Longitudinal profiles of the vaginal microbiota of pre-, peri-, and postmenopausal women: preliminary insights from a secondary data analysis.

OBJECTIVE: Menopause is often accompanied by lowered Lactobacillus spp. relative abundance and increased abundance of diverse anaerobic/aerobic bacteria in the vaginal microbiota due in part to declines in estrogen. These microbiota are associated with urogenital symptoms and infections. In premenopause, vaginal microbiota can fluctuate rapidly, particularly with menstrual cycles and sexual activity; however, the longitudinal dynamics of vaginal microbiota are understudied in peri- and postmenopause. We described vaginal community stability across reproductive stages. METHODS: Pre- (n = 83), peri- (n = 8), and postmenopausal (n = 11) participants provided twice-weekly mid-vaginal samples (total, 1,556; average, 15 per participant) over 8 weeks in an observational study. Composition of the vaginal microbiota was characterized by 16S rRNA gene amplicon sequencing, and a community state type (CST) was assigned to each sample. Clustering of longitudinal CST profiles, CST transition rates, duration of low-Lactobacillus/high bacterial diversity CSTs, and other metrics of bacterial community dynamics were assessed across reproductive stages. RESULTS: The proportion of participants with longitudinal CST profiles characterized by low-Lactobacillus CSTs was similar among pre- (38.6%), peri- (37.5%), and postmenopausal (36.4%) participants (P = 0.69). CST transition rates between consecutive samples were 21.1%, 16.7%, and 14.6% for pre-, peri-, and postmenopausal participants, respectively (P = 0.49). Low-Lactobacillus CST tended to persist for at least 4 weeks, irrespective of reproductive stage. CONCLUSIONS: Findings from this small yet frequently sampled cohort revealed vaginal bacterial fluctuations over 8 weeks that were similar across reproductive stages. Larger and longer-term studies based on these preliminary data could provide insights into the influence of microbiota dynamics on urogenital outcomes during menopause.

Extracellular vesicles from vaginal Gardnerella vaginalis and Mobiluncus mulieris contain distinct proteomic cargo and induce inflammatory pathways.

Colonization of the vaginal space with bacteria such as Gardnerella vaginalis and Mobiluncus mulieris is associated with increased risk for STIs, bacterial vaginosis, and preterm birth, while Lactobacillus crispatus is associated with optimal reproductive health. Although host-microbe interactions are hypothesized to contribute to reproductive health and disease, the bacterial mediators that are critical to this response remain unclear. Bacterial extracellular vesicles (bEVs) are proposed to participate in host-microbe communication by providing protection of bacterial cargo, delivery to intracellular targets, and ultimately induction of immune responses from the host. We evaluated the proteome of bEVs produced in vitro from G. vaginalis, M. mulieris, and L. crispatus, identifying specific proteins of immunologic interest. We found that bEVs from each bacterial species internalize within cervical and vaginal epithelial cells, and that epithelial and immune cells express a multi-cytokine response when exposed to bEVs from G. vaginalis and M. mulieris but not L. crispatus. Further, we demonstrate that the inflammatory response induced by G. vaginalis and M. mulieris bEVs is TLR2-specific. Our results provide evidence that vaginal bacteria communicate with host cells through secreted bEVs, revealing a mechanism by which bacteria lead to adverse reproductive outcomes associated with inflammation. Elucidating host-microbe interactions in the cervicovaginal space will provide further insight into the mechanisms contributing to microbiome-mediated adverse outcomes and may reveal new therapeutic targets.

Sexual transmission of urogenital bacteria: whole metagenome sequencing evidence from a sexual network study.

