Human Vaginal Microbiota Research Articles

Gonorrhea caused due to antimicrobial-resistant bacteria Neisseria gonorrhoeae treated using probiotic peptide.

Neisseria gonorrhea is a sexually transmitted disease from gonorrhea that lacks treatment; despite the urgency, the absence of adequate drugs, lack of human correlates of protection, and inadequate animal models of infection have delayed progress toward the prevention of gonococcal infection. Lactobacillus crispatus is a lactic acid bacterium typically found in the human vaginal microbiota. Peptides from L. crispatus have shown a potential therapeutic option for targetting N. gonorrhea. Bioinformatics analysis is important for speeding up drug target acquisition, screening refinement, and evaluating adverse effects and drug resistance prediction. Therefore, this study identified an antimicrobial peptide from the bacteriocin immunity protein (BIP) of L. crispatus using the bioinformatics tool and Collection of Antimicrobial Peptide (CAMPR3). Based on the AMP score and highest ADMET properties, the peptide SM20 was chosen for docking analysis. SM20 was docked against multiple proteins from the genome of the AMR bacterium N. gonorrhea using an online tool; protein-peptide interactions were established and visualized using the PyMol visualizing tool. Molecular docking was carried out using the CABSdock tool, and multiple conformations were obtained against the membrane proteins of N. gonorrhoea. The peptide SM20 exhibited higher docking scores and ADMET properties. Therefore, SM20 could be further encapsulated with cellulose; it can be applied topically to the genital tract to target N. gonorrhea infection. The controlled release of the antimicrobial peptide from the gel can provide sustained delivery of the treatment, increasing its efficacy and reducing the risk of resistance development.

Investigating the Metabolism of Estrogens in Ligilactobacillus salivarius Strains Isolated from Human Milk and Vaginal Microbiota.

The interplay between enterohepatic circulation and the gut microbiota is the main driver determining systemic levels of estrogens and their metabolites. Nevertheless, the role of potentially probiotic microorganisms in estrogen metabolism has not been investigated so far. In this work, we have explored the ability of six Ligilactobacillus salivarius strains isolated from human milk and vaginal samples to degrade and/or conjugate parental estrogens in vitro and under aerobic conditions. The quantification of estrogens and their derivatives was carried out in cell-free supernatants by LC-QQQ-MS. All the tested L. salivarius strains achieved an average degradation rate of estrone and estriol of 98% and 55%, respectively, whereas 17β-estradiol was preferentially conjugated (up to 40%). The presence of seven out of ten genes encoding enzymes relevant for estrogen metabolism was further confirmed by PCR, highlighting their genetic potential for degrading, conjugating and/or deconjugating estrogens. The tested L. salivarius strains may be considered potential probiotics affecting the fate of endogenous estrogens. Clinical trials targeting populations with estrogen-dependent conditions will be required to elucidate the true potential of these strains for the restoration and maintenance of a healthy host estrobolome.

Characterizing the vaginal microbiome in a sexually fluid primate (Pan paniscus).

While there have been recent breakthroughs in human vaginal microbiome research, very few non-human primate (NHP) vaginal microbiome studies exist due to difficulty in obtaining samples. In this study, we sought to: (1) characterize the bonobo vaginal microbiota for the first time, and (2) determine the relationship between vaginal pH and swelling size. During a 21-day study period, we collected observational data and 71 vaginal swabs from three cohoused adult females at the Ape Cognition and Conservation Initiative. After filtering and classification, a total of 3452 amplicon sequence variants were recruited from 12 vaginal samples. The most dominant phylum represented was Actinobacteria, and the most abundant genera were Gardnerella, Atopobium, and Prevotella. The mean pH score was 6.1 (range: 5.1-7.0), and pH levels varied with relative swelling size. This is the first study to examine the vaginal microbial composition in this species, and it conforms to previous NHP studies in that there was not the same bacterial dominance of Lactobacillus spp. often highlighted in human vaginal microbiota studies. Our findings suggest there may be other factors contributing to the protection of the bonobo vaginal environment, but future analysis of a larger sample is needed to fully understand how sociality and sexuality shape vaginal microbiota and host health.

Description of the vaginal microbiota in nulliparous ewes during natural mating and pregnancy: preliminary signs of the male preputial microbiota modulation.

