The vaginal microbiome of South African pregnant women living with human immunodeficiency virus (HIV) with and without Chlamydia trachomatis infection.

BACKGROUND: Chlamydia genital infections continue to be a serious health concern globally. Previous studies have reported that Chlamydia trachomatis infection alters the vaginal microbiota of infected women. This study investigated differences in the vaginal microbiome of South African pregnant women living with HIV with and without C. trachomatis infection. METHODS: This was a cross-sectional study among 385 pregnant women, recruited from the King Edward VIII Hospital in Durban, South Africa. C. trachomatis was detected using the Applied Biosystems™ TaqMan® Assays. A total of 40 samples, 20 C. trachomatis positive and 20 C. trachomatis negative, were selected for sequencing. The sequencing of the vaginal microbiome was performed using the PacBio platform. Statistical analysis was performed on IBM SPSS version 26. RESULTS: The prevalence of C. trachomatis infection was 12.2% (47/385). The genus Gardnerella (32.14% vs. 24.02%) and species in the genus Gardnerella (31.97% vs. 24.03%) were more abundant in the C. trachomatis-infected group compared to the uninfected group. Lactobacillus iners were also more abundant in the C. trachomatis-infected women (28.30%) compared to the uninfected women. However, these observed patterns did not reach statistical significance. Discriminant analysis showed that the class Alpha-Proteobacteria; order Bacillales; family Enterococcaceae; the genera Enhydrobacter, Enterococcus, and Parabacteroides; Enterococcus spp.; and Pseudomonas stutzeri significantly contributed to a model separating C. trachomatis-infected women from the uninfected group (p < 0.05). CONCLUSION: The organisms and taxa that significantly contributed to separating the vaginal microbiota of C. trachomatis-infected women from the uninfected women in this study cohort have not been previously observed in association with C. trachomatis infection or the vaginal microbiota. Future studies in larger cohorts that will investigate the role of these microorganisms in C. trachomatis infection and the vaginal microbiota are required.

Gardnerella Species and Their Association With Bacterial Vaginosis.

BACKGROUND: Bacterial vaginosis (BV) is a condition marked by high vaginal bacterial diversity. Gardnerella vaginalis has been implicated in BV but is also detected in healthy women. The Gardnerella genus has been expanded to encompass 6 validly named species and several genomospecies. We hypothesized that particular Gardnerella species may be more associated with BV. METHODS: Quantitative polymerase chain reaction (PCR) assays were developed targeting the cpn60 gene of species groups including G. vaginalis, G. piotii/pickettii, G. swidsinskii/greenwoodii, and G. leopoldii. These assays were applied to vaginal swabs from individuals with (n = 101) and without BV (n = 150) attending a sexual health clinic in Seattle, Washington. Weekly swabs were collected from 42 participants for up to 12 weeks. RESULTS: Concentrations and prevalence of each Gardnerella species group were significantly higher in participants with BV; 91.1% of BV-positive participants had 3 or more Gardnerella species groups detected compared to 32.0% of BV-negative participants (P < .0001). BV-negative participants with 3 or more species groups detected were more likely to develop BV within 100 days versus those with fewer (60.5% vs 3.7%, P < .0001). CONCLUSIONS: These results suggest that BV reflects a state of high Gardnerella species diversity. No Gardnerella species group was a specific marker for BV.

Phase variable colony variants are conserved across Gardnerella spp. and exhibit different virulence-associated phenotypes.