Sexual transmission of the urogenital microbiota may contribute to adverse sexual and reproductive health outcomes. The extent of sexual transmission of the urogenital microbiota is unclear as prior studies largely investigated specific pathogens. We used epidemiologic data and whole metagenome sequencing to characterize urogenital microbiota strain concordance between participants of a sexual network study. Individuals who screened positive for genital Chlamydia trachomatis were enrolled and referred their sexual contacts from the prior 60-180 days. Snowball recruitment of sexual contacts continued for up to four waves. Vaginal swabs and penile urethral swabs were collected for whole metagenome sequencing. We evaluated bacterial strain concordance using inStrain and network analysis. We defined concordance as ≥99.99% average nucleotide identity over ≥50% shared coverage; we defined putative sexual transmission as concordance between sexual contacts with <5 single-nucleotide polymorphisms per megabase. Of 138 participants, 74 (54%) were female; 120 (87%) had genital chlamydia; and 43 (31%) were recruited contacts. We identified 115 strain-concordance events among 54 participants representing 25 bacterial species. Seven events (6%) were between sexual contacts including putative heterosexual transmission of Fannyhessea vaginae, Gardnerella leopoldii, Prevotella amnii, Sneathia sanguinegens, and Sneathia vaginalis (one strain each), and putative sexual transmission of Lactobacillus iners between female contacts. Most concordance events (108, 94%) were between non-contacts, including eight female participants connected through 18 Lactobacillus crispatus and 3 Lactobacillus jensenii concordant strains, and 14 female and 2 male participants densely interconnected through 52 Gardnerella swidsinskii concordance events.IMPORTANCEEpidemiologic evidence consistently indicates bacterial vaginosis (BV) is sexually associated and may be sexually transmitted, though sexual transmission remains subject to debate. This study is not capable of demonstrating BV sexual transmission; however, we do provide strain-level metagenomic evidence that strongly supports heterosexual transmission of BV-associated species. These findings strengthen the evidence base that supports ongoing investigations of concurrent male partner treatment for reducing BV recurrence. Our data suggest that measuring the impact of male partner treatment on F. vaginae, G. leopoldii, P. amnii, S. sanguinegens, and S. vaginalis may provide insight into why a regimen does or does not perform well. We also observed a high degree of strain concordance between non-sexual-contact female participants. We posit that this may reflect limited dispersal capacity of vaginal bacteria coupled with individuals' comembership in regional transmission networks where transmission may occur between parent and child at birth, cohabiting individuals, or sexual contacts.

Rapid intestinal and systemic metabolic reprogramming in an immunosuppressed environment.

Intrinsic metabolism shapes the immune environment associated with immune suppression and tolerance in settings such as organ transplantation and cancer. However, little is known about the metabolic activities in an immunosuppressive environment. In this study, we employed metagenomic, metabolomic, and immunological approaches to profile the early effects of the immunosuppressant drug tacrolimus, antibiotics, or both in gut lumen and circulation using a murine model. Tacrolimus induced rapid and profound alterations in metabolic activities within two days of treatment, prior to alterations in gut microbiota composition and structure. The metabolic profile and gut microbiome after seven days of treatment was distinct from that after two days of treatment, indicating continuous drug effects on both gut microbial ecosystem and host metabolism. The most affected taxonomic groups are Clostriales and Verrucomicrobiae (i.e., Akkermansia muciniphila), and the most affected metabolic pathways included a group of interconnected amino acids, bile acid conjugation, glucose homeostasis, and energy production. Highly correlated metabolic changes were observed between lumen and serum metabolism, supporting their significant interactions. Despite a small sample size, this study explored the largely uncharacterized microbial and metabolic events in an immunosuppressed environment and demonstrated that early changes in metabolic activities can have significant implications that may serve as antecedent biomarkers of immune activation or quiescence. To understand the intricate relationships among gut microbiome, metabolic activities, and immune cells in an immune suppressed environment is a prerequisite for developing strategies to monitor and optimize alloimmune responses that determine transplant outcomes.

Sub-communities of the vaginal microbiota in pregnant and non-pregnant women.