The vaginal microbiota plays a key role in animals' health. Understanding its diversity and composition and associated changes occurring through the reproductive cycle represents valuable knowledge to disclose the mechanisms leading to dysbiosis and eventually to infection. Even if the human vaginal microbiota has been thoroughly studied, scarce research has been conducted on the vaginal microbiota of livestock. In this study, 16S rRNA gene-based sequencing was performed on vaginal samples of ten nulliparous ewes at three different sampling points: before the estrus synchronization protocol (T0), at the time of estrus before mating (Testrus), and the day of the pregnancy diagnosis (Tpreg). Preputial samples from the three males collected pre and post-mating were also analyzed. Firmicutes, Proteobacteria, Bacteroidetes, and Actinobacteria were the most abundant phyla in vaginal samples. The most abundant genera were Porphyromonas, Anaerococcus, and Peptinophilius. Vaginal microbiota biodiversity decreased during pregnancy. Tenericutes (Ureaplasma spp.) increased significantly at Tpreg in both pregnant and non-pregnant ewes. Differences were observed between pregnant and non-pregnant ewes at Tpreg where pregnant ewes had a significantly higher abundance of Actinobacillus spp. and Ureaplasma spp. Ewes that were diagnosed with pregnancy at Tpreg showed a decreased abundance of gram-negative bacteria such as Bacteroidales, Campylobacterales, and Enterobacteriales. In addition, a significant decrease in the relative abundances of genera within Firmicutes, such as Alloicoccus (Lactobacillales), Atopostipes (Lactobacillales), and an uncultured bacteria W5053 from Family XI (Firmicutes, Clostridiales) was observed in non-pregnant ewes at Tpreg. The four most abundant phyla in the rams' prepuce were the same as in the ewes' vagina. The most abundant genus was Corynebacterium. No major differences were observed in the ram's preputial microbiota between pre and post-mating samples. Nevertheless, the differences in the taxonomic composition of ewes' vaginal microbiota between Testrus and Tpreg could be explained by the exposure to the preputial microbiota. This study offers new insights into the effects of several key steps of the ewe's reproductive cycle such as estrus-synchronization protocol, mating, and pregnancy on ovine vaginal microbiota. The knowledge of the microbiota dynamics during the reproductive cycle can help improve the reproductive outcomes of dams by identifying biomarkers and putative probiotics.

Human vaginal microbiota colonization is regulated by female sex hormones in a mouse model.

Clinically, a Lactobacillus rich vaginal microbiota (VMB) is considered optimal for reproductive outcomes, while a VMB populated by anaerobes is associated with dysbiosis and the clinical condition bacterial vaginosis (BV), which is linked to increased susceptibility to sexually transmitted infections and adverse reproductive outcomes. Mouse models that mimic eubiotic and dysbiotic VMB are currently lacking but could play a critical role in improving protective interventions. In this study, probiotic, eubiotic, and dysbiotic models were developed in C57BL/6 mice, using probiotic strains Lactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC-14, eubiotic Lactobacillus crispatus, or dysbiotic Gardnerella vaginalis strains. Endogenous sex hormones were manipulated by either ovariectomizing (OVX) mice or administering 17β-estradiol or progesterone pellets in OVX mice. Hormone-altered mice were inoculated with probiotic Lactobacillus species, L. crispatus, or G. vaginalis, and colonization was tracked using quantitative plating assays. Glycogen and MUC-1 levels in hormone-treated mice were determined with ELISA and MUC-1 staining. Following a single administration, L. rhamnosus and L. reuteri persisted in the mouse vaginal tract for up to eight days, L. crispatus persisted for up to three days, and G. vaginalis persisted for up to two days, as measured by quantitative plating assays and qPCR. Colonization of G. vaginalis was facilitated by the presence of mucin. The lack of endogenous hormones in OVX mice dramatically decreased VMB bacterial load compared to normal mice. None of the exogenous bacteria including Lactobacilli could colonize OVX mice for more than 24 hours. Treatment with 17β-estradiol but not progesterone restored the endogenous VMB and colonization with Lactobacilli and G. vaginalis. Interestingly, 17β-estradiol treated mice had significantly increased levels of glycogen compared to OVX and progesterone-treated mice. Based on the results, we have shown that estrogen played a significant role in the ability for human VMB species to colonize in our mouse models, potentially through a glycogen mediated mechanism. These results suggest there is a dynamic interaction between sex hormones and the VMB, which can affect bacterial diversity and the ability for a VMB to colonize.