The Gardnerella genus, comprising at least 13 species, is associated with the polymicrobial disorder bacterial vaginosis (BV). However, the details of BV pathogenesis are poorly defined, and the contributions made by individual species, including Gardnerella spp., are largely unknown. We report here that colony phenotypes characterized by size (large and small) and opacity (opaque and translucent) are phase variable and are conserved among all tested Gardnerella strains, representing at least 10 different species. With the hypothesis that these different variants could be an important missing piece to the enigma of how BV develops in vivo, we characterized their phenotypic, proteomic, and genomic differences. Beyond increased colony size, large colony variants showed reduced vaginolysin secretion and faster growth rate relative to small colony variants. The ability to inhibit the growth of Neisseria gonorrhoeae and commensal Lactobacillus species varied by strain and, in some instances, differed between variants. Proteomics analyses indicated that 127-173 proteins were differentially expressed between variants. Proteins with increased expression in large variants of both strains were associated with amino acid and protein synthesis and protein folding, whereas those increased in small variants were related to nucleotide synthesis, phosphate transport, ABC transport, and glycogen breakdown. Furthermore, whole genome sequencing analyses revealed an abundance of genes associated with variable homopolymer tracts, implicating slipped strand mispairing in Gardnerella phase variation and illuminating the potential for previously unrecognized heterogeneity within clonal populations. Collectively, these results suggest that phase variants may be primed to serve different roles in BV pathogenesis.IMPORTANCEBacterial vaginosis is the most common gynecological disorder in women of childbearing age. Gardnerella species are crucial to the development of this dysbiosis, but the mechanisms involved in the infection are not understood. We discovered that Gardnerella species vary between two different forms, reflected in bacterial colony size. A slow-growing form makes large amounts of the toxin vaginolysin and is better able to survive in human cervix tissue. A fast-growing form is likely the one that proliferates to high numbers just prior to symptom onset and forms the biofilm that serves as a scaffold for multiple BV-associated anaerobic bacteria. Identification of the proteins that vary between different forms of the bacteria as well as those that vary randomly provides insight into the factors important for Gardnerella infection and immune avoidance.

Gardnerella diversity and ecology in pregnancy and preterm birth.

The vaginal microbiome has been linked to negative health outcomes including preterm birth. Specific taxa, including Gardnerella spp., have been identified as risk factors for these conditions. Historically, microbiome analysis methods have treated all Gardnerella spp. as one species, but the broad diversity of Gardnerella has become more apparent. We explore the diversity of Gardnerella clades and genomic species in the vaginal microbiome of pregnant women and their associations with microbiome composition and preterm birth. Relative abundance of Gardnerella clades and genomic species and other taxa was quantified in shotgun metagenomic sequencing data from three distinct cohorts of pregnant women. We also assessed the diversity and abundance of Gardnerella variants in 16S rRNA gene amplicon sequencing data from seven previously conducted studies in differing populations. Individual microbiomes often contained multiple Gardnerella variants, and the number of clades was associated with increased microbial load, or the ratio of non-human reads to human reads. Taxon co-occurrence patterns were largely consistent across Gardnerella clades and among cohorts. Some variants previously described as rare were prevalent in other cohorts, highlighting the importance of surveying a diverse set of populations to fully capture the diversity of Gardnerella. The diversity of Gardnerella both across populations and within individual vaginal microbiomes has long been unappreciated, as has been the intra-species diversity of many other members of the vaginal microbiome. The broad genomic diversity of Gardnerella has led to its reclassification as multiple species; here we demonstrate the diversity of Gardnerella found within and between vaginal microbiomes.IMPORTANCEThe present study shows that single microbiomes can contain all currently known species of Gardnerella and that multiple similar species can exist within the same environment. Furthermore, surveys of demographically distinct populations suggest that some species appear more commonly in certain populations. Further studies in broad and diverse populations will be necessary to fully understand the ecological roles of each Gardnerella sp., how they can co-exist, and their distinct impacts on microbial communities, preterm birth, and other health outcomes.

Microbial interactions among Gardnerella, Prevotella and Fannyhessea prior to incident bacterial vaginosis: protocol for a prospective, observational study.