Diverse and non-Lactobacillus-dominated vaginal microbial communities are associated with adverse health outcomes such as preterm birth and the acquisition of sexually transmitted infections. Despite the importance of recognizing and understanding the key risk-associated features of these communities, their heterogeneous structure and properties remain ill-defined. Clustering approaches are commonly used to characterize vaginal communities, but they lack sensitivity and robustness in resolving substructures and revealing transitions between potential sub-communities. Here, we address this need with an approach based on mixed membership topic models. Using longitudinal data from cohorts of pregnant and non-pregnant study participants, we show that topic models more accurately describe sample composition, longitudinal changes, and better predict the loss of Lactobacillus dominance. We identify several non-Lactobacillus-dominated sub-communities common to both cohorts and independent of reproductive status. In non-pregnant individuals, we find that the menstrual cycle modulates transitions between and within sub-communities, as well as the concentrations of half of the cytokines and 18% of metabolites. Overall, our analyses based on mixed membership models reveal substructures of vaginal ecosystems which may have important clinical and biological associations.

Integrating compositional and functional content to describe vaginal microbiomes in health and disease.

BACKGROUND: A Lactobacillus-dominated vaginal microbiome provides the first line of defense against adverse genital tract health outcomes. However, there is limited understanding of the mechanisms by which the vaginal microbiome modulates protection, as prior work mostly described its composition through morphologic assessment and marker gene sequencing methods that do not capture functional information. To address this gap, we developed metagenomic community state types (mgCSTs) which use metagenomic sequences to describe and define vaginal microbiomes based on both composition and functional potential. RESULTS: MgCSTs are categories of microbiomes classified using taxonomy and the functional potential encoded in their metagenomes. MgCSTs reflect unique combinations of metagenomic subspecies (mgSs), which are assemblages of bacterial strains of the same species, within a microbiome. We demonstrate that mgCSTs are associated with demographics such as age and race, as well as vaginal pH and Gram stain assessment of vaginal smears. Importantly, these associations varied between mgCSTs predominated by the same bacterial species. A subset of mgCSTs, including three of the six predominated by Gardnerella vaginalis mgSs, as well as mgSs of L. iners, were associated with a greater likelihood of bacterial vaginosis diagnosed by Amsel clinical criteria. This L. iners mgSs, among other functional features, encoded enhanced genetic capabilities for epithelial cell attachment that could facilitate cytotoxin-mediated cell lysis. Finally, we report a mgSs and mgCST classifier for which source code is provided and may be adapted for use by the microbiome research community. CONCLUSIONS: MgCSTs are a novel and easily implemented approach to reduce the dimension of complex metagenomic datasets while maintaining their functional uniqueness. MgCSTs enable the investigation of multiple strains of the same species and the functional diversity in that species. Future investigations of functional diversity may be key to unraveling the pathways by which the vaginal microbiome modulates the protection of the genital tract. Importantly, our findings support the hypothesis that functional differences between vaginal microbiomes, including those that may look compositionally similar, are critical considerations in vaginal health. Ultimately, mgCSTs may lead to novel hypotheses concerning the role of the vaginal microbiome in promoting health and disease, and identify targets for novel prognostic, diagnostic, and therapeutic strategies to improve women's genital health. Video Abstract.

Rapid intestinal and systemic metabolic reprogramming in an immunosuppressed environment.

Intrinsic metabolism shapes the immune environment associated with immune suppression and tolerance in settings such as organ transplantation and cancer. However, little is known about the metabolic activities in an immunosuppressive environment. In this study, we employed metagenomic, metabolomic, and immunological approaches to profile the early effects of the immunosuppressant drug tacrolimus, antibiotics, or both in gut lumen and circulation using a murine model. Tacrolimus induced rapid and profound alterations in metabolic activities within two days of treatment, prior to alterations in gut microbiota composition and structure. The metabolic profile and gut microbiome after seven days of treatment was distinct from that after two days of treatment, indicating continuous drug effects on both gut microbial ecosystem and host metabolism. The most affected taxonomic groups are Clostriales and Verrucomicrobiae (i.e., Akkermansia muciniphila), and the most affected metabolic pathways included a group of interconnected amino acids, bile acid conjugation, glucose homeostasis, and energy production. Highly correlated metabolic changes were observed between lumen and serum metabolism, supporting their significant interactions. Despite a small sample size, this study explored the largely uncharacterized microbial and metabolic events in an immunosuppressed environment and demonstrated that early changes in metabolic activities can have significant implications that may serve as antecedent biomarkers of immune activation or quiescence. To understand the intricate relationships among gut microbiome, metabolic activities, and immune cells in an immune suppressed environment is a prerequisite for developing strategies to monitor and optimize alloimmune responses that determine transplant outcomes.