Vaginal microbial dynamics and pathogen colonization in a humanized microbiota mouse model

Vaginal microbial composition is associated with differential risk of urogenital infection. Although Lactobacillus spp. are thought to confer protection against infection, the lack of in vivo models resembling the human vaginal microbiota remains a prominent barrier to mechanistic discovery. Using 16S rRNA amplicon sequencing of C57BL/6J female mice, we found that vaginal microbial composition varies within and between colonies across three vivaria. Noting vaginal microbial plasticity in conventional mice, we assessed the vaginal microbiome of humanized microbiota mice (HMbmice). Like the community structure in conventional mice, HMbmice vaginal microbiota clustered into community state types but, uniquely, HMbmice communities were frequently dominated by Lactobacillus or Enterobacteriaceae. Compared to conventional mice, HMbmice were less susceptible to uterine ascension by urogenital pathobionts group B Streptococcus (GBS) and Prevotella bivia. Although Escherichia and Lactobacillus both correlated with the absence of uterine GBS, vaginal pre-inoculation with exogenous HMbmouse-derived E. coli, but not Ligilactobacillus murinus, reduced vaginal GBS burden. Overall, HMbmice serve as a useful model to elucidate the role of endogenous microbes in conferring protection against urogenital pathogens.

Bacterial amylases enable glycogen degradation by the vaginal microbiome.

The human vaginal microbiota is frequently dominated by lactobacilli and transition to a more diverse community of anaerobic microbes is associated with health risks. Glycogen released by lysed epithelial cells is believed to be an important nutrient source in the vagina. However, the mechanism by which vaginal bacteria metabolize glycogen is unclear, with evidence implicating both bacterial and human enzymes. Here we biochemically characterize six glycogen-degrading enzymes (GDEs), all of which are pullanases (PulA homologues), from vaginal bacteria that support the growth of amylase-deficient Lactobacillus crispatus on glycogen. We reveal variations in their pH tolerance, substrate preferences, breakdown products and susceptibility to inhibition. Analysis of vaginal microbiome datasets shows that these enzymes are expressed in all community state types. Finally, we confirm the presence and activity of bacterial and human GDEs in cervicovaginal fluid. This work establishes that bacterial GDEs can participate in the breakdown of glycogen, providing insight into metabolism that may shape the vaginal microbiota.

Physicochemical considerations in the formulation development of silicone elastomer vaginal rings releasing 5-nitroimidazole drugs for the treatment of bacterial vaginosis

Bacterial vaginosis (BV) is a common dysbiosis of the human vaginal microbiota characterized by depletion of hydrogen peroxide and lactic acid-producing Lactobacillus bacteria and an overgrowth of certain facultative anaerobic bacteria. Although short-term cure rates following treatment with frontline antibiotics (most notably oral metronidazole (MNZ), clindamycin vaginal cream, and MNZ vaginal gel) are generally high, longer-term recurrence rates are an issue. The development of vaginal formulations offering continuous/sustained administration of antibiotic drugs over one or more weeks might prove useful in reducing recurrence. Here, we report the manufacture and preclinical testing of matrix-type vaginal rings offering sustained release of four 5-nitroimidazole antimicrobial drugs either being used clinically or having potential in treatment of BV – MNZ, tinidazole (TNZ), secnidazole (SNZ) and ornidazole (ONZ). All four drugs showed good compatibility with a medical-grade addition-cure silicone elastomer based upon thermal analysis experiments, and matrix-type rings containing 250 mg (3.125 %w/w) of each drug were successfully manufactured by reaction injection molding. 28-day in vitro drug release studies demonstrated root-time kinetics, with daily release rates of 25, 22, 9 and 6 mg/day½ for SNZ, ONZ, MNZ and TNZ, respectively. The rank order of drug release from rings correlated with the simple molecular permeability parameter S/V, where S is the measured drug solubility in silicone fluid and V is the drug molecular volume. The relative merits of SNZ and ONZ over MNZ (the current reference treatment) are discussed. The data support development of vaginal rings for sustained release of 5-nitroimidazole compounds for treatment of BV.

