Genetic predictors for bacterial vaginosis in women living with and at risk for HIV infection.

PROBLEM: Bacterial vaginosis (BV) disproportionally impacts Black and Hispanic women, placing them at risk for HIV, sexually transmitted infections and preterm birth. It is unknown whether there are differences by genetic ancestry in BV risk or whether polymorphisms associated with BV risk differ by ancestry. METHODS: Women's Interagency HIV Study (WIHS) participants with longitudinal Nugent scores were dichotomized as having (n = 319, Nugent 7-10) or not having BV (n = 367, Nugent 0-3). Genetic ancestry was defined by clustering of principal components from ancestry informative markers and further stratified by BV status. 627 single nucleotide polymorphisms (SNPs) across 41 genes important in mucosal defense were identified in the WIHS GWAS. A logistic regression analysis was adjusted for nongenetic predictors of BV and self-reported race/ethnicity to assess associations between genetic ancestry and genotype. RESULTS: Self-reported race and genetic ancestry were associated with BV risk after adjustment for behavioral factors. Polymorphisms in mucosal defense genes including syndecans, cytokines and toll-like receptors (TLRs) were associated with BV in all ancestral groups. CONCLUSIONS: The common association of syndecan, cytokine and TLR genes and the importance of immune function and inflammatory pathways in BV, suggests these should be targeted for further research on BV pathogenesis and therapeutics.

Genome-wide association study in Estonia reveals importance of vaginal epithelium associated genes in case of recurrent vaginitis.

Recurrent vaginitis is a leading reason for visiting a gynaecologist, with bacterial vaginosis (BV) and vulvovaginal candidiasis (VVC) being the most common diagnoses. Reasons and mechanisms behind their recurrent nature are poorly understood. We conducted a genome-wide association study (GWAS) to find possible genetic risk factors for recurrent vaginitis using data from a large population-based biobank, the Estonian Biobank. The study included 6870 cases (at least two episodes of vaginitis) and 5945 controls (no vaginitis episodes). GWAS approach included single marker and gene-based analyses, followed by functional annotation of associated variants and candidate gene mapping.In single marker analysis, one statistically significant (P = 7.8 × 10-9) variant rs1036732378 was identified on chromosome 10. The gene-based association analysis identified one gene, KRT6A, that exceeded the recommended significance threshold (P = 2.6 × 10-6). This is a member of the keratin protein family and is expressed during differentiation in epithelial tissues.Functional mapping and annotation of genetic associations by using adjusted significance level identified 22 potential risk loci that may be associated with recurrent vaginitis phenotype. Comparison of our results with previous studies provided nominal support for LBP (associated with immune response to vaginal bacteria) and PRKCH genes (possible role in keratinocyte differentiation and susceptibility to candidiasis).In conclusion, this study is the first highlighting a potential role of the vaginal epithelium in recurrent vaginitis.

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.

Species-Specific Analysis of Bacterial Vaginosis-Associated Bacteria.

Vaginal dysbiosis in women reduces the abundance of Lactobacillus species and increases that of anaerobic fastidious bacteria. This dysbiotic condition in the vagina, called bacterial vaginosis (BV), can be symptomatic with odorous vaginal discharges or asymptomatic and affects a third of women of reproductive age. Three unclassified bacterial species designated BV-associated bacteria 1, 2, and 3 (BVAB-1, -2, and -3) in 2005 were found to be highly preponderant in the vagina of females with BV. Here, we used sequence homology and phylogenetics analyses to identify the actual species of BVAB-1, -2, and -3 and found BVAB-1 to be Clostridiales genomosp. BVAB-1, BVAB-2 to be Oscillospiraceae bacterium strain CHIC02, and BVAB-3 to be Mageeibacillus indolicus, respectively. These are anaerobic and uncultured species that can be identified only through metagenomics. Long-read sequencing of BV specimens can also enable a genomic reassembly of these species' genomes from metagenomes. Species-specific identification of these pathogens and the availability of their genomes from assembled metagenomes will advance our understanding of their biology, facilitate the design of sensitive diagnostics and drugs, and enhance the treatment of BV. IMPORTANCE For many years since 2005, BVAB, an important pathogen of the female vaginal tract that is associated with BV, has been identified using PCR without knowing its actual species. Without a full genome of these pathogens, a better understanding of their pathogenicity, treatment, resistance, and diagnostics cannot be reached. In this analysis, we use the DNA of BVAB-1, -2, and -3 to determine their actual species to enhance further research into their pathogenicity, resistance, diagnosis, and treatment.