Evaluation of vaginal microbiome equilibrium states identifies microbial parameters linked to resilience after menses and antibiotic therapy.

The vaginal microbiome (VMB) is a complex microbial community that is closely tied to reproductive health. Optimal VMB communities have compositions that are commonly defined by the dominance of certain Lactobacillus spp. and can remain stable over time or transition to non-optimal states dominated by anaerobic bacteria and associated with bacterial vaginosis (BV). The ability to remain stable or undergo transitions suggests a system with either single (mono-stable) or multiple (multi-stable) equilibrium states, though factors that contribute to stability have been difficult to determine due to heterogeneity in microbial growth characteristics and inter-species interactions. Here, we use a computational model to determine whether differences in microbial growth and interaction parameters could alter equilibrium state accessibility and account for variability in community composition after menses and antibiotic therapies. Using a global uncertainty and sensitivity analysis that captures parameter sets sampled from a physiologically relevant range, model simulations predicted that 79.7% of microbial communities were mono-stable (gravitate to one composition type) and 20.3% were predicted to be multi-stable (can gravitate to more than one composition type, given external perturbations), which was not significantly different from observations in two clinical cohorts (HMP cohort, 75.2% and 24.8%; Gajer cohort, 78.1% and 21.9%, respectively). The model identified key microbial parameters that governed equilibrium state accessibility, such as the importance of non-optimal anaerobic bacteria interactions with Lactobacillus spp., which is largely understudied. Model predictions for composition changes after menses and antibiotics were not significantly different from those observed in clinical cohorts. Lastly, simulations were performed to illustrate how this quantitative framework can be used to gain insight into the development of new combinatorial therapies involving altered prebiotic and antibiotic dosing strategies. Altogether, dynamical models could guide development of more precise therapeutic strategies to manage BV.

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.

High-resolution functional description of vaginal microbiomes in health and disease.

Background: A Lactobacillus-dominated vaginal microbiome provides the first line of defense against numerous adverse genital tract health outcomes. However, there is limited understanding of the mechanisms by which the vaginal microbiome modulates protection, as prior work mostly described its composition through morphologic assessment and marker gene sequencing methods that do not capture functional information. To address this limitation, we developed metagenomic community state types (mgCSTs) which uses metagenomic sequences to describe and define vaginal microbiomes based on both composition and function. Results: MgCSTs are categories of microbiomes classified using taxonomy and the functional potential encoded in their metagenomes. MgCSTs reflect unique combinations of metagenomic subspecies (mgSs), which are assemblages of bacterial strains of the same species, within a microbiome. We demonstrate that mgCSTs are associated with demographics such as age and race, as well as vaginal pH and Gram stain assessment of vaginal smears. Importantly, these associations varied between mgCSTs predominated by the same bacterial species. A subset of mgCSTs, including three of the six predominated by Gardnerella mgSs, as well as a mgSs of L. iners, were associated with a greater likelihood of Amsel bacterial vaginosis diagnosis. This L. iners mgSs, among other functional features, encoded enhanced genetic capabilities for epithelial cell attachment that could facilitate cytotoxin-mediated cell lysis. Finally, we report a mgSs and mgCST classifier as an easily applied, standardized method for use by the microbiome research community. Conclusions: MgCSTs are a novel and easily implemented approach to reducing the dimension of complex metagenomic datasets, while maintaining their functional uniqueness. MgCSTs enable investigation of multiple strains of the same species and the functional diversity in that species. Future investigations of functional diversity may be key to unraveling the pathways by which the vaginal microbiome modulates protection to the genital tract. Importantly, our findings support the hypothesis that functional differences between vaginal microbiomes, including those that may look compositionally similar, are critical considerations in vaginal health. Ultimately, mgCSTs may lead to novel hypotheses concerning the role of the vaginal microbiome in promoting health and disease, and identify targets for novel prognostic, diagnostic, and therapeutic strategies to improve women's genital health.