Partial convergence of the human vaginal and rectal maternal microbiota in late gestation and early post-partum

The human vaginal and fecal microbiota change during pregnancy. Because of the proximity of these perineal sites and the evolutionarily conserved maternal-to-neonatal transmission of the microbiota, we hypothesized that the microbiota of these two sites (rectal and vaginal) converge during the last gestational trimester as part of the preparation for parturition. To test this hypothesis, we analyzed 16S rRNA sequences from vaginal introitus and rectal samples in 41 women at gestational ages 6 and 8 months, and at 2 months post-partum. The results show that the human vaginal and rectal bacterial microbiota converged during the last gestational trimester and into the 2nd month after birth, with a significant decrease in Lactobacillus species in both sites, as alpha diversity progressively increased in the vagina and decreased in the rectum. The microbiota convergence of the maternal vaginal-anal sites perinatally might hold significance for the inter-generational transmission of the maternal microbiota.

Single-Molecule Approach to 16S rRNA for Vaginal Microbiome Signatures in Response to Metronidazole Treatment.

Bacterial vaginosis (BV) is the most common infection of the lower reproductive tract among women of reproductive age, characterized by a depletion of health-associated Lactobacillus and an overgrowth of anaerobes. Metronidazole has been recommended as a first-line therapy for treating BV for decades. Although most cases are cured by the treatment, recurrent infections of BV seriously affect women's reproductive health. Until now, limited information on the vaginal microbiota has been explored at the species level. Here, we adopted a single molecular sequencing approach for the 16S rRNA gene, named FLAST (full-length assembly sequencing technology), to analyze the human vaginal microbiota that improved species-level resolution for taxonomy and identified microbiota alterations in the vaginal tract in response to treatment with metronidazole. Appling high-throughput sequencing, we identified 96 and 189 novel full-length 16S rRNA gene sequences in Lactobacillus and Prevotella, respectively, which had not previously been reported in vaginal samples. Moreover, we found that Lactobacillus iners was significantly enriched in the cured group before metronidazole treatment, and that was maintained in a high frequency after the treatment, suggesting an important role for this species in response to metronidazole treatment. Our research also highlights the importance of the single-molecule paradigm for progressing the field of microbiology and applying these insights to better understand the dynamic microbiota during BV treatment. Subsequent novel treatment approaches should be proposed to improve BV treatment outcomes, optimize the vaginal microbiome, and reduce gynecological and obstetric sequelae. IMPORTANCE Bacterial vaginosis (BV) is a common infectious disease of the reproductive tract. Metronidazole treatment, as the first line of treatment, frequently fails at recovery of the microbiome. However, the precise types of Lactobacillus and other bacteria involved in BV remain unclear, and this has resulted in a failure to identify potential markers to predict clinic outcomes. In this study, we adopted a 16S rRNA gene full-length assembly sequencing technology for the taxonomy analysis and evaluation of vaginal microbiota before and after treatment with metronidazole. We additionally identified 96 and 189 novel 16S rRNA gene sequences in Lactobacillus and Prevotella species, respectively, in vaginal samples, which improves our understanding of the vaginal microbiota. Moreover, we found that the abundance of Lactobacillus iners and Prevotella bivia before treatment was associated with a lack of cure. These potential biomarkers will help to facilitate future studies aimed at improving BV treatment outcomes, optimize the vaginal microbiome, and reduce adverse sexual and reproductive outcomes.

Inhibitory effects of vaginal Lactobacilli on Candida albicans growth, hyphal formation, biofilm development, and epithelial cell adhesion.