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.

Vaginal colonization of women after oral administration of Lactobacillus crispatus strain NTCVAG04 from the human microbiota.

BACKGROUND: The genomic approach has deeply changed the microbiology perspective, mainly concerning the microbioma identification. In this regard, some microbes colonize the healthy vagina. Vaginitis is a common gynecological ailment and includes bacterial vaginosis (BV), usually caused by local dysbiosis, such as a microbiota imbalance. Lactobacilli are the most prevalent bacteria colonizing the healthy vagina, so guaranteeing local eubiosis. In particular, vaginal colonization by L. crispatus is associated with low susceptibility to BV. Therefore, probiotics, such as life bacteria providing health advantages, are a current strategy in the prevention or treatment of vaginitis, including BV. However, there is a low level of evidence that probiotics after ingestion could really colonize the vagina. In particular, no study evidenced that L. crispatus after ingestion can colonize vagina. Therefore, the current study explored the capacity of Biovaginil® (NTC, Milan, Italy) dietary supplement containing Lactobacillus crispatus NTCVAG04 and vitamin A to colonize the gut and vagina in women with a history of vaginitis/vaginosis. METHODS: Twenty fertile females (mean age 34.0 years) were enrolled in the study. Rectal and vaginal swabs were collected at baseline and after the first and second cycle of Biovaginil®. Each cycle lasted 14 days within two consecutive menstrual periods. RESULTS: Seven women were excluded from the analysis because the samples were technically not evaluable. One woman dropped out because of mild adverse event. At the end of the study, nine women (75%) had positive rectal swab for L. crispatus NTCVAG04, and 8 of them also had positive vaginal swab. CONCLUSIONS: The current study provided the first evidence that L. crispatus NTCVAG04, administered by two Biovaginil® courses, colonized both the gut and vagina. Moreover, the L. crispatus NTCVAG04 strain could be considered the archetype of a new class of oral probiotics that actively colonize the vagina, and that could be called "colpobiotics."

Novel insights in bacterial vaginosis etiology through genomic approaches.

Bacterial vaginosis (BV) has been considered as dysbiosis state whose etiology is not fully understood. This condition affects a large number of women of reproductive age and its study has been highly relevant due to the growing association of BV with and gynecological and obstetric complications and diseases, in addition to a greater susceptibility to sexually transmitted diseases, including HIV. The vaginal microbiota composition presents high variability among different ethnic groups of women, although, generally, the prevalence of lactobacilli species has been reported. Several studies suggest they may play a protective role, especially Lactobacillus crispatus whose population is typically present in low proportions in women with BV. This review article describes the contributions and limitations of genomic approaches in elucidating protective characteristics and mechanisms associated with colonization and persistence of lactobacilli strains. Although some genetic features were associated with resilience of L. crispatus during BV, furher studies are required to uncover their functions.

Slipped-Strand Mispairing in the Gene Encoding Sialidase NanH3 in Gardnerella spp.

Cell wall proteins with sialidase activity are involved in carbohydrate assimilation, adhesion to mucosal surfaces, and biofilm formation. Gardnerella spp. inhabit the human vaginal microbiome and encode up to three sialidase enzymes, two of which are suspected to be cell wall associated. Here, we demonstrate that the gene encoding extracellular sialidase NanH3 is found almost exclusively in Gardnerella piotii and the closely related species Gardnerella genome sp. 3, and its presence correlates with a sialidase-positive phenotype in a collection of 112 Gardnerella isolates. The nanH3 gene sequence includes a homopolymeric repeat of cytosines that varies in length within cell populations, indicating that this gene is subject to slipped-strand mispairing, a mechanism of phase variation in bacteria. Variation in the length of the homopolymer sequence results in production of either the full-length sialidase protein or truncated peptides lacking the sialidase domain due to introduction of reading-frame shifts and premature stop codons. Phase variation in NanH3 may be involved in immune evasion or modulation of adhesion to host epithelial cells and formation of biofilms characteristic of the vaginal dysbiosis known as bacterial vaginosis.