INTRODUCTION: The aetiology of bacterial vaginosis (BV), a biofilm-associated vaginal infection, remains unknown. Epidemiologic data suggest that it is sexually transmitted. BV is characterised by loss of lactic acid-producing lactobacilli and an increase in facultative and strict anaerobic bacteria. Gardnerella spp are present in 95%-100% of cases; Gardnerella vaginalis has been found to be more virulent than other BV-associated bacteria (BVAB) in vitro. However, G. vaginalis is found in women with normal vaginal microbiota and colonisation is not sufficient for BV development. We hypothesise that Gardnerella spp initiate BV biofilm formation, but incident BV (iBV) requires incorporation of other key BVAB (ie, Prevotella bivia, Fannyhessea vaginae) into the biofilm that alter the transcriptome of the polymicrobial consortium. This study will investigate the sequence of microbiologic events preceding iBV. METHODS AND ANALYSIS: This study will enrol 150 women aged 18-45 years with normal vaginal microbiota and no sexually transmitted infections at a sexual health research clinic in Birmingham, Alabama. Women will self-collect twice daily vaginal specimens up to 60 days. A combination of 16S rRNA gene sequencing, qPCR for Gardnerella spp, P. bivia and F. vaginae, and broad range 16S rRNA gene qPCR will be performed on twice daily vaginal specimens from women with iBV (Nugent score 7-10 on at least 2 consecutive days) and controls (with comparable age, race, contraceptive method and menstrual cycle days) maintaining normal vaginal microbiota to investigate changes in the vaginal microbiota over time for women with iBV. Participants will complete daily diaries on multiple factors including sexual activity. ETHICS AND DISSEMINATION: This protocol is approved by the University of Alabama at Birmingham Institutional Review Board (IRB-300004547) and written informed consent will be obtained from all participants. Findings will be presented at scientific conferences and published in peer-reviewed journals as well as disseminated to providers and patients in communities of interest.

Under conditions closely mimicking vaginal fluid, Lactobacillus jensenii strain 62B produces a bacteriocin-like inhibitory substance that targets and eliminates Gardnerella species.

Within the vaginal ecosystem, lactobacilli and Gardnerella spp. likely interact and influence each other's growth, yet the details of this interaction are not clearly defined. Using medium simulating vaginal fluid and a two-chamber co-culturing system to prevent cell-to-cell contact between the bacteria, we examined the possibility that Lactobacillus jensenii 62B (Lj 62B) and/or G. piotii (Gp) JCP8151B produce extracellular factors through which they influence each other's viability. By 24 h post-inoculation (hpi) in the co-culture system and under conditions similar to the vaginal environment - pH 5.0, 37 °C, and 5% CO2, Lj 62B viability was not affected but Gp JCP8151B had been eliminated. Cell-free supernatant harvested from Lj 62B cultures (Lj-CFS) at 20 hpi, but not 16 hpi, also eliminated Gp JCP8151B growth. Neither lactic acid nor H2O2 production by Lj 62B was responsible for this effect. The Lj-CFS did not affect viability of three species of lactobacilli or eight species of Gram-positive and Gram-negative uropathogens but eliminated viability of eight different strains of Gardnerella spp. Activity of the inhibitory factor within Lj-CFS was abolished by protease treatment and reduced by heat treatment suggesting it is most likely a bacteriocin-like protein; fractionation revealed that the factor has a molecular weight within the 10-30 kDa range. These results suggest that, in medium mimicking vaginal fluid and growth conditions similar to the vaginal environment, Lj 62B produces a potential bacteriocin-like inhibitory substance (Lj-BLIS) that clearly targets Gardnerella spp. strains. Once fully characterized, Lj-BLIS may be a potential treatment for Gardnerella-related BV that does not alter the vaginal microflora.

Gardnerella pickettii sp. nov. (formerly Gardnerella genomic species 3) and Gardnerella greenwoodii sp. nov. (formerly Gardnerella genomic species 8) isolated from female urinary microbiome.