Antifungal agents are not always efficient in resolving vulvovaginal candidiasis (VVC), a common genital infection caused by the overgrowth of Candida spp., including Candida albicans, or in preventing recurrent infections. Although lactobacilli (which are dominant microorganisms constituting healthy human vaginal microbiota) are important barriers against VVC, the Lactobacillus metabolite concentration needed to suppress VVC is unknown. We quantitatively evaluated Lactobacillus metabolite concentrations to determine their effect on Candida spp., including 27 vaginal strains of Lactobacillus crispatus, L. jensenii, L. gasseri, Lacticaseibacillus rhamnosus, and Limosilactobacillus vaginalis, with inhibitory abilities against biofilms of C. albicans clinical isolates. Lactobacillus culture supernatants suppressed viable fungi by approximately 24%-92% relative to preformed C. albicans biofilms; however, their suppression differed among strains and not species. A moderate negative correlation was found between Lactobacillus lactate production and biofilm formation, but no correlation was observed between hydrogen peroxide production and biofilm formation. Both lactate and hydrogen peroxide were required to suppress C. albicans planktonic cell growth. Lactobacillus strains that significantly inhibited biofilm formation in culture supernatant also inhibited C. albicans adhesion to epithelial cells in an actual live bacterial adhesion competition test. Healthy human microflora and their metabolites may play important roles in the development of new antifungal agent against C. albicans-induced VVC.

Vaginal microbiota of adolescents and their mothers: A preliminary study of vertical transmission and persistence

IntroductionFactors that influence vaginal microbiota composition, including its source, are not well understood.MethodsTo determine if vaginal microbiota transmission from mother to daughter at birth could possibly influence the human vaginal microbiota composition in adolescence, we conducted a preliminary study to investigate the relationship between the vaginal microbiota of 13 adolescents (ages 15-21) and their mothers and the daughter’s birth mode.Results and discussionBased on analysis of bacterial 16S rRNA gene sequences, the vaginal microbiotas of mother/daughter pairs were more similar to each other if the daughter was born by vaginal delivery rather than by C-section. Specifically, within pair β-diversity, measured by the Yue and Clayton θ (θYC) distance metric, was significantly lower if the daughter was born by vaginal delivery. Additionally, genome sequences from an important member of the vaginal microbiota, Lactobacillus crispatus, isolated from one mother/daughter pair in which the daughter was born by vaginal delivery, were highly similar based on recombination-filtered single nucleotide polymorphisms (SNPs). Both community-level analysis and isolate genome sequence analysis are consistent with birth-mode dependent transmission and persistence of at least some members of the vaginal microbiota.

The First Lanthipeptide from Lactobacillus iners, Inecin L, Exerts High Antimicrobial Activity against Human Vaginal Pathogens.

Vaginal infections continue to be a serious public health issue, and developing new approaches to address antibiotic-resistant pathogens is an urgent task. The dominant vaginal Lactobacillus species and their active metabolites (e.g., bacteriocins) have the potential to defeat pathogens and help individuals recover from disorders. Here, we describe for the first time a novel lanthipeptide, inecin L, a bacteriocin from Lactobacillus iners with posttranslational modifications. The biosynthetic genes of inecin L were actively transcribed in the vaginal environment. Inecin L was active against the prevailing vaginal pathogens, such as Gardnerella vaginalis and Streptococcus agalactiae, at nanomolar concentrations. We demonstrated that the antibacterial activity of inecin L was closely related to the N terminus and the positively charged His13 residue. In addition, inecin L was a bactericidal lanthipeptide that showed little effect on the cytoplasmic membrane but inhibited the cell wall biosynthesis. Thus, the present work characterizes a new antimicrobial lanthipeptide from a predominant species of the human vaginal microbiota. IMPORTANCE The human vaginal microbiota plays essential roles in preventing pathogenic bacteria, fungi, and viruses from invading. The dominant vaginal Lactobacillus species show great potential to be developed as probiotics. However, the molecular mechanisms (such as bioactive molecules and their modes of action) involved in the probiotic properties remain to be determined. Our work describes the first lanthipeptide molecule from the dominant Lactobacillus iners. Additionally, inecin L is the only lanthipeptide found among the vaginal lactobacilli thus far. Inecin L shows strong antimicrobial activity toward the prevalent vaginal pathogens and antibiotic-resistant strains, suggesting that inecin L is a potent antibacterial molecule for drug development. In addition, our results show that inecin L exhibits specific antibacterial activity related to the residues in the N-terminal region and ring A, which will contribute to structure-activity relationship studies in lacticin 481-like lanthipeptides.

Role of the human vaginal microbiota in the regulation of inflammation and sexually transmitted infection acquisition: Contribution of the non-human primate model to a better understanding?

The human vaginal microbiota has a central role in the regulation of the female reproductive tract (FRT) inflammation. Indeed, on one hand an optimal environment leading to a protection against sexually transmitted infections (STI) is associated with a high proportion of Lactobacillus spp. (eubiosis). On the other hand, a more diverse microbiota with a high amount of non-Lactobacillus spp. (dysbiosis) is linked to a higher local inflammation and an increased STI susceptibility. The composition of the vaginal microbiota is influenced by numerous factors that may lead to a dysbiotic environment. In this review, we first discuss how the vaginal microbiota composition affects the local inflammation with a focus on the cytokine profiles, the immune cell recruitment/phenotype and a large part devoted on the interactions between the vaginal microbiota and the neutrophils. Secondly, we analyze the interplay between STI and the vaginal microbiota and describe several mechanisms of action of the vaginal microbiota. Finally, the input of the NHP model in research focusing on the FRT health including vaginal microbiota or STI acquisition/control and treatment is discussed.

Three-dimensional models of the cervicovaginal epithelia to study host-microbiome interactions and sexually transmitted infections.

2D cell culture systems have historically provided controlled, reproducible means to analyze host–pathogen interactions observed in the human reproductive tract. Although inexpensive, straightforward, and requiring a very short time commitment, these models recapitulate neither the functionality of multilayered cell types nor the associated microbiome that occurs in a human. Animal models have commonly been used to recreate the complexity of human infections. However, extensive modifications of animal models are required to recreate interactions that resemble those in the human reproductive tract. 3D cell culture models have emerged as alternative means of reproducing vital elements of human infections at a fraction of the cost of animal models and on a scale that allows for replicative experiments. Here, we describe a new 3D model that utilizes transwells with epithelial cells seeded apically and a basolateral extracellular matrix (ECM)-like layer. The model produced tissues with morphologic and physiological resemblance to human cervical and vaginal epithelia, including mucus levels produced by cervical cells. Infection by Chlamydia trachomatis and Neisseria gonorrhoeae was demonstrated, as well as the growth of bacterial species observed in the human vaginal microbiota. This enabled controlled mechanistic analyses of the interactions between host cells, the vaginal microbiota, and STI pathogens. Affordable and semi high-throughput 3D models of the cervicovaginal epithelia that are physiologically relevant by sustaining vaginal bacterial colonization, and facilitate studies of chlamydial and gonococcal infections.

Complete Genome Sequences of Ezakiella coagulans C0061C1 and Fenollaria massiliensis C0061C2

ABSTRACTEzakiella coagulans and Fenollaria massiliensis are two obligate anaerobic bacteria in the family Peptoniphilaceae and are both uncommon members of the human vaginal microbiota. We isolated a strain of each bacterium from the same vaginal swab specimen and here report the first complete genome sequences of the two species.

Dynamics of vaginal microbiome in female beagles at different ages

Age-related changes in human vaginal microbiota composition have been reported, and such changes might be influenced by humidity, external stimuli, hormone levels, drug to use, and other factors. However, there is no report about the vaginal microbiota composition of female beagles at different ages. To investigate the effects of aging on the vaginal microbiota independent of other effects, we analyzed the vaginal microbiomes of 23 beagles at a wide range of ages from 1 year to 7 years old (except the 3rd year), 1–2 y were categorized into youth stage (YS), 4–5 y were categorized into middle stage (MS), and 6–7 y were categorized into elderly stage (ES) based on age. Samples were collected by scraping the vaginal mucosa of YS (n = 8), MS (n = 5) and ES (n = 10), and analyzed by 16S-rRNA gene high-throughput-sequencing. The diversity of the vaginal microbiome in female beagles was found to continuously change with age. We also found associations between age and specific members and functions of the vaginal microbiome. The metabolism of terpenoids and polyketide and the cell motility are significantly enhanced with age. Our results suggest that the proportion of Tenericutes might be a biomarker which could distinguish between YS and others

Characterization of a novel type of glycogen-degrading amylopullulanase from Lactobacillus crispatus.