Genetic Variation in Toll-Like Receptor 5 and Colonization with Flagellated Bacterial Vaginosis-Associated Bacteria.

Bacterial vaginosis (BV) is a vaginal dysbiotic condition linked to negative gynecological and reproductive sequelae. Flagellated bacteria have been identified in women with BV, including Mobiluncus spp. and BV-associated bacterium-1 (BVAB1), an uncultivated, putatively flagellated species. The host response to flagellin mediated through Toll-like receptor 5 (TLR5) has not been explored in BV. Using independent discovery and validation cohorts, we examined the hypothesis that TLR5 deficiency-defined by a dominant negative stop codon polymorphism, rs5744168-is associated with an increased risk for BV and increased colonization with flagellated bacteria associated with BV (BVAB1, Mobiluncus curtisii, and Mobiluncus mulieris). TLR5 deficiency was not associated with BV status, and TLR5-deficient women had decreased colonization with BVAB1 in both cohorts. We stimulated HEK-hTLR5-overexpressing NF-κB reporter cells with whole, heat-killed M. mulieris or M. curtisii and with partially purified flagellin from these species; as BVAB1 is uncultivated, we used cervicovaginal lavage (CVL) fluid supernatant from women colonized with BVAB1 for stimulation. While heat-killed M. mulieris and CVL fluid from women colonized with BVAB1 stimulate a TLR5-mediated response, heat-killed M. curtisii did not. In contrast, partially purified flagellin from both Mobiluncus species stimulated a TLR5-mediated response in vitro We observed no correlation between vaginal interleukin 8 (IL-8) and flagellated BVAB concentrations among TLR5-sufficient women. Interspecies variation in accessibility of flagellin recognition domains may be responsible for these observations, as reflected in the potentially novel flagellin products encoded by Mobiluncus species versus those encoded by BVAB1.

Comparison between a novel molecular tool and conventional methods for diagnostic classification of bacterial vaginosis: is integration of the two approaches necessary for a better evaluation?

The etiological cause of bacterial vaginosis (BV) is the change of the vaginal ecosystem characterized by a decrease of lactobacilli and an increase of other germs, such as Gardnerella vaginalis and Atopobium vaginae. Molecular tools have revolutionized the diagnosis of these conditions. The aim of this paper was to compare results obtained from 158 vaginal swabs collected from women aged between 18 and 59 years old and subjected to microscopic evaluation (Nugent Score), culture and to the multiparametric molecular assay Vaginitis and Vaginosis Multiplex-Tandem (MT) PCR (AU27117) - Nuclear Laser Medicine. In 50 samples we also used matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for bacterial microbiome identification. Our results showed a moderate concordance between traditional and molecular methods for diagnosis of candidiasis and a lower concordance for BV and normal flora. MALDI TOF MS allowed us to discriminate more than 10 species of lactobacilli with a greater abundance of Lactobacillus gasseri, Lactobacillus paracasei spp. paracasei, Lactobacillus pentosus and Lactobacillus crispatus in BV and altered flora. This work underlined how the integration of different assays and metagenomics studies can greatly expand our current understanding of vaginal microbial diversity, providing more reliable diagnostic criteria for BV and its intermediate condition diagnosis.

Exploring potential of vaginal Lactobacillus isolates from South African women for enhancing treatment for bacterial vaginosis.

Antibiotics continue to be the standard-of-care for bacterial vaginosis (BV), although recurrence rates are high. Vaginal probiotics may improve durability of BV treatment, although few probiotics for vaginal health contain Lactobacillus spp. that commonly colonize the lower female genital tract. Characteristics of vaginal Lactobacillus strains from South African women were evaluated for their probiotic potential in vitro compared to strains from commercial vaginal products, including growth at varying pHs, ability to lower pH, produce D-/L-lactate and H2O2, influence growth of BV-associated Gardnerella vaginalis and Prevotella bivia, adherence to cervical cells and susceptibility to antibiotics. Fifty-seven Lactobacillus strains were purified from cervico-vaginal fluid, including L. crispatus, L. jensenii, L. gasseri, L. mucosae, and L. vaginalis. L crispatus strains grew better at pHs below 4.5 and lowered pH more effectively than other strains. Production of D-/L-lactate and H2O2 varied between Lactobacillus species and strains. Lactobacillus strains generally inhibited P. bivia more uniformly than G. vaginalis isolates. All vaginal Lactobacillus isolates were resistant to metronidazole while susceptibility to clindamycin varied. Furthermore, vaginal Lactobacillus strains tended to be broadly susceptible to penicillin, amoxicillin, rifampicin and rifabutin. Whole-genome-sequencing of five of the best-performing vaginal Lactobacillus strains confirmed their likely safety, due to antimicrobial resistance elements being largely absent, while putative intact prophages were present in the genomes of two of the five strains. Overall, vaginal Lactobacillus strains largely performed better in these in vitro assays than probiotic strains currently used in probiotics for vaginal health. Including the best-performing vaginal Lactobacillus isolates in a region-specific probiotic for vaginal health may result in improved BV treatment options.