During an ongoing female urinary microbiome research study, strains c17Ua_112T and c31Ua_26T isolated from urine samples of a patient diagnosed with overactive bladder and a healthy postmenopausal woman, respectively, could not be allocated to any Gardnerella species with valid names. In this work, we aimed to characterize these strains. The 16S rRNA gene sequences confirmed that these strains are members of the genus Gardnerella. Phylogenetic analysis based on cpn60 strongly supported two clades, one encompassing c17Ua_112T and nine other strains from the public database, and the other including c31Ua_26T and three other strains, which were distinct from currently recognized species of the genus Gardnerella. Likewise, the phylogenomic tree also showed that strains c17Ua_112T and c31Ua_26T formed independent and robust clusters. Average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between c17Ua_112T and c31Ua_26T were 79.27 and 27.4 %, respectively. Strain c17Ua_112T showed the highest ANI (94.8 %) and dDDH values (59.8 %) with Gardnerella piotii UGent 18.01T, and strain c31Ua_26T revealed highest ANI (84.2 %) and dDDH (29.1 %) values with Gardnerella swidsinskii GS 9838-1T. Based on the data presented here, the two strains c17Ua_112T and c31Ua_26T represent two novel species of the genus Gardnerella, for which the names Gardnerella pickettii (c17Ua_112T=DSM 113414T=CCP 71T) and Gardnerella greenwoodii (c31Ua_26T=DSM 113415T=CCP 72T) are proposed.

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.

Cervicovaginal Gardnerella sialidase-encoding gene in persistent human papillomavirus infection.

Disturbed vaginal microbiota have a role in the persistence of high-oncogenic-risk human papillomavirus (hrHPV) and Gardnerella spp. is closely related with this condition. Such bacteria are the major source of cervicovaginal sialidases, important for microbiota alterations. The sialidase-encoding gene nanH3 is account for their sialidase activity. Thus, a subset of 212 women positive for hrHPV at the first visit were included in the analysis of the current study aiming to compare the loads of nanH3 in cervicovaginal fluid (CFV) of women with persistent hrHPV infection and with those cleared the infection after a year. Participants were assigned to two study groups named "persistence" (n = 124, 53.22%) or "clearance" (n = 88, 37.77%), according to the HPV status upon enrollment and follow-up. Absolute quantification of nanH3 gene was performed using quantitative real-time PCR (qPCR). Persistence and clearance group did not show statistical difference in the load of nanH3 gene (p = 0.19). When considering the subset of women with HPV16, differences in number of copies of nanh3 gene was observed between the persistent (7.39E+08 copies/µL) and clearance group (2.85E+07 copies/µL) (p = 0.007). Therefore, baseline loads of nanH3 gene is increased in women that persist with cervical HPV16 infection after 12 months.

Vaginal microbiome community state types and high-risk human papillomaviruses in cervical precancer and cancer in North-central Nigeria.

BACKGROUND: High risk human papillomaviruses (HR-HPV) have a causal role in cervical oncogenesis, and HIV-mediated immune suppression allows HR-HPV to persist. We studied whether vaginal microbiome community state types (CSTs) are associated with high-grade precancer and/or invasive cervical cancer (HSIL/ICC). METHODS: This was a cross-sectional study of adult women with cervical cancer screening (CCS) at the Jos University Teaching Hospital (JUTH) in Jos, Nigeria, between January 2020 and February 2022. Cervical swabs underwent HPV genotyping (Anyplex™ II HPV28). Cervico-vaginal lavage (CVL) sample was collected for 16 S rRNA gene amplicon sequencing. We used multivariable logistic regression modelling to assess associations between CSTs and other factors associated with HSIL/ICC. RESULTS: We enrolled 155 eligible participants, 151 with microbiome data for this analysis. Women were median age 52 (IQR:43-58), 47.7% HIV positive, and 58.1% with HSIL/ICC. Of the 138 with HPV data, 40.6% were negative for HPV, 10.1% had low-risk HPV, 26.8% had single HR-HPV, and 22.5% had multiple HR-HPV types. The overall prevalence of any HR-HPV type (single and multiple) was 49.3%, with a higher proportion in women with HSIL/ICC (NILM 31.6%, LSIL 46.5%, HSIL 40.8%, and 81.5% ICC; p = 0.007). Women with HIV were more likely to have HSIL/ICC (70.3% vs. 29.7% among women without HIV). In crude and multivariable analysis CST was not associated with cervical pathology (CST-III aOR = 1.13, CST-IV aOR = 1.31). However, in the presence of HR-HPV CST-III (aOR = 6.7) and CST-IV (aOR = 3.6) showed positive association with HSIL/ICC. CONCLUSION: Vaginal microbiome CSTs were not significantly associated with HSIL/ICC. Our findings suggest however, that CST could be helpful in identifying women with HSIL/ICC and particularly those with HR-HPV. Characterization of CSTs using point-of-care molecular testing in women with HR-HPV should be studied as an approach to improve early detection and cervical cancer prevention. Future longitudinal research will improve our understanding of the temporal effect of non-optimal CST, HR-HPV, and other factors in cervical cancer development, prevention, and control.

