gram-negative anaerobes. Women with BV are at increased risk of secondary reproductive tract infections and adverse pregnancy outcomes. However, which specific bacteria cause clinical features of BV is unclear. METHODS: We previously demonstrated that Gardnerella vaginalis could elicit many BV features in mice. In this study, we established a BV model in which we coinfected mice with G. vaginalis and another species commonly found in women with BV: Prevotella bivia. RESULTS: This coinfection model recapitulates several aspects of human BV, including vaginal sialidase activity (a diagnostic BV feature independently associated with adverse outcomes), epithelial exfoliation, and ascending infection. It is notable that G. vaginalis facilitated uterine infection by P. bivia. CONCLUSIONS: Taken together, our model provides a framework for advancing our understanding of the role of individual or combinations of BV-associated bacteria in BV pathogenesis.

Association between obesity and bacterial vaginosis as assessed by Nugent score.

BACKGROUND: Bacterial vaginosis is 1 of the most common vaginal conditions in the United States. Recent studies have suggested that obese women have an abnormal microbiota reminiscent of bacterial vaginosis; however, few studies have investigated the prevalence of bacterial vaginosis in overweight and obese populations. Moreover, despite the increased prevalence of obesity and bacterial vaginosis in black women, it is not known whether racial disparities exist in the relationship between obesity and bacterial vaginosis. OBJECTIVE: The objective of this study was to examine the relationship between body mass index and bacterial vaginosis as determined by Nugent score and to determine the influence of race in this context. STUDY DESIGN: We performed a cross-sectional study using patient data and vaginal smears from 5918 participants of the Contraceptive CHOICE Project. Gram-stained vaginal smears were scored with the Nugent method and categorized as bacterial vaginosis-negative (Nugent score, 0-3), bacterial vaginosis-intermediate (Nugent score, 4-6), or bacterial vaginosis-positive (Nugent score, 7-10). Body mass index was determined with Centers for Disease Control and Prevention guidelines, and obese individuals were categorized as class I, II, or III obese based on National Institutes of Health and World Health Organization body mass index parameters. Linear regression was used to model mean differences in Nugent scores; Poisson regression with robust error variance was used to model prevalence of bacterial vaginosis. RESULTS: In our cohort, 50.7% of participants were black; 41.5% were white, and 5.1% were of Hispanic ethnicity; the average age of 25.3 years old. Overall, 28.1% of participants were bacterial vaginosis-positive. Bacterial vaginosis was prevalent in 21.3% of lean, 30.4% of overweight, and 34.5% of obese women (P<.001). The distribution of bacterial vaginosis-intermediate individuals was similar across all body mass index categories. Compared with the scores of lean women, Nugent scores were highest among overweight and obese class I women (adjusted mean difference: overweight women, 0.33 [95% confidence interval, 0.14-0.51] and obese women, 0.51 [95% confidence interval, 0.29-0.72]). Consistent with this, overweight and obese women had a higher frequency of bacterial vaginosis compared with lean women, even after adjustment for variables that included race. Among white women, the prevalence of bacterial vaginosis was higher for overweight and class I and class II/III obese white women compared with lean white women, which is a phenomenon not observed among black women and suggests an effect modification. CONCLUSION: Overweight and obese women have higher Nugent scores and a greater occurrence of bacterial vaginosis compared with lean women. Black women have a greater prevalence of bacterial vaginosis independent of their body mass index compared with white women.

Relationship between nugent score and vaginal epithelial exfoliation.