Differential Expression of Local Immune Response Genes in the Vagina: Implication for the Diagnosis of Vaginal Infections.

Transcription profiles of genes of local immune response were determined in the vagina of women with bacterial vaginosis, aerobic vaginitis, and vulvovaginal candidosis for detection of the most specific immune markers for these vaginal infections. Laboratory diagnosis of the vaginal infections was performed microscopically; the inflammatory reaction in the vagina (leukorrhea) was defined as the presence of >10 white blood cells per field of view. Transcription profiles of IL1b, IL10, IL18, TNFα, TLR4, GATA3, and CD68 were determined using reverse-transcription quantitative real-time PCR. The strongest predictors of aerobic vaginitis were increased levels of IL1b and IL10 mRNA. Bacterial vaginosis was strongly associated with reduced levels of IL18 and GATA3 mRNA. Increased levels of IL1b and TLR4 transcripts showed significant discriminatory power for vulvovaginal candidosis and leukorrhea. The results of this study suggest differential expression of local immune response genes in the vagina of women with different vaginal infections. Detection of specific immune markers in the vagina using reverse-transcriptase PCR could supplement PCR detection of abnormal vaginal microflora for the diagnosis of vaginal infections.

Structure determination of CAMP factor of Mobiluncus curtisii and insights into structural dynamics.

Bacterial vaginosis (BV) is a common type of vaginal inflammation caused by a proliferation of pathogenic bacteria, among which Mobiluncus curtisii. In our previous studies on M. curtisii genome, we identified the presence of a genomic fragment encoding a 25 kDa pore-forming toxin, the CAMP factor, which is known to be involved in the synergistic lysis of erythrocytes namely CAMP reaction. However, whether this hypothetical gene product has hemolytic activity is unknown. Moreover, its relative structure and function are not yet solved. Here we found that the M. curtisii CAMP factor is a monomer at pH 4.4 and oligomer at pH > 4.6. Hemolysis assays showed that M. curtisii CAMP factor could lyse sheep red blood cells efficiently in pH 5.4-7.4. Negative staining electron microscope analysis of the CAMP factor revealed ring-like structures at pH above 4.6. Additionally, the crystal structure of M. curtisii CAMP factor, determineded at 1.85 Å resolution, reveals a 5 + 3 helix motif. Further functional analysis suggested that the structural rearrangement of the N-terminal domain might be required for protein function. In conclusion, this structure-function relationship study of CAMP factor provides a new perspective of the M. curtisii role in BV development.

Sexual practices have a significant impact on the vaginal microbiota of women who have sex with women.

Women-who-have-sex-with-women (WSW) are at increased risk of bacterial vaginosis (BV). We investigated the impact of practices and past BV on the vaginal microbiota within a two-year longitudinal cohort of Australian WSW. Self-collected vaginal swabs were used to characterise the vaginal microbiota using 16S-rRNA gene sequencing. Hierarchical clustering defined community state types (CSTs). Bacterial diversity was calculated using the Shannon diversity index and instability of the vaginal microbiota was assessed by change of CST and Bray-Curtis dissimilarity. Sex with a new partner increased the bacterial diversity (adjusted-coefficient = 0.41, 95%CI: 0.21,0.60, p < 0.001) and instability of the vaginal microbiota, in terms of both change of CST (adjusted-odds-ratio = 2.65, 95%CI: 1.34,5.22, p = 0.005) and increased Bray-Curtis dissimilarity (adjusted-coefficient = 0.21, 95%CI: 0.11,0.31, p < 0.001). Women reporting sex with a new partner were more likely than women reporting no new partner to have a vaginal microbiota characterised by Gardnerella vaginalis (adjusted-relative-risk-ratio[aRRR] = 3.45, 95%CI: 1.42,8.41, p = 0.006) or anaerobic BV-associated bacteria (aRRR = 3.62, 95%CI: 1.43,9.14, p = 0.007) relative to a Lactobacillus crispatus dominated microbiota. Sex with a new partner altered the vaginal microbiota of WSW by increasing the diversity and abundance of BV-associated bacteria. These findings highlight the influence of practices on the development of a non-optimal vaginal microbiota and provide microbiological support for the sexual exchange of bacteria between women.