A novel Gardnerella, Prevotella, and Lactobacillus standard that improves accuracy in quantifying bacterial burden in vaginal microbial communities.

Bacterial vaginosis (BV) is the most common vaginal dysbiosis. In this condition, a polymicrobial biofilm develops on vaginal epithelial cells. Accurately quantifying the bacterial burden of the BV biofilm is necessary to further our understanding of BV pathogenesis. Historically, the standard for calculating total bacterial burden of the BV biofilm has been based on quantifying Escherichia coli 16S rRNA gene copy number. However, E. coli is improper for measuring the bacterial burden of this unique micro-environment. Here, we propose a novel qPCR standard to quantify bacterial burden in vaginal microbial communities, from an optimal state to a mature BV biofilm. These standards consist of different combinations of vaginal bacteria including three common BV-associated bacteria (BVAB) Gardnerella spp. (G), Prevotella spp. (P), and Fannyhessea spp. (F) and commensal Lactobacillus spp. (L) using the 16S rRNA gene (G:P:F:L, G:P:F, G:P:L and 1G:9L). We compared these standards to the traditional E. coli (E) reference standard using known quantities of mock vaginal communities and 16 vaginal samples from women. The E standard significantly underestimated the copy numbers of the mock communities, and this underestimation was significantly greater at lower copy numbers of these communities. The G:P:L standard was the most accurate across all mock communities and when compared to other mixed vaginal standards. Mixed vaginal standards were further validated with vaginal samples. This new G:P:L standard can be used in BV pathogenesis research to enhance reproducibility and reliability in quantitative measurements of BVAB, spanning from the optimal to non-optimal (including BV) vaginal microbiota.

Prevotella and Gardnerella Are Associated With Treatment Failure Following First-line Antibiotics for Bacterial Vaginosis.

BACKGROUND: Bacterial vaginosis (BV) is a common vaginal dysbiosis that often recurs following first-line antibiotics. We investigated if vaginal microbiota composition was associated with BV recurrence. METHODS: We analyzed samples and data from 121 women who participated in 3 published trials evaluating novel interventions for improving BV cure, including concurrent antibiotic treatment of regular sexual partners (RSPs). Women diagnosed with BV received first-line antibiotics and self-collected vaginal swabs pretreatment and the day after finishing antibiotics (immediately posttreatment). 16S rRNA gene sequencing was performed on vaginal samples. Logistic regression explored associations between BV recurrence and features of the vaginal microbiota pre- and posttreatment. RESULTS: Sixteen women (13% [95% confidence interval {CI}, 8%-21%]) experienced BV recurrence within 1 month of treatment. Women with an untreated RSP were more likely to experience recurrence than women with no RSP (P = .008) or an RSP who received treatment (P = .011). A higher abundance of Prevotella pretreatment (adjusted odds ratio [AOR], 1.35 [95% CI, 1.05-1.91]) and Gardnerella immediately posttreatment (AOR, 1.23 [95% CI, 1.03-1.49]) were associated with increased odds of BV recurrence. CONCLUSIONS: Having specific Prevotella spp prior to recommended treatment and persistence of Gardnerella immediately posttreatment may contribute to the high rates of BV recurrence. Interventions that target these taxa are likely required to achieve sustained BV cure.

Bacterial Vaginosis-Vaginal Polymicrobial Biofilms and Dysbiosis.

BACKGROUND: Bacterial vaginosis (BV) is the most common genital disease worldwide in women of sexually active age, with a prevalence of 23-29%. Its traditional definition as dysbiosis, i.e., a disruption of the normal balance of the vaginal microbiota, with a massive increase of facultative and obligate anaerobic bacteria (mainly Gardnerella spp.) and a loss of lactobacilli, accurately describes the change in the vaginal microbiota, but does not explain the underlying pathophysiology. METHODS: This review is based on information in pertinent articles retrieved by a selective literature search and on the authors' own research findings. RESULTS: Fluorescent in situ hybridization (FISH) has revealed Gardnerella spp.-dominated polymicrobial vaginal biofilm as a cause of ascending gynecologic and pregnancy-related infections, preterm birth, and infertility in patients with BV. The biofilm-induced disturbance of epithelial homeostasis favors co-infection with pathogens of sexually transmitted infection (STI). Standard antibiotic therapy is ineffective against biofilms, and there is thus a recurrence rate above 50%. The characteristic biofilm can be followed as a diagnostic marker and is considered evidence of sexual transmission when heterosexual couples and ejaculate samples are examined. FISH studies have shown that, in addition to biofilm-related vaginosis, there are other dysbiotic changes in the vaginal microbiota that have not yet been characterized in detail. It is therefore justified to speak of a "bacterial vaginosis syndrome." CONCLUSION: The simplistic view of BV as dysbiosis, characterizable by microscopic reference methods, has so far led to inadequate therapeutic success. An evaluation of molecular genetic testing methods that would be suitable for routine use and the development of therapeutic agents that are effective against biofilms are urgently needed if the "bacterial vaginosis syndrome" is to be effectively treated.

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.

Gardnerella Revisited: Species Heterogeneity, Virulence Factors, Mucosal Immune Responses, and Contributions to Bacterial Vaginosis.

Gardnerella species are associated with bacterial vaginosis (BV) and have been investigated as etiological agents of the condition. Nonetheless, the isolation of this taxon from healthy individuals has raised important questions regarding its etiological role. Recently, using advanced molecular approaches, the Gardnerella genus was expanded to include several different species that exhibit differences in virulence potential. Understanding the significance of these different species with respect to mucosal immunity and the pathogenesis and complications of BV could be crucial to solving the BV enigma. Here, we review key findings regarding the unique genetic and phenotypic diversity within this genus, virulence factors, and effects on mucosal immunity as they stand. We also comment on the relevance of these findings to the proposed role of Gardnerella in BV pathogenesis and in reproductive health and identify key gaps in knowledge that should be explored in the future.

Glycogen-Degrading Activities of Catalytic Domains of α-Amylase and α-Amylase-Pullulanase Enzymes Conserved in Gardnerella spp. from the Vaginal Microbiome.

Gardnerella spp. are associated with bacterial vaginosis in which normally dominant lactobacilli are replaced with facultative and anaerobic bacteria, including Gardnerella spp. Co-occurrence of multiple species of Gardnerella is common in the vagina, and competition for nutrients such as glycogen likely contributes to the differential abundances of Gardnerella spp. Glycogen must be digested into smaller components for uptake, a process that depends on the combined action of glycogen-degrading enzymes. In this study, the ability of culture supernatants of 15 isolates of Gardnerella spp. to produce glucose, maltose, maltotriose, and maltotetraose from glycogen was demonstrated. Carbohydrate-active enzymes (CAZymes) were identified bioinformatically in Gardnerella proteomes using dbCAN2. Identified proteins included a single-domain α-amylase (EC 3.2.1.1) (encoded by all 15 isolates) and an α-amylase-pullulanase (EC 3.2.1.41) containing amylase, carbohydrate binding modules, and pullulanase domains (14/15 isolates). To verify the sequence-based functional predictions, the amylase and pullulanase domains of the α-amylase-pullulanase and the single-domain α-amylase were each produced in Escherichia coli. The α-amylase domain from the α-amylase-pullulanase released maltose, maltotriose, and maltotetraose from glycogen, and the pullulanase domain released maltotriose from pullulan and maltose from glycogen, demonstrating that the Gardnerella α-amylase-pullulanase is capable of hydrolyzing α-1,4 and α-1,6 glycosidic bonds. Similarly, the single-domain α-amylase protein also produced maltose, maltotriose, and maltotetraose from glycogen. Our findings show that Gardnerella spp. produce extracellular amylase enzymes as "public goods" that can digest glycogen into maltose, maltotriose, and maltotetraose that can be used by the vaginal microbiota. IMPORTANCE Increased abundance of Gardnerella spp. is a diagnostic characteristic of bacterial vaginosis, an imbalance in the human vaginal microbiome associated with troubling symptoms, and negative reproductive health outcomes, including increased transmission of sexually transmitted infections and preterm birth. Competition for nutrients is likely an important factor in causing dramatic shifts in the vaginal microbial community, but little is known about the contribution of bacterial enzymes to the metabolism of glycogen, a major food source available to vaginal bacteria. The significance of our research is characterizing the activity of enzymes conserved in Gardnerella species that contribute to the ability of these bacteria to utilize glycogen.