Strain-specific alterations in gut microbiome and host immune responses elicited by tolerogenic Bifidobacterium pseudolongum.

The beneficial effects attributed to Bifidobacterium are largely attributed to their immunomodulatory capabilities, which are likely to be species- and even strain-specific. However, their strain-specificity in direct and indirect immune modulation remain largely uncharacterized. We have shown that B. pseudolongum UMB-MBP-01, a murine isolate strain, is capable of suppressing inflammation and reducing fibrosis in vivo. To ascertain the mechanism driving this activity and to determine if it is specific to UMB-MBP-01, we compared it to a porcine tropic strain B. pseudolongum ATCC25526 using a combination of cell culture and in vivo experimentation and comparative genomics approaches. Despite many shared features, we demonstrate that these two strains possess distinct genetic repertoires in carbohydrate assimilation, differential activation signatures and cytokine responses signatures in innate immune cells, and differential effects on lymph node morphology with unique local and systemic leukocyte distribution. Importantly, the administration of each B. pseudolongum strain resulted in major divergence in the structure, composition, and function of gut microbiota. This was accompanied by markedly different changes in intestinal transcriptional activities, suggesting strain-specific modulation of the endogenous gut microbiota as a key to immune modulatory host responses. Our study demonstrated a single probiotic strain can influence local, regional, and systemic immunity through both innate and adaptive pathways in a strain-specific manner. It highlights the importance to investigate both the endogenous gut microbiome and the intestinal responses in response to probiotic supplementation, which underpins the mechanisms through which the probiotic strains drive the strain-specific effect to impact health outcomes.

Biofilms preserve the transmissibility of a multi-drug resistance plasmid.

Self-transmissible multidrug resistance (MDR) plasmids are a major health concern because they can spread antibiotic resistance to pathogens. Even though most pathogens form biofilms, little is known about how MDR plasmids persist and evolve in biofilms. We hypothesize that (i) biofilms act as refugia of MDR plasmids by retaining them in the absence of antibiotics longer than well-mixed planktonic populations and that (ii) the evolutionary trajectories that account for the improvement of plasmid persistence over time differ between biofilms and planktonic populations. In this study, we evolved Acinetobacter baumannii with an MDR plasmid in biofilm and planktonic populations with and without antibiotic selection. In the absence of selection, biofilm populations were better able to maintain the MDR plasmid than planktonic populations. In planktonic populations, plasmid persistence improved rapidly but was accompanied by a loss of genes required for the horizontal transfer of plasmids. In contrast, in biofilms, most plasmids retained their transfer genes, but on average, plasmid, persistence improved less over time. Our results showed that biofilms can act as refugia of MDR plasmids and favor the horizontal mode of plasmid transfer, which has important implications for the spread of MDR.

Vaginal microbiome-host interactions modeled in a human vagina-on-a-chip.