Glycogen is one of the major carbohydrates utilized by the human vaginal microbiota, which is commonly dominated by Lactobacillus, especially L. crispatus. An in silico analysis predicted that a type I pullulanase was involved in glycogen degradation in L. crispatus. The biochemical and genetic properties of the pullulanase still need to be determined. Here, we de novo identified the glycogen (Glg)-utilization enzyme (named GlgU) from L. crispatus through a biochemical assay. GlgU was optimally active at acidic pH, approximately 4.0 ~ 4.5, and was able to hydrolyze glycogen into low-molecular-weight malto-oligosaccharides. Actually, GlgU was a type II pullulanase (amylopullulanase) with just one catalytic domain that possessed substrate specificity toward both α-1,4 and α-1,6-glucosidic bonds. Phylogenetically, GlgU was obviously divergent from the known amylases and pullulanases (including amylopullulanases) in lactobacilli. In addition, we confirmed the catalytic activity of glgU in a nonglycogen-utilizing lactobacilli strain, demonstrating the essential role of glgU in glycogen metabolism. Overall, this study characterized a novel type of amylopullulanases, contributing to the knowledge of the glycogen utilization mechanism of the dominant species of human vaginal microbiota. KEY POINTS: • GlgU was a type II pullulanase, not a type I pullulanase predicted before. • GlgU was able to completely hydrolyze glycogen into malto-oligosaccharides. • GlgU played a key role in the metabolism of extracellular glycogen.

How were Lactobacillus species selected as single dominant species in the human vaginal microbiota? Coevolution of humans and Lactobacillus

The optimal microbiota in the vagina for women’s health is dominated by single species of Lactobacillus. However, the mechanism by which these species become dominant remains unknown. Therefore, understanding this mechanism is important to establish new treatments, such as probiotics, for vaginal dysbiosis-related conditions. To date, at least 5 hypotheses have been proposed to explain the vaginal dominance of Lactobacillus. However, these hypotheses do not address why a “single” species of Lactobacillus becomes dominant in the vaginal microbiota. First, we evaluated the effect of Lactobacillus culture supernatant on the biomass concentration of each different strain and species of Lactobacillus. The results indicated that the biomass concentrations were not significantly affected by the culture supernatant from other vaginal Lactobacillus species. Then, we discussed host factors that select the vaginal Lactobacillus species and the antagonism of vaginal bacteria. We also assess the mechanism of coevolution of humans and Lactobacillus. Here, we present the hypothesis that host factors such as antimicrobial peptides and proteins (AMPs) and immunoglobulins could play important roles in the selection of specific species of Lactobacillus in the vagina.

Antibiotic Therapy and Vaginal Microbiota Transplantation Reduce Endometriosis Disease Progression in Female Mice via NF-κB Signaling Pathway.

Endometriosis (EMS) is a disease characterized by estrogen-dependent, chronic inflammatory, and annoying symptoms, which inflicts about 10% reproductive-age women. The diagnosis of endometriosis mainly depends on pathological examination after surgical resection while the pathogenesis of EMS is not clear enough. Surgical resection and drug therapy (including painkillers and hormone therapy, especially gonadotropin-releasing hormone analogs, GnRH-a) are widely used, but they are expensive and have many side effects. There are few studies on vaginal microorganisms in women with endometriosis. We collected vaginal secretions from women with EMS confirmed by pathology and demonstrated that they were different from that of healthy women by 16s rRNA high-throughput sequencing. Additionally, we established the EMS model in female mice by intraperitoneally injecting fragments from donor mice (3-week growth). Then, the mice were treated with mixed antibiotics (vagina) and NF-κB signaling pathway inhibitors (intraperitoneal injection), respectively. The result suggested that the ectopic lesions were inhibited. In addition, inflammatory cytokines IL-1β, IL-6, and TNF-α in peritoneal fluid, cell proliferation marker ki-67, and macrophage marker Iba-1 in ectopic lesions decreased significantly from that of mock mice. We also observed similar results as above by vaginal microbiota transplantation (VMT) and subcutaneous injection of leuprorelin acetate (LA, one of GnRH-a) for mice with EMS. These results showed that vaginal use of antibiotics or VMT is helpful to treat endometriosis in mice. However, due to the great difference between human and mouse vaginal microbiota, its mechanism and clinical transformation application still need to be further studied in the future.