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.

Characterization of the GU microbiome in women with self-perceived bladder health over the life course.

BACKGROUND: A variety of factors influence bladder health, including environmental factors, life experiences, biologic foundations, and coexistent medical conditions. A biologically diverse microbial community exists in the urine that is likely influenced by the microbial inhabitants of the vagina. The relationship between the genitourinary (GU) microbiome and self-perceived bladder health is unknown. OBJECTIVE: To longitudinally define the GU microbiome in women with self-percieved bladder health sampled across multiple time points over a year. STUDY DESIGN: Women with no reported lower urinary tract dysfunction or symptoms (LUTS) were recruited from six clinical sites and assessed every 6 weeks for 1 year. Voided urine and vaginal samples were longitudinally collected. Self-perceived bladder health was assessed with select items from the LURN comprehensive assessment of self-reported urinary symptoms (CASUS) tool. We defined four life phases as follows: young (18-34 years, nulliparous), midlife (35-45 years, menstruating), transitional (46-60 years, perimenopausal), mature (>60 years, not using vaginal and/or systemic hormone replacement therapy). DNA was extracted from samples, and the V4 region of the 16S rRNA gene was amplified with region-specific primers. The 16S rRNA sequencing on an Illumina NovaSeq. Microbial beta-diversity was calculated using DEICODE to identify microbial taxa that cluster in the samples. Longitudinal volatility analysis was performed using the gemelli plugin. Log-abundance ratios of microbial features were explored and visualized in Qurro. RESULTS: Fifty-four (N = 16 young, N = 16 midlife, N = 15 transitional, N = 7 mature) women were enrolled and provided baseline data. Most women in each life phase (93%-98%) continued to report self-perceived bladder health throughout the 1-year follow-up as assessed by CASUS items. Temporal-based microbial diversity of urinary and vaginal microbiome remained relatively stable over 1 year in all subjects. The GU microbiomes of mature women were distinct and microbially diverse from that of young, midlife, and transitional women, with genera of Gardnerella, Cupriavidus, and Dialister contributory to the microbial features of the mature microbiome. The mature GU microbiome was statistically different (p < 0.0001) from the midlife, transitional, and young microbiome for the log ratio of Gardnerella and Cupriavidus (in the numerator) and Lactobacillus (in the denominator) for voided samples and Gardnerella and Dialister (in the numerator) and Lactobacillus (in the denominator) for vaginal samples. Differences in the GU microbiome were also demonstrated via longitudinal beta-diversity between women developing urinary frequency as reported by CASUS responses or objectively on bladder diary compared to women without urinary frequency. CONCLUSION: In women with a self-perceived healthy bladder, the GU microbiome remained stable in all age groups over a 1 year period. Differences were seen with respect to life phase, where mature women were distinct from all other groups, and with respect to self-reported LUTS.

Unraveling the impact of Lactobacillus spp. and other urinary microorganisms on the efficacy of mirabegron in female patients with overactive bladder.

