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.

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.

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.

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.

Gardnerella vaginalis promotes group B Streptococcus vaginal colonization, enabling ascending uteroplacental infection in pregnant mice.

BACKGROUND: Group B Streptococcus is a common vaginal bacterium and the leading cause of invasive fetoplacental infections. Group B Streptococcus in the vagina can invade through the cervix to cause ascending uteroplacental infections or can be transmitted to the neonate during vaginal delivery. Some studies have found that women with a "dysbiotic" polymicrobial or Lactobacillus-depleted vaginal microbiota are more likely to harbor group B Streptococcus. Gardnerella vaginalis is often the most abundant bacteria in the vaginas of women with dysbiosis, while being detected at lower levels in most other women, and has been linked with several adverse pregnancy outcomes. Mouse models of group B Streptococcus and Gardnerella vaginalis colonization have been reported but, to the best of our knowledge, the two have not been studied together. The overarching idea driving this study is that certain members of the dysbiotic vaginal microbiota, such as Gardnerella vaginalis, may directly contribute to the increased rate of group B Streptococcus vaginal colonization observed in women with vaginal dysbiosis. OBJECTIVE: We used a mouse model to test the hypothesis that vaginal exposure to Gardnerella vaginalis may facilitate colonization and/or invasive infection of the upper reproductive tract by group B Streptococcus during pregnancy. STUDY DESIGN: Timed-pregnant mice were generated using an allogeneic mating strategy with BALB/c males and C57Bl/6 females. Dams were vaginally inoculated at gestational day 14 with group B Streptococcus alone (using a 10-fold lower dose than previously reported models) or coinoculated with group B Streptococcus and Gardnerella vaginalis. Bacterial titers were enumerated in vaginal, uterine horn, and placental tissues at gestational day 17. The presence (Fisher exact tests) and levels (Mann-Whitney U tests) of bacterial titers were compared between mono- and coinoculated dams in each compartment. Relative risks were calculated for outcomes that occurred in both groups. Tissue samples were also examined for evidence of pathophysiology. RESULTS: Inoculation of pregnant mice with 107 group B Streptococcus alone did not result in vaginal colonization or ascending infection. In contrast, coinoculation of group B Streptococcus with Gardnerella vaginalis in pregnant mice resulted in a 10-fold higher risk of group B Streptococcus vaginal colonization (relative risk, 10.31; 95% confidence interval, 2.710-59.04; P=.0006 [Fisher exact test]). Ascending group B Streptococcus infection of the uterus and placenta occurred in approximately 40% of coinoculated animals, whereas none of those receiving group B Streptococcus alone developed uterine or placental infections. Immunofluorescence microscopy revealed group B Streptococcus in both the maternal and fetal sides of the placenta. Histologic inflammation and increased proinflammatory cytokines were evident in the setting of group B Streptococcus placental infection. Interestingly, placentas from dams exposed to group B Streptococcus and Gardnerella vaginalis, but without recoverable vaginal or placental bacteria, displayed distinct histopathologic features and cytokine signatures. CONCLUSION: These data suggest that Gardnerella vaginalis vaginal exposure can promote group B Streptococcus vaginal colonization, resulting in a greater likelihood of invasive perinatal group B Streptococcus infections. These findings suggest that future clinical studies should examine whether the presence of Gardnerella vaginalis is a risk factor for group B Streptococcus vaginal colonization in women. Because Gardnerella vaginalis can also be present in women without bacterial vaginosis, these findings may be relevant both inside and outside of the context of vaginal dysbiosis.

Treatment decision-making among men with lower urinary tract symptoms: A qualitative study of men's experiences with recommendations for patient-centred practice.

AIMS: To inform and guide patient-centred care for men with lower urinary tract symptoms (LUTS), by providing in-depth qualitative evidence regarding men's perspectives on treatment decision-making for LUTS. METHODS: An interview study of men recruited from 26 English urology departments. Purposive sampling captured surgical/nonsurgical treatment decisions, and diversity in demographics and symptom burden, in men who had urodynamics and those who did not. After diagnostic assessments, men were interviewed either pre-treatment or after LUTS surgery. Thematic analysis was conducted. Participants' descriptions of how LUTS treatment decisions were made were categorised as patient-led, doctor-led, or shared. RESULTS: A total of 41 men participated (25 pre-treatment, 16 post-surgery), ages 52-89. Twenty out of 41 described the treatment decision as shared with their consultant, 14 as doctor-led, and seven as patient-led. There was no obvious association between treatment decision-making style and patients' satisfaction with either clinicians' role in their decision or their treatment decision. Incomplete or rushed discussions and misperceptions of LUTS and its treatment were reported, indicating a risk of suboptimal decision-making support by clinicians. As well as clinician opinion, men's treatment decision-making was influenced by the results of urological assessments, comparing current symptoms with possible side-effects of surgery, and others' experiences and opinions. CONCLUSIONS: Men with LUTS report and prefer different kinds of decision-making support from their clinicians, who must tailor their input to patients' preferences and needs. Patients' treatment decision-making involves multiple factors and can be challenging, and areas of inadequate clinician support were identified. Recommendations for patient-centred consultations about LUTS treatment are presented.