BACKGROUND: A dominance of non-iners Lactobacillus species in the vaginal microbiome is optimal and strongly associated with gynecological and obstetric health, while the presence of diverse obligate or facultative anaerobic bacteria and a paucity in Lactobacillus species, similar to communities found in bacterial vaginosis (BV), is considered non-optimal and associated with adverse health outcomes. Various therapeutic strategies are being explored to modulate the composition of the vaginal microbiome; however, there is no human model that faithfully reproduces the vaginal epithelial microenvironment for preclinical validation of potential therapeutics or testing hypotheses about vaginal epithelium-microbiome interactions. RESULTS: Here, we describe an organ-on-a-chip (organ chip) microfluidic culture model of the human vaginal mucosa (vagina chip) that is lined by hormone-sensitive, primary vaginal epithelium interfaced with underlying stromal fibroblasts, which sustains a low physiological oxygen concentration in the epithelial lumen. We show that the Vagina Chip can be used to assess colonization by optimal L. crispatus consortia as well as non-optimal Gardnerella vaginalis-containing consortia, and to measure associated host innate immune responses. Co-culture and growth of the L. crispatus consortia on-chip was accompanied by maintenance of epithelial cell viability, accumulation of D- and L-lactic acid, maintenance of a physiologically relevant low pH, and down regulation of proinflammatory cytokines. In contrast, co-culture of G. vaginalis-containing consortia in the vagina chip resulted in epithelial cell injury, a rise in pH, and upregulation of proinflammatory cytokines. CONCLUSION: This study demonstrates the potential of applying human organ chip technology to create a preclinical model of the human vaginal mucosa that can be used to better understand interactions between the vaginal microbiome and host tissues, as well as to evaluate the safety and efficacy of live biotherapeutics products. Video Abstract.

Persistence and In Vivo Evolution of Vaginal Bacterial Strains over a Multiyear Time Period.

It is not clear whether the bacterial strains that comprise our microbiota are mostly long-term colonizers or transient residents. Studies have demonstrated decades-long persistence of bacterial strains within the gut, but persistence at other body sites has yet to be determined. The vaginal microbiota (VMB) is often dominated by Lactobacillus, although it is also commonly comprised of a more diverse set of other facultative and obligate anaerobes. Longitudinal studies have demonstrated that these communities can be stable over several menstrual cycles or can fluctuate temporally in species composition. We sought to determine whether the bacterial strains that comprise the VMB were capable of persisting over longer time periods. We performed shotgun metagenomics on paired samples from 10 participants collected 1 and 2 years apart. The resulting sequences were de novo assembled and binned into high-quality metagenome assembled genomes. Persistent strains were identified based on the sequence similarity between the genomes present at the two time points and were found in the VMB of six of the participants, three of which had multiple persistent strains. The VMB of the remaining four participants was similar in species composition at the two time points but was comprised of different strains. For the persistent strains, we were able to identify the mutations that were fixed in the populations over the observed time period, giving insight into the evolution of these bacteria. These results indicate that bacterial strains can persist in the vagina for extended periods of time, providing an opportunity for them to evolve in the host microenvironment. IMPORTANCE The stability of strains within the vaginal microbiota is largely uncharacterized. Should these strains be capable of persisting for extended periods of time, they could evolve within their host in response to selective pressures exerted by the host or by other members of the community. Here, we present preliminary findings demonstrating that bacterial strains can persist in the vagina for at least 1 year. We further characterized in vivo evolution of the persistent strains. Several participants were also found to not have persistent strains, despite having a vaginal microbiota (VMB) with similar species composition at the two time points. Our observations motivate future studies that collect samples from more participants, at more time points, and over even longer periods of time. Understanding which strains persist, what factors drive their persistence, and what selective pressures they face will inform the development and delivery of rationally designed live biotherapeutics for the vagina.

Identification of shared bacterial strains in the vaginal microbiota of related and unrelated reproductive-age mothers and daughters using genome-resolved metagenomics.

It has been suggested that the human microbiome might be vertically transmitted from mother to offspring and that early colonizers may play a critical role in development of the immune system. Studies have shown limited support for the vertical transmission of the intestinal microbiota but the derivation of the vaginal microbiota remains largely unknown. Although the vaginal microbiota of children and reproductive age women differ in composition, the vaginal microbiota could be vertically transmitted. To determine whether there was any support for this hypothesis, we examined the vaginal microbiota of daughter-mother pairs from the Baltimore metropolitan area (ages 14-27, 32-51; n = 39). We assessed whether the daughter's microbiota was similar in composition to their mother's using metataxonomics. Permutation tests revealed that while some pairs did have similar vaginal microbiota, the degree of similarity did not exceed that expected by chance. Genome-resolved metagenomics was used to identify shared bacterial strains in a subset of the families (n = 22). We found a small number of bacterial strains that were shared between mother-daughter pairs but identified more shared strains between individuals from different families, indicating that vaginal bacteria may display biogeographic patterns. Earlier-in-life studies are needed to demonstrate vertical transmission of the vaginal microbiota.