Objective: Overactive bladder (OAB) is a disease that seriously affects patients' quality of life and mental health. To address this issue, more and more researchers are examining the relationship between OAB treatment and urinary microecology. In this study, we sought to determine whether differences in treatment efficacy were related to microbiome diversity and composition as well as the abundance of specific genera. Machine learning algorithms were used to construct predictive models for urine microbiota-based treatment of OAB. Methods: Urine samples were obtained from 64 adult female OAB patients for 16S rRNA gene sequencing. Patients' overactive bladder symptom scores (OABSS) were collected before and after mirabegron treatment and patients were divided into effective and ineffective groups. The relationship between the relative abundance of certain genera and OABSS were analyzed. Three machine learning algorithms, including random forest (RF), supporting vector machine (SVM) and eXtreme gradient boosting (XGBoost) were utilized to predict the therapeutic effect of mirabegron based on the relative abundance of certain genera in OAB patients' urine microbiome. Results: The species composition of the two groups differed. For one, the relative abundance of Lactobacillus was significantly higher in the effective group than in the ineffective group. In addition, the relative abundance of Gardnerella and Prevotella in the effective group was significantly lower than in the ineffective group. Alpha-diversity and beta-diversity differed significantly between the two groups. LEfSe analysis revealed that Lactobacillus abundance increased while Prevotella and Gardnerella abundance decreased in the effective group. The Lactobacillus abundance ROC curve had high predictive accuracy. The OABSS after treatment was negatively correlated with the abundance of Lactobacillus, whereas the relationship between OABSS and Prevotella and Gardnerella showed the opposite trend. In addition, RF, SVM and XGBoost models demonstrated high predictive ability to assess the effect of mirabegron in OAB patients in the test cohort. Conclusions: The results of this study indicate that urinary microbiota might influence the efficacy of mirabegron, and that Lactobacillus might be a potential marker for evaluating the therapeutic efficacy of mirabegron in OAB patients.

Insights into the vaginal microbiome in a diverse group of women of African, Asian and European ancestries.

Background: Intra-continentally, vaginal microbiome signatures are reported to be significantly different between Black and Caucasian women, with women of African ancestry having the less well defined heterogenous bacterial community state type (CST) deficient of Lactobacillus species (CST IV). The objective of this study was to characterize the vaginal microbiomes across a more diverse intercontinental group of women (N = 151) of different ethnicities (African American, African Kenyan, Afro-Caribbean, Asian Indonesian and Caucasian German) using 16S rRNA gene sequence analysis to determine their structures and offer a comprehensive description of the non-Lactobacillus dominant CSTs and subtypes. Results: In this study, the bacterial composition of the vaginal microbiomes differed significantly among the ethnic groups. Lactobacillus spp. (L. crispatus and L. iners) dominated the vaginal microbiomes in African American women (91.8%) compared to European (German, 42.4%), Asian (Indonesian, 45.0%), African (Kenyan, 34.4%) and Afro-Caribbean (26.1%) women. Expanding on CST classification, three subtypes of CST IV (CST IV-A, IV-B and IV-C) (N = 56, 37.1%) and four additional CSTs were described: CST VI Gardnerella vaginalis-dominant (N = 6, 21.8%); CST VII (Prevotella-dominant, N = 1, 0.66%); CST VIII (N = 9, 5.96%), resembling aerobic vaginitis, was differentiated by a high proportion of taxa such as Enterococcus, Streptococcus and Staphylococcus (relative abundance [RA] > 50%) and CST IX (N = 7, 4.64%) dominated by genera other than Lactobacillus, Gardnerella or Prevotella (e.g., Bifidobacterium breve and Anaerococcus vaginalis). Within the vaginal microbiomes, 32 "taxa with high pathogenic potential" (THPP) were identified. Collectively, THPP (mean RA ~5.24%) negatively correlated (rs = -0.68, p < 2.2e-16) with Lactobacillus species but not significantly with Gardnerella/Prevotella spp. combined (r = -0.13, p = 0.1). However, at the individual level, Mycoplasma hominis exhibited moderate positive correlations with Gardnerella (r = 0.46, p = 2.6e-09) and Prevotella spp. (r = 0.47, p = 1.4e-09). Conclusions: These findings while supporting the idea that vaginal microbiomes vary with ethnicity, also suggest that CSTs are more wide-ranging and not exclusive to any particular ethnic group. This study offers additional insight into the structure of the vaginal microbiome and contributes to the description and subcategorization of non-Lactobacillus-dominated CSTs.