Aerococcus urinae Isolated from Women with Lower Urinary Tract Symptoms: In Vitro Aggregation and Genome Analysis.

Aerococcus urinae is increasingly recognized as a potentially significant urinary tract bacterium. A. urinae has been isolated from urine collected from both males and females with a wide range of clinical conditions, including urinary tract infection (UTI), urgency urinary incontinence (UUI), and overactive bladder (OAB). A. urinae is of particular clinical concern because it is highly resistant to many antibiotics and, when undiagnosed, can cause invasive and life-threatening bacteremia, sepsis, or soft tissue infections. Previous genomic characterization studies have examined A. urinae strains isolated from patients experiencing UTI episodes. Here, we analyzed the genomes of A. urinae strains isolated as part of the urinary microbiome from patients with UUI or OAB. Furthermore, we report that certain A. urinae strains exhibit aggregative in vitro phenotypes, including flocking, which can be modified by various growth medium conditions. Finally, we performed in-depth genomic comparisons to identify pathways that distinguish flocking and nonflocking strains.IMPORTANCEAerococcus urinae is a urinary bacterium of emerging clinical interest. Here, we explored the ability of 24 strains of A. urinae isolated from women with lower urinary tract symptoms to display aggregation phenotypes in vitro We sequenced and analyzed the genomes of these A. urinae strains. We performed functional genomic analyses to determine whether the in vitro hyperflocking aggregation phenotype displayed by certain A. urinae strains was related to the presence or absence of certain pathways. Our findings demonstrate that A. urinae strains have different propensities to display aggregative properties in vitro and suggest a potential association between phylogeny and flocking.

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.

Grading Severity and Bother Using the International Prostate Symptom Score and International Consultation on Incontinence Questionnaire Male Lower Urinary Tract Symptoms Score in Men Seeking Lower Urinary Tract Symptoms Therapy.

PURPOSE: We established severity banding ranges, bother assessment and key item content in principal patient reported outcomes measures in men seeking therapy for lower urinary tract symptoms. MATERIALS AND METHODS: Data for International Prostate Symptom Score (I-PSS) and International Consultation on Incontinence Questionnaire Male Lower Urinary Tract Symptoms (ICIQ-MLUTS) were derived from a study evaluating 820 men at 26 United Kingdom hospitals. ROC curves were used to establish severity bandings. RESULTS: Classification tree showed that thresholds between mild-moderate and moderate-severe severity bands were 15 and 27 for I-PSS, 16 and 26 for ICIQ-MLUTS/severity, and 22 and 81 for ICIQ-MLUTS/bother, respectively. Highest area under the ROC curve and lowest Akaike's information criteria of univariate logistic regression indicated that ICIQ-MLUTS/bother was more related to global quality of life than were I-PSS and ICIQ-MLUTS/severity. The symptoms affecting I-PSS-quality of life (QoL) were only fully identified by ICIQ-MLUTS, because 2 key symptoms (urinary incontinence and post-micturition dribble) are not measured by I-PSS. ICIQ-MLUTS demonstrated that bother of some lower urinary tract symptoms is disproportionate to severity, and that persisting high bother levels following surgery are more likely due to storage (18% to 25%) and post-voiding (18% to 28%) lower urinary tract symptoms than voiding lower urinary tract symptoms (5% to 13%). Symptom improvement after surgery was uncertain if baseline I-PSS-QoL score was less than 3. CONCLUSIONS: The severity threshold scores were measured for the 2 key lower urinary tract symptoms patient reported outcomes measures, and the results indicate suitable categories of symptom severity for use in men referred for urological care. The ICIQ-MLUTS measures all the lower urinary tract symptoms affecting quality of life and includes individual symptom bother scores.

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.

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.

Structural and functional characterization of a modified legionaminic acid involved in glycosylation of a bacterial lipopolysaccharide.