Interplay Between the Host, the Human Microbiome, and Drug Metabolism.

The human microbiome is composed of four major areas including intestinal, skin, vaginal, and oral microbiomes, with each area containing unique species and unique functionalities. The human microbiome may be modulated with prebiotics, probiotics, and postbiotics to potentially aid in the treatment of diseases like irritable bowel syndrome, bacterial vaginosis, atopic dermatitis, gingivitis, obesity, or cancer. There is also potential for many of the inhabitants of the human microbiome to directly modulate host gene expression and modulate host detoxifying enzyme activity like cytochrome P450s (CYPs), dehydrogenases, and carboxylesterases. Therefore, the microbiome may be important to consider during drug discovery, risk assessment, and dosing regimens for various diseases given that the human microbiome has been shown to impact host detoxification processes.

Vaginal microbiota of asymptomatic bacterial vaginosis and vulvovaginal candidiasis: Are they different from normal microbiota?

Vaginal microbiota contributes in maintaining and protecting the urogenital niche from infections and their sequelae. Despite extensive research, microbiome studies have often ignored asymptomatic bacterial vaginosis (BV) and vulvovaginal candidiasis (VVC). The present study aimed to explore the cultivable vaginal bacterial and mycological communities in women asymptomatic for BV and VVC using multiplex PCR and species-specific PCR. Vaginal swabs collected from 199 participants asymptomatic for urogenital infections, scored by Nugent criteria indicated 73.9% had normal microbiota, 11.6% intermediate and 14.5% BV. The most frequent Lactobacillus species in normal women were L. iners (69.4%), L. crispatus (24.5%), L. reuteri (20.4%). Women with BV colonized L. iners (62.1%); L. rhamnosus (41.4%); L. salivarius (13.7%) and L. reuteri (7.2%). Furthermore, L. crispatus was associated with normal microbiota, whereas L. iners was a frequent member of normal and dysbiotic microbiota. Lactobacillus abundance and species richness reduced in asymptomatic BV. Also L. crispatus, L. fermentum, L. acidophilus and L. delbruckii were absent in these women. L. iners significantly co-existed with other Lactobacillus species, indicating its failure in independently maintaining the healthy vaginal niche. Of 30.4% women detected with Candida, 72.1% constituted non-albicans Candida. Predominance of C. albicans increased from 18.4% in healthy to 60% in women with asymptomatic BV; whereas distribution of BV related bacteria did not vary across the groups. Heterogeneous population of lactobacilli in 80.8% of normal women calls attention towards cumulative effects of these species in safeguarding the vaginal microenvironment. Since the microbiota of asymptomatic BV was different from healthy, screening and management could be encouraged to avoid further complications of infections.

Vaginal Microbiota Composition Correlates Between Pap Smear Microscopy and Next Generation Sequencing and Associates to Socioeconomic Status.

Recent research on vaginal microbiota relies on high throughput sequencing while microscopic methods have a long history in clinical use. We investigated the correspondence between microscopic findings of Pap smears and the vaginal microbiota composition determined by next generation sequencing among 50 asymptomatic women. Both methods produced coherent results regarding the distinction between Lactobacillus-dominant versus mixed microbiota, reassuring gynaecologists for the use of Pap smear or wet mount microscopy for rapid evaluation of vaginal bacteria as part of diagnosis. Cytologic findings identified women with bacterial vaginosis and revealed that cytolysis of vaginal epithelial cells is associated to Lactobacillus crispatus-dominated microbiota. Education and socio-economic status were associated to the vaginal microbiota variation. Our results highlight the importance of including socio-economic status as a co-factor in future vaginal microbiota studies.