OBJECTIVE: Clue cells characteristic of bacterial vaginosis (BV) are thought to arise due to exfoliation of the vaginal epithelium; however, there is little published data connecting total numbers of epithelial cells to markers of BV. The purpose of this study was to enumerate exfoliated epithelial cells (independent of clue cells) and examine the relationship to Nugent score. STUDY DESIGN: We conducted a cross-sectional sub-study of the Contraceptive CHOICE Project cohort. Vaginal swabs were used to create vaginal smears for Gram staining and these smears were later scored using the Nugent method, and then two blinded observers used microscopy to enumerate exfoliated epithelial cells. The degree of epithelial cell exfoliation was compared between women diagnosed as BV-negative (Nugent score 0-3), BV-intermediate (Nugent score 4-6), and BV-positive (Nugent score 7-10). BV specimens (Nugent 7-10) were randomly matched to specimens in the two other groups (Nugent low and Nugent-intermediate), in order to avoid comparing groups of women with potentially confounding baseline demographics. RESULTS: Exfoliated epithelial cell counts were higher in the vaginal smears from BV-positive women compared with BV-negative women. Higher levels of epithelial exfoliation were also evident in BV-intermediate women compared to those with low Nugent scores. After adjustment for clustering introduced by matching, the incidence ratio of increased epithelial cell counts was 2.09 (95% CI 1.50-2.90) for the BV-intermediate women and 1.71 (95% CI 1.23-2.38) for the BV positive women. CONCLUSION: A vaginal epithelial exfoliation phenotype was measured in both Nugent-defined BV-positive and BV-intermediate women. Bacterial vaginosis and intermediate status (Nugent score >3) was associated with significantly more vaginal epithelial exfoliation compared to women with Lactobacillus-dominated microbiotas (Nugent 0-3).

The sialate O-acetylesterase EstA from gut Bacteroidetes species enables sialidase-mediated cross-species foraging of 9-O-acetylated sialoglycans.

The gut harbors many symbiotic, commensal, and pathogenic microbes that break down and metabolize host carbohydrates. Sialic acids are prominent outermost carbohydrates on host glycoproteins called mucins and protect underlying glycan chains from enzymatic degradation. Sialidases produced by some members of the colonic microbiota can promote the expansion of several potential pathogens (e.g. Clostridium difficile, Salmonella, and Escherichia coli) that do not produce sialidases. O-Acetyl ester modifications of sialic acids help resist the action of many sialidases and are present at high levels in the mammalian colon. However, some gut bacteria, in turn, produce sialylate-O-acetylesterases to remove them. Here, we investigated O-acetyl ester removal and sialic acid degradation by Bacteroidetes sialate-O-acetylesterases and sialidases, respectively, and subsequent utilization of host sialic acids by both commensal and pathogenic E. coli strains. In vitro foraging studies demonstrated that sialidase-dependent E. coli growth on mucin is enabled by Bacteroides EstA, a sialate O-acetylesterase acting on glycosidically linked sialylate-O-acetylesterase substrates, particularly at neutral pH. Biochemical studies suggested that spontaneous migration of O-acetyl esters on the sialic acid side chain, which can occur at colonic pH, may serve as a switch controlling EstA-assisted sialic acid liberation. Specifically, EstA did not act on O-acetyl esters in their initial 7-position. However, following migration to the 9-position, glycans with O-acetyl esters became susceptible to the sequential actions of bacterial esterases and sialidases. We conclude that EstA specifically unlocks the nutritive potential of 9-O-acetylated mucus sialic acids for foraging by bacteria that otherwise are prevented from accessing this carbon source.

Genome Sequences of 15 Gardnerella vaginalis Strains Isolated from the Vaginas of Women with and without Bacterial Vaginosis.

Gardnerella vaginalis is a predominant species in bacterial vaginosis, a dysbiosis of the vagina that is associated with adverse health outcomes, including preterm birth. Here, we present the draft genome sequences of 15 Gardnerella vaginalis strains (now available through BEI Resources) isolated from women with and without bacterial vaginosis.

Genome Sequences of 12 Bacterial Isolates Obtained from the Urine of Pregnant Women.

The presence of bacteria in urine can pose significant risks during pregnancy. However, there are few reference genome strains for many common urinary bacteria. We isolated 12 urinary strains of Streptococcus, Staphylococcus, Citrobacter, Gardnerella, and Lactobacillus These strains and their genomes are now available to the research community.

Genome Sequences of 11 Human Vaginal Actinobacteria Strains.

The composition of the vaginal microbiota is an important health determinant. Several members of the phylum Actinobacteria have been implicated in bacterial vaginosis, a condition associated with many negative health outcomes. Here, we present 11 strains of vaginal Actinobacteria (now available through BEI Resources) along with draft genome sequences.