Nonulosonic acids (NulOs) are a diverse family of α-keto acid carbohydrates present across all branches of life. Bacteria biosynthesize NulOs among which are several related prokaryotic-specific isomers and one of which, N-acetylneuraminic acid (sialic acid), is common among all vertebrates. Bacteria display various NulO carbohydrates on lipopolysaccharide (LPS), and the identities of these molecules tune host-pathogen recognition mechanisms. The opportunistic bacterial pathogen Vibrio vulnificus possesses the genes for NulO biosynthesis; however, the structures and functions of the V. vulnificus NulO glycan are unknown. Using genetic and chemical approaches, we show here that the major NulO produced by a clinical V. vulnificus strain CMCP6 is 5-N-acetyl-7-N-acetyl-d-alanyl-legionaminic acid (Leg5Ac7AcAla). The CMCP6 strain could catabolize modified legionaminic acid, whereas V. vulnificus strain YJ016 produced but did not catabolize a NulO without the N-acetyl-d-alanyl modification. In silico analysis suggested that Leg5Ac7AcAla biosynthesis follows a noncanonical pathway but appears to be present in several bacterial species. Leg5Ac7AcAla contributed to bacterial outer-membrane integrity, as mutant strains unable to produce or incorporate Leg5Ac7AcAla into the LPS have increased membrane permeability, sensitivity to bile salts and antimicrobial peptides, and defects in biofilm formation. Using the crustacean model, Artemia franciscana, we demonstrate that Leg5Ac7AcAla-deficient bacteria have decreased virulence potential compared with WT. Our data indicate that different V. vulnificus strains produce multiple NulOs and that the modified legionaminic acid Leg5Ac7AcAla plays a critical role in the physiology, survivability, and pathogenicity of V. vulnificus CMCP6.

Transient microbiota exposures activate dormant Escherichia coli infection in the bladder and drive severe outcomes of recurrent disease.

Pathogens often inhabit the body asymptomatically, emerging to cause disease in response to unknown triggers. In the bladder, latent intracellular Escherichia coli reservoirs are regarded as likely origins of recurrent urinary tract infection (rUTI), a problem affecting millions of women worldwide. However, clinically plausible triggers that activate these reservoirs are unknown. Clinical studies suggest that the composition of a woman's vaginal microbiota influences her susceptibility to rUTI, but the mechanisms behind these associations are unclear. Several lines of evidence suggest that the urinary tract is routinely exposed to vaginal bacteria, including Gardnerella vaginalis, a dominant member of the vaginal microbiota in some women. Using a mouse model, we show that bladder exposure to G. vaginalis triggers E. coli egress from latent bladder reservoirs and enhances the potential for life-threatening outcomes of the resulting E. coli rUTI. Transient G. vaginalis exposures were sufficient to cause bladder epithelial apoptosis and exfoliation and interleukin-1-receptor-mediated kidney injury, which persisted after G. vaginalis clearance from the urinary tract. These results support a broader view of UTI pathogenesis in which disease can be driven by short-lived but powerful urinary tract exposures to vaginal bacteria that are themselves not "uropathogenic" in the classic sense. This "covert pathogenesis" paradigm may apply to other latent infections, (e.g., tuberculosis), or for diseases currently defined as noninfectious because routine culture fails to detect microbes of recognized significance.

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.

Recommendations for conducting invasive urodynamics for men with lower urinary tract symptoms: Qualitative interview findings from a large randomized controlled trial (UPSTREAM).

AIMS: To capture in-depth qualitative evidence regarding attitudes to and experiences of urodynamic testing among men with lower urinary tract symptoms (LUTS) at each end of the clinical pathway. METHODS: Semi-structured interview study conducted within the Urodynamics for Prostate Surgery: Randomized Evaluation of Assessment Methods (UPSTREAM) trial, which randomized men to a care pathway including urodynamics or routine non-invasive tests from 26 secondary care urology sites across England. Men were interviewed after assessments but prior to treatment, or after surgery for LUTS. Men were purposively sampled to include those who had urodynamics and those who did not, and diversity in demographic characteristics and symptom burden. Interviews were analyzed using inductive thematic analysis. RESULTS: Forty-one men participated (25 pre-treatment, 16 post-surgery), ages 52-89. The 16 men who had not previously experienced urodynamics said they would accept the test in their assessment, but some were apprehensive or wanted more information. The 25 men who had experienced urodynamics all found it acceptable, though some reported pain, infection, or embarrassment. Embarrassment was minimized by informing patients what the procedure would be like, and ensuring privacy. Urodynamics was valued for its perceived diagnostic insight. Information deficits were reported before, during, and after the test. How and when results were explained and the adequacy of explanations varied. CONCLUSIONS: Urodynamics is acceptable to men with LUTS and generally well-tolerated. To ensure patients are prepared and informed, good communication before and during the procedure is essential. Privacy should be prioritized, and test results discussed promptly and in sufficient detail. Staff require training and guidance in these areas.

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.

Human milk oligosaccharides inhibit growth of group B Streptococcus.

Streptococcus agalactiae (group B Streptococcus, GBS) is a leading cause of invasive bacterial infections in newborns, typically acquired vertically during childbirth secondary to maternal vaginal colonization. Human milk oligosaccharides (HMOs) have important nutritional and biological activities that guide the development of the immune system of the infant and shape the composition of normal gut microbiota. In this manner, HMOs help protect against pathogen colonization and reduce the risk of infection. In the course of our studies of HMO-microbial interactions, we unexpectedly uncovered a novel HMO property to directly inhibit the growth of GBS independent of host immunity. By separating different HMO fractions through multidimensional chromatography, we found the bacteriostatic activity to be confined to specific non-sialylated HMOs and synergistic with a number of conventional antibiotic agents. Phenotypic screening of a GBS transposon insertion library identified a mutation within a GBS-specific gene encoding a putative glycosyltransferase that confers resistance to HMOs, suggesting that HMOs may function as an alternative substrate to modify a GBS component in a manner that impairs growth kinetics. Our study uncovers a unique antibacterial role for HMOs against a leading neonatal pathogen and expands the potential therapeutic utility of these versatile molecules.

Discovery and characterization of de novo sialic acid biosynthesis in the phylum Fusobacterium.

Sialic acids are nine-carbon backbone carbohydrates found in prominent outermost positions of glycosylated molecules in mammals. Mimicry of sialic acid (N-acetylneuraminic acid, Neu5Ac) enables some pathogenic bacteria to evade host defenses. Fusobacterium nucleatum is a ubiquitous oral bacterium also linked with invasive infections throughout the body. We employed multidisciplinary approaches to test predictions that F. nucleatum engages in de novo synthesis of sialic acids. Here we show that F. nucleatum sbsp. polymorphum ATCC10953 NeuB (putative Neu5Ac synthase) restores Neu5Ac synthesis to an Escherichia coli neuB mutant. Moreover, purified F. nucleatum NeuB participated in synthesis of Neu5Ac from N-acetylmannosamine and phosphoenolpyruvate in vitro Further studies support the interpretation that F. nucleatum ATCC10953 NeuA encodes a functional CMP-sialic acid synthetase and suggest that it may also contain a C-terminal sialic acid O-acetylesterase. We also performed BLAST queries of F. nucleatum genomes, revealing that only 4/31 strains encode a complete pathway for de novo Neu5Ac synthesis. Biochemical studies including mass spectrometry were consistent with the bioinformatic predictions, showing that F. nucleatum ATCC10953 synthesizes high levels of Neu5Ac, whereas ATCC23726 and ATCC25586 do not express detectable levels above background. While there are a number of examples of sialic acid mimicry in other phyla, these experiments provide the first biochemical and genetic evidence that a member of the phylum Fusobacterium can engage in de novo Neu5Ac synthesis.

Host-like carbohydrates promote bloodstream survival of Vibrio vulnificus in vivo.

Sialic acids are found on all vertebrate cell surfaces and are part of a larger class of molecules known as nonulosonic acids. Many bacterial pathogens synthesize related nine-carbon backbone sugars; however, the role(s) of these non-sialic acid molecules in host-pathogen interactions is poorly understood. Vibrio vulnificus is the leading cause of seafood-related death in the United States due to its ability to quickly access the host bloodstream, which it can accomplish through gastrointestinal or wound infection. However, little is known about how this organism persists systemically. Here we demonstrate that sialic acid-like molecules are present on the lipopolysaccharide of V. vulnificus, are required for full motility and biofilm formation, and also contribute to the organism's natural resistance to polymyxin B. Further experiments in a murine model of intravenous V. vulnificus infection demonstrated that expression of nonulosonic acids had a striking benefit for bacterial survival during bloodstream infection and dissemination to other tissues in vivo. In fact, levels of bacterial persistence in the blood corresponded to the overall levels of these molecules expressed by V. vulnificus isolates. Taken together, these results suggest that molecules similar to sialic acids evolved to facilitate the aquatic lifestyle of V. vulnificus but that their emergence also resulted in a gain of function with life-threatening potential in the human host.