Comparative analysis of virulence factors & biotypes of Gardnerella vaginalis isolated from the genital tract of women with & without bacterial vaginosis.

BACKGROUND & OBJECTIVES: : Bacterial vaginosis (BV) involves the presence of a thick vaginal multispecies biofilm, where Gardnerella vaginalis is the predominant species. The reason for an increase in the number of G. vaginalis which are usually present as normal flora of the female genital tract in cases of BV, is not known. Hence, the objective of the present study was to compare the biotypes and virulence factors of G. vaginalis isolated from the genital tract of women with and without BV. METHODS: : High vaginal swabs collected from 811 women of reproductive age were cultured. G. vaginalis isolates were biotyped and tested for adherence to vaginal epithelial cells, biofilm formation, agglutination of human red blood cells (RBCs), protease production, phospholipase production and surface hydrophobicity. RESULTS: : Of the isolates from women with BV, 83.3 per cent (60/72) showed good adherence, 78.4 per cent (58/74) produced biofilm, 82.9 per cent (63/76) produced phospholipase, 67.1 per cent (51/76) produced protease, 77.3 per cent (58/75) were positive for surface hydrophobicity and 61.6 per cent (45/73) were positive for haemagglutination of human RBC. In case of G. vaginalis from non-BV women, 25 per cent (15/60) isolates showed good adherence, 18.4 per cent (9/49) biofilm production, 35 per cent (21/60) phospholipase, 36.6 per cent (22/60) protease, 41.7 per cent (25/60) surface hydrophobicity and 10.1 per cent (6/59) agglutination of human RBCs. Maximum number of isolates belonged to biotypes 6, 2 and 3. Biotype 3 was more associated with non-BV rather than BV; biotype 6, 2 and 1 were more associated with cases of BV. Maximum virulence factors were expressed by biotypes 6, 2 and 1. INTERPRETATION & CONCLUSIONS: : Virulence factors were more expressed by G. vaginalis isolates obtained from women with BV rather than from non-BV. Biotypes 6, 2 and 1 were more associated with cases of BV and expressed maximum virulence factors.

Quantitation of all Four Gardnerella vaginalis Clades Detects Abnormal Vaginal Microbiota Characteristic of Bacterial Vaginosis More Accurately than Putative G. vaginalis Sialidase A Gene Count.

BACKGROUND: Bacterial vaginosis (BV) is a vaginal disorder characterized by a depletion of the normal lactobacillus-dominant microbiota and overgrowth of mainly anaerobic bacteria. OBJECTIVES: The study aimed to evaluate the distribution and abundance of the Gardnerella vaginalis clades and sialidase A gene in vaginal samples from Russian women, and investigate if the G. vaginalis sialidase A gene count detects an abnormal vaginal microbiota characteristic of BV more accurately than G. vaginalis load. METHODS: Vaginal samples from 299 non-pregnant patients of gynecological clinics were examined using Nugent scores and G. vaginalis clade and sialidase A gene quantitative real-time polymerase chain reactions (PCRs). Discriminatory power for BV microbiota was evaluated with receiver operating characteristic (ROC) analysis. RESULTS: The vaginal microbiota was characterized by Nugent scores as normal, intermediate, and BV microbiota in 162, 58, and 79 women, respectively. G. vaginalis clades 1, 2, 3, 4, and the sialidase A gene were detected in 56% (51-62%), 40% (34-45%), 20% (16-25%), 94% (91-96%), and 70% (64-75%) of vaginal samples, respectively. The frequency and abundance of clades 1, 2, 4, and the sialidase A gene as well as clade multiplicity were significantly associated with abnormal microbiota. The sialidase A gene was present in all multi-clade samples, in all single-clade samples comprising clades 1, 2, and 3, and in four of 84 (5% [2-12%]) samples comprising clade 4 only. Total G. vaginalis load showed significantly higher discriminatory power for abnormal microbiota than sialidase A gene count (areas under ROC curves 0.933 vs. 0.881; p = 0.0306). CONCLUSIONS: Quantifying all four G. vaginalis clades discriminates between BV microbiota and normal microbiota more accurately than measuring G. vaginalis sialidase A gene. Clade 4 is strongly associated with BV microbiota, despite most clade 4 strains lacking the sialidase A gene.

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.