Complete Genome Sequences of Ezakiella coagulans C0061C1 and Fenollaria massiliensis C0061C2.

Ezakiella 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.

Highly Specialized Carbohydrate Metabolism Capability in Bifidobacterium Strains Associated with Intestinal Barrier Maturation in Early Preterm Infants.

"Leaky gut," or high intestinal barrier permeability, is common in preterm newborns. The role of the microbiota in this process remains largely uncharacterized. We employed both short- and long-read sequencing of the 16S rRNA gene and metagenomes to characterize the intestinal microbiome of a longitudinal cohort of 113 preterm infants born between 240/7 and 326/7 weeks of gestation. Enabled by enhanced taxonomic resolution, we found that a significantly increased abundance of Bifidobacterium breve and a diet rich in mother's breastmilk were associated with intestinal barrier maturation during the first week of life. We combined these factors using genome-resolved metagenomics and identified a highly specialized genetic capability of the Bifidobacterium strains to assimilate human milk oligosaccharides and host-derived glycoproteins. Our study proposes mechanistic roles of breastmilk feeding and intestinal microbial colonization in postnatal intestinal barrier maturation; these observations are critical toward advancing therapeutics to prevent and treat hyperpermeable gut-associated conditions, including necrotizing enterocolitis (NEC). IMPORTANCE Despite improvements in neonatal intensive care, necrotizing enterocolitis (NEC) remains a leading cause of morbidity and mortality. "Leaky gut," or intestinal barrier immaturity with elevated intestinal permeability, is the proximate cause of susceptibility to NEC. Early detection and intervention to prevent leaky gut in "at-risk" preterm neonates are critical for decreasing the risk of potentially life-threatening complications like NEC. However, the complex interactions between the developing gut microbial community, nutrition, and intestinal barrier function remain largely uncharacterized. In this study, we reveal the critical role of a sufficient breastmilk feeding volume and the specialized carbohydrate metabolism capability of Bifidobacterium in the coordinated postnatal improvement of the intestinal barrier. Determining the clinical and microbial biomarkers that drive the intestinal developmental disparity will inform early detection and novel therapeutic strategies to promote appropriate intestinal barrier maturation and prevent NEC and other adverse health conditions in preterm infants.

HIV-associated vaginal microbiome and inflammation predict spontaneous preterm birth in Zambia.

A Lactobacillus-deficient, anaerobe-rich vaginal microbiome has been associated with local inflammation and spontaneous preterm birth (sPTB), but few studies have assessed this association in the setting of HIV. We performed metagenomic sequencing and inflammatory marker assays on vaginal swabs collected in pregnancy. We grouped samples into 7 metagenomic clusters (mgClust) using the non-redundant VIRGO catalogue, and derived inflammatory scores by factor analysis. Of 221 participants, median Shannon diversity index (SDI) was highest in HIV+ with detectable viral load (1.31, IQR: 0.85-1.66; p < 0.001) and HIV+ with undetectable virus (1.17, IQR: 0.51-1.66; p = 0.01) compared to HIV- (0.74, IQR: 0.35-1.26). Inflammatory scores positively correlated with SDI (+ 0.66, 95%CI 0.28, 1.03; p = 0.001), highest among anaerobe-rich mgClust2-mgClust6. HIV was associated with predominance of anaerobe-rich mgClust5 (17% vs. 6%; p = 0.02) and mgClust6 (27% vs. 11%; p = 0.002). Relative abundance of a novel Gardnerella metagenomic subspecies > 50% predicted sPTB (RR 2.6; 95%CI: 1.1, 6.4) and was higher in HIV+ (23% vs. 10%; p = 0.001). A novel Gardnerella metagenomic subspecies more abundant in women with HIV predicted sPTB. The risk of sPTB among women with HIV may be mediated by the vaginal microbiome and inflammation, suggesting potential targets for prevention.