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IntroductionThe cervicovaginal microbiota is influenced by host physiology, immunology, lifestyle, and ethnicity. We hypothesized that there would be differences in the cervicovaginal microbiota among pregnant, non-pregnant and menopausal women living in Puerto Rico with and without Human Papillomavirus (HPV) infection and cervical cancer. We specifically wanted to determine if the microbiota associated with variation in cervical cytology. A total of 294 women comprised of reproductive-age non-pregnant (N=196), pregnant (N=37), and menopausal (N=61) women were enrolled. The cervicovaginal bacteria was characterized by 16S rRNA amplicon sequencing, the HPV were genotyped with SPF10-LiPA, and cervical cytology was quantified. High-risk HPV (HR-HPV, 67.3%) was prevalent, including genotypes not covered by the 9vt HPV vaccine. Cervical lesions (34%) were also common. The cervical microbiota was dominated byLactobacillus iners. Pregnant women in the 2ndand 3rdtrimesters had decreased diversity and a decreased abundance of microbes associated with bacterial vaginosis. Women in menopause had greater alpha diversity, a greater proportion of facultative and strictly anaerobic bacteria, and higher cervicovaginal pH than pre-menopausal women. Cervical lesions were associated with greater alpha diversity. However, no significant associations between the microbiota and HPV infection (HR or LR-HPV types) were found. The cervicovaginal microbiota women living in Puerto Rican were either dominated byL. inersor diverse microbial communities regardless of a woman’s physiological stage. We postulate that the microbiota and the high prevalence of HR-HPV, increase the risk of cervical lesions of women living in Puerto Rico.

To investigate associations between bacterial species in the vagina in mid-trimester pregnant women from Brazil. The vaginal microbiome in 613 subjects was identified by analysis of the V1–V3 region of bacterial 16S ribosomal RNA and the relative prevalence of individual bacteria were determined. The bacterial species present in the greatest number of women were Lactobacillus crispatus (306 women), L. iners (298 women) and Gardnerella vaginalis (179 women). When present in the vagina, L. crispatus was the most abundant bacterium more than 85% of the time. In contrast, L. iners and G. vaginalis were most abundant in 63% and 41% of women who were positive for these microorganisms, respectively (p

Substantial changes in the composition of the vaginal microbiome occur following the end of pregnancy. To identify potential drivers of microbiome changes in individual women during the pregnancy to postpartum transition, we evaluated vaginal samples from 48 pregnant women during their first and third trimesters and postpartum. We determined the species composition of vaginal communities and the vaginal fluid levels of compounds involved in mediating changes in host physiology and the immune system at each time point. We used linear mixed-effects models to characterize associations. Consistent with previous reports, but with a larger sample size, a US population, and variations in the dominant bacteria, the vaginal microbiome was found to be more diverse during the postpartum period. There was a lower abundance of Lactobacillus and significantly higher proportions of Streptococcus anginosus and Prevotella bivia. Moreover, we uniquely demonstrated that postpartum vaginal secretions were also altered postpartum. There were elevated levels of hyaluronan and Hsp70 and decreased levels of the D- and L-lactic acid isomers. We posit that these variations are consequences of alterations in the vagina after delivery that profoundly alter the host environment and, thus, lead to changes in the capability of different bacterial species to survive and proliferate.

Objectives Short cervical length is a predictor of preterm birth. We evaluated if there were racial differences in variables associated with cervical length in pregnant Brazilian women. Methods Cervical length was determined by vaginal ultrasound in 414 women at 21 weeks gestation. All women were seen at the same clinic and analyzed by the same investigators. Women found to have a short cervix (≤25 mm) received vaginal progesterone throughout gestation. Composition of the vaginal microbiome was determined by analysis of the V1–V3 region of the gene coding for bacterial 16S ribosomal RNA. Demographic, clinical and outcome variables were determined by chart review. Subjects were 53.4% White, 37.2% mixed race and 9.4% Black. Results Pregnancy, medical history and education level were similar in all groups. Mean cervical length was shorter in Black women (28.4 mm) than in White (32.4 mm) or mixed race (32.8 mm) women (p≤0.016) as was the percentage of women with a short cervix (23.1, 12.2, 7.8% in Black, White, mixed race respectively) (p≤0.026). Mean cervical length increased with maternal age in White (p=0.001) and mixed race (p=0.045) women but not Black women. There were no differences in bacterial dominance in the vaginal microbiota between groups. Most women with a short cervix delivered at term. Conclusions We conclude that Black women in Brazil have a shorter cervical length than White or mixed race women independent of maternal age, pregnancy and demographic history or composition of the vaginal microbiome.

The vaginal microbiota of healthy women typically has low diversity, which increases after perturbations. Among these, lifestyle associated with certain sexual and antimicrobial practices may be associated with higher diversity. To test this hypothesis, we characterized the vaginal microbiota in the cervicovaginal and introital sites in sexually active Amerindians (N = 82) spanning urbanization, and in urban mestizos (N = 29), in the Venezuelan Amazonas. HPV status was also considered. Sampling was performed in an urban gradient from remote villages to a town, and women were individually classified by the degree of urbanization (low, medium, and high). Amerindian cervicovaginal and introital microbiota diversity were not associated with major changes in urbanization or ethnicity. There was a non-significant trend of increased diversity with urbanization, with a few taxa found overrepresented in urban Amerindians (Brevibacterium linens and Peptoniphilus lacrimalis) or mestizos (Mobiluncus mulieris and Prevotella sp.). Among all women, cervicovaginal and introital samples clustered, respectively, in four and two community state types (CSTs), where most profiles were dominated by Lactobacillus iners, Gardnerella vaginalis or were highly diverse profiles. HPV status did not associate with microbial diversity. In conclusion, no association was found between urban level and the vaginal microbiome in Amerindian women, and little difference was found between ethnicities. L. iners and high diversity profiles, associated with vaginal health outcomes, prevail in these populations.

Bacterial plasmids substantially contribute to the rapid spread of antibiotic resistance, which is a crisis in healthcare today. Coevolution of plasmids and their hosts promotes this spread of resistance by ameliorating the cost of plasmid carriage. However, our knowledge of plasmid–bacteria coevolution is solely based on studies done in well-mixed liquid cultures, even though biofilms represent the main way of bacterial life on Earth and are responsible for most infections. The spatial structure and the heterogeneity provided by biofilms are known to lead to increased genetic diversity as compared with well-mixed liquids. Therefore, we expect that growth in this complex environment could affect the evolutionary trajectories of plasmid–host dyads. We experimentally evolved Shewanella oneidensis MR-1 with plasmid pBP136Gm in biofilms and chemostats and sequenced the genomes of clones and populations. Biofilm populations not only maintained a higher diversity of mutations than chemostat populations but contained a few clones with markedly more persistent plasmids that evolved via multiple distinct trajectories. These included the acquisition of a putative toxin–antitoxin transposon by the plasmid and chromosomal mutations. Some of these genetic changes resulted in loss of plasmid transferability or decrease in plasmid cost. Growth in chemostats led to a higher proportion of variants with decreased plasmid persistence, a phenomenon not detected in biofilms. We suggest that the presence of more stable plasmid–host dyads in biofilms reflects higher genetic diversity and possibly unknown selection pressures. Overall, this study underscores the importance of the mode of growth in the evolution of antibiotic-resistant bacteria.

Torquetenovirus (TTV) is a nonpathogenic endogenous virus whose abundance varies with the extent of immune system activation. We determined if the TTV titer in the vagina of pregnant women was associated with vaginal microbiota composition and levels of compounds in vaginal secretions. Vaginal TTV and microbiota composition in 494 second trimester pregnant women were identified by gene amplification and analysis. Vaginal matrix metalloproteinases (MMPs), tissue inhibitors of MMP (TIMP) and lactic acid isomers were measured by ELISA. Dominance was defined as the relative abundance of a specific bacterium or species at >50% of the total number of bacteria identified. Clinical data were obtained by chart review. The median log10 TTV titer was lowest when Lactobacillus species other than L. iners were dominant (L. iners (4.1, p = 0.0001), bacteria other than lactobacilli (4.5, p = 0.0016) or no bacterium (4.7, p = 0.0009) dominated. The TTV titer was inversely proportional to L. crispatus abundance (pG. vaginalis (p = 0.0008) and L. iners (p = 0.0010). The TTV titer was proportional to TIMP-1, TIMP-2, MMP-8 and MMP-9 abundance (p≤0.0002) and inversely proportional to the level of D-lactic acid (p = 0.0024). We conclude that the association between variations in the TTV titer and the relative abundance of specific bacterial species and vaginal compounds indicates that local changes in immune status likely influence vaginal fluid composition.

Two-dimensional (2D) cell culture systems have provided controlled, reproducible means to analyze host-pathogen interactions. Although inexpensive, straightforward, and requiring very short time commitment, these models recapitulate neither the functionality of multi-layered cell types nor the microbial diversity of an infected human. Animal models have commonly been used to recreate the complexity of human infections. However, extensive modifications are commonly required to recreate interactions that resemble those in the human reproductive tract microbiologically and physiologically. Three-dimensional (3D) cell culture models have emerged as alternative means of reproducing key elements of human infections at a fraction of the cost of animal models and on a scale that allows for replicative experiments to be readily performed. Here we describe a new 3D model that utilizes transwells with epithelial cells seeded apically and a basolateral extra cellular matrix (ECM)-like layer containing collagen and fibroblasts. In this system, basal feeding creates a liquid/air interface on the apical side. The model produced tissues with close morphologic and physiological resemblance to human cervical and vaginal epithelia, including observable levels of mucus produced by cervical cells. Infection by both Chlamydia trachomatis and Neisseria gonorrhoeae was demonstrated as well as the growth of bacterial species observed in the human vaginal microbiota, enabling controlled mechanistic analyses of the interactions between host cells, vaginal microbiota and STI pathogens. Future experiments may include immune cells to mimic more closely the genital environment. Finally, the modular set up of the model makes it fully applicable to the analysis of non-genital host-microbiome-pathogen interactions.IMPORTANCEInfected sites in humans are a complex mix of host and microbial cell types interacting with each other to perform specific and necessary functions. The ability to understand the mechanism(s) that facilitate these interactions, and interactions with external factors is paramount to being able to develop preventative therapies. Models that attempt to faithfully replicate the complexity of these interactions are time intensive, costly, and not conducive to high throughput analysis. Two-dimensional (2D) models that have been used as a platform to understand these interactions, while cost effective, are generally limiting in experimental flexibility and structural/physiological relevance. Our three-dimensional (3D) models of the cervicovaginal epithelium can facilitate analysis of interactions between the host epithelium, sexually transmitted pathogens and bacteria present in the vaginal microbiota. Due to the modular design, additional cell types and environmental modulators can be introduced to the system to provide added complexity, approaching conditions in the infected human host.

Spatial structure is pervasive in the microbial world, yet we know little about how it influences the evolution of microbial populations. It is thought that spatial structure limits the scale of competitive interactions and protracts selective sweeps. This may allow microbial populations to simultaneously explore multiple evolutionary paths. But how structured a microbial population must be before this effect is realized is not known. We used empirical and simulation studies to explore the relationship between spatial structure and the maintenance of diversity. The degree of spatial structure experienced by Escherichia coli metapopulations was manipulated by varying the migration rate between its component subpopulations. Each subpopulation was inoculated with an equal number of two equally fit genotypes, and their frequencies in 12 subpopulations were determined during 150 generations of evolution. We observed that the frequency of the "loser" genotypes decreased exponentially as the migration rate between the subpopulations was increased and that higher frequencies of the loser genotypes were maintained in structured metapopulations. These results demonstrate that structured microbial populations can evolve along multiple evolutionary trajectories even when migration rates between the subpopulations are relatively high.

Diabetic patients suffer from gastrointestinal disorders associated with dysmotility, enteric neuropathy and dysbiosis of gut microbiota; however, gender differences are not fully known. Previous studies have shown that a high-fat diet (HFD) causes type two diabetes (T2D) in male mice after 4-8 weeks but only does so in female mice after 16 weeks. This study seeks to determine whether sex influences the development of intestinal dysmotility, enteric neuropathy and dysbiosis in mice fed HFD. We fed 8-week-old C57BL6 male and female mice a standard chow diet (SCD) or a 72% kcal HFD for 8 weeks. We analyzed the associations between sex and intestinal dysmotility, neuropathy and dysbiosis using motility assays, immunohistochemistry and next-generation sequencing. HFD ingestion caused obesity, glucose intolerance and insulin resistance in male but not female mice. However, HFD ingestion slowed intestinal propulsive motility in both male and female mice. This was associated with decreased inhibitory neuromuscular transmission, loss of myenteric inhibitory motor neurons and axonal swelling and loss of cytoskeletal filaments. HFD induced dysbiosis and changed the abundance of specific bacteria, especially Allobaculum, Bifidobacterium and Lactobacillus, which correlated with dysmotility and neuropathy. Female mice had higher immunoreactivity and numbers of myenteric inhibitory motor neurons, matching larger amplitudes of inhibitory junction potentials. This study suggests that sex influences the development of HFD-induced metabolic syndrome but dysmotility, neuropathy and dysbiosis occur independent of sex and prior to T2D conditions. Gastrointestinal dysmotility, neuropathy and dysbiosis might play a crucial role in the pathophysiology of T2D in humans irrespective of sex.

BACKGROUND: A survey was developed to characterize disease incidence, common pathology lesions, environmental characteristics, and nutrition programs within captive research marmoset colonies. METHODS: 17 research facilities completed the electronic survey. RESULTS: Nutritional management programs varied amongst research institutions housing marmosets; eight primary base diets were reported. The most common clinical syndromes reported were gastrointestinal (GI) disease (i.e. inflammatory bowel disease (IBD) like disease, chronic lymphocytic enteritis (CLE), chronic malabsorption, chronic diarrhea), metabolic bone disease or fracture, infectious diarrhea, and oral disease (tooth root abscesses, gingivitis, tooth root resorption). The five most common pathology morphologic diagnoses were colitis, nephropathy/ nephritis, enteritis, chronic lymphoplasmacytic enteritis, and cholecystitis. Obesity was more common (average 20% of a reporting institution’s population) than thin body condition (average 5%). CONCLUSIONS: Through review of current practices, we aim to inspire development of evidence based practices to standardize husbandry and nutrition practices for marmoset research colonies.

Bacterial vaginosis (BV) is the most common vaginal disorder of reproductive-aged women, yet its etiology remains enigmatic. One clinical symptom of BV, malodor, is linked to the microbial production of biogenic amines (BA). Using targeted liquid chromatography mass spectrometry, we analyzed 149 longitudinally collected vaginal samples to determine the in vivo concentrations of the most common BAs and then assessed their relationship to BV and effect upon the growth kinetics of axenically cultured vaginal Lactobacillus species. Increases in cadaverine, putrescine, and tyramine were associated with greater odds of women transitioning from L. crispatus-dominated vaginal microbiota to microbiota that have a paucity of Lactobacillus spp. and from Nugent scores of 0 to 3 to Nugent scores of 7 to 10, consistent with BV. Exposure to putrescine lengthened the lag time and/or slowed the growth of all vaginal Lactobacillus spp. except L. jensenii 62G. L. iners AB107’s lag time was lengthened by cadaverine but reduced in the presence of spermidine and spermine. The growth rate of L. crispatus VPI 3199 was slowed by cadaverine and tyramine, and strain-specific responses to spermine and spermidine were observed. BAs were associated with reduced production of d- and l-lactic acid by vaginal Lactobacillus spp., and this effect was independent of their effect upon Lactobacillus species growth. The exceptions were higher levels of d- and l-lactic acid by two strains of L. crispatus when grown in the presence of spermine. Results of this study provide evidence of a direct impact of common biogenic amines on vaginal Lactobacillus spp. IMPORTANCELactobacillus spp. are credited with providing the primary defense against gynecological conditions, including BV, most notably through the acidification of the vaginal microenvironment, which results from their production of lactic acid. The microbial production of BAs has been hypothesized to play a mechanistic role in diminishing Lactobacillus species-mediated protection, enabling the colonization and outgrowth of diverse anaerobic bacterial species associated with BV. Here, we demonstrate that in vivo increases in the most commonly observed BAs are associated with a loss of Lactobacillus spp. and the development of BV, measured by Nugent score. Further, we show that BAs formed by amino acid decarboxylase enzymes negatively affect the growth of type strains of the most common vaginal Lactobacillus spp. and separately alter their production of lactic acid. These results suggest that BAs destabilize vaginal Lactobacillus spp. and play an important and direct role in diminishing their protection of the vaginal microenvironment.

Made available in DSpace on 2021-06-25T12:36:01Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-01-27 Bill and Melinda Gates Foundation Brazilian Ministry of Health (DECIT) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) NIH COBRE grant Background: Predictors of spontaneous preterm birth in primigravid women remain undetermined. Aim: We evaluated whether biomarkers in vaginal secretions and/or differences in the dominant bacterium in the vaginal microbiome predicted the risk for spontaneous preterm birth in primigravid women with a cervical length >25mm. Study design: In a prospective study, 146 second trimester pregnant women with their first conception and a cervix >25mm were enrolled. The vaginal microbiome composition was characterized by analysis of 16S ribosomal RNA gene sequences. The concentrations of d- and l-lactic acid, matrix metalloproteinase (MMP) 2, 8 and 9 and tissue inhibitor of metalloproteinase (TIMP) 1 and 2 in vaginal secretions were measured by ELISA. Cervical length was determined by vaginal ultrasonography. Pregnancy outcome data were subsequently collected. There was a spontaneous preterm birth (SPTB) in 13 women (8.9%) while in an additional 8 women (5.5%) preterm delivery was medically indicated. Lactobacillus iners was the dominant vaginal bacterium in 61.5% of women with a SPTB but only in 31.2% of those who delivered at term (p = .0354). The vaginal concentration of TIMP-1 (p = .0419) and L-lactic acid (p = .0495) was higher in women with a SPTB as compared to those who delivered at term. Lactobacillus iners dominance was associated with elevated levels of TIMP-1 (p = .0434) and TIMP-2 (p = .0161) and lower levels of D-lactic acid (p < .0001) compared to when L. crispatus was dominant. Conclusion: In this exploratory study of primigravid women, elevations in vaginal TIMP-1 and L- lactic acid and L. iners dominance in the vaginal microbiome are associated with an increased occurrence of SPTB. Fed Univ Jundiai, Dept Obstet & Gynecol, Sao Paulo, Brazil Univ Fed Sao Paulo, Dept Obstet, Sao Paulo, Brazil Univ Sao Paulo, Inst Trop Med, Sao Paulo, Brazil Univ Idaho, Dept Biol Sci, Moscow, ID 83843 USA Univ Idaho, Inst Bioinformat & Evolutionary Studies, Moscow, ID 83843 USA Univ Fed Ceara, Dept Obstet & Gynecol, Fortaleza, Ceara, Brazil Univ Sao Paulo, Dept Obstet & Gynecol, Sch Med, Sao Paulo, Brazil Weill Cornell Med, Dept Obstet & Gynecol, New York, NY USA Univ Fed Sao Paulo, Dept Prevent Med, Sao Paulo, Brazil Sao Paulo State Univ, Dept Obstet & Gynecol, Sch Med, Botucatu, SP, Brazil Univ Sao Paulo, Inst Trop Med, Virol Lab LIM 52, Sao Paulo, Brazil Sao Paulo State Univ, Dept Obstet & Gynecol, Sch Med, Botucatu, SP, Brazil CNPq: 401626/2013-0 NIH COBRE grant: P30GM103324

In this study, we show that multiple bacteria in the vaginal community produce amylases that hydrolyze glycogen into simpler sugars (i.e., maltose and maltotriose). These sugars serve as “common goods” that sustain bacterial populations in vaginal communities., Dominance of Lactobacillus species in vaginal communities is a hallmark of healthy conditions in the female genital tract. Key nutrients for lactobacilli include sugars produced when glycogen is degraded by α-amylase in the vagina. While α-amylase activity has been demonstrated in vaginal fluids, it is unclear whether α-amylases are produced solely by the host, bacteria in the vagina, or both. We screened cervicovaginal mucus from 23 reproductive-age women, characterized the species composition of vaginal communities, measured vaginal pH, and determined levels of amylase activity, glycogen, and lactic acid. Based on differences in these measured variables, one sample from each of four individual donors was selected for metagenomic and proteomic analyses. Of eight putative bacterial amylases identified in the assembled bacterial metagenomes, we detected four in vaginal fluids. These amylases were produced by various bacteria in different vaginal communities. Moreover, no two communities were the same in terms of which bacteria were producing amylases. Although we detected bacterial amylases in vaginal fluids, there was no clear association between the bacterial species that was dominant in a community and the level of amylase activity. This association was likely masked by the presence of human α-amylase, which was also detected in vaginal fluids. Finally, the levels of amylase activity and glycogen were only weakly associated. Our findings show, for the first time, that multiple amylases from both bacterial and human origins can be present simultaneously in the vagina. This work also suggests that the link between glycogen, amylase, and Lactobacillus in the vagina is complex. IMPORTANCE In this study, we show that multiple bacteria in the vaginal community produce amylases that hydrolyze glycogen into simpler sugars (i.e., maltose and maltotriose). These sugars serve as “common goods” that sustain bacterial populations in vaginal communities. Given the temporal changes that are observed in the human vaginal microbiome, we expect the kinds of bacterial amylases produced will also vary over time. These differences influence the pool of resources that are broadly shared and shape the species composition of the vaginal bacterial community.

By characterizing the trajectories of antibiotic resistance gene transfer in bacterial communities such as the gut microbiome, we will better understand the factors that influence this spread of resistance. Our aim was to investigate the host network of a multi-drug resistance broad-host-range plasmid in the culturable gut microbiome of zebrafish. This was done throughin vitroandin vivoconjugation experiments withEscherichia colias donor of the plasmid pB10::gfp. When this donor was mixed with the extracted gut microbiome, only transconjugants ofAeromonas veroniiwere detected. In separate matings between the same donor and four prominent isolates from the gut microbiome, the plasmid transferred to two of these four isolates,A. veronii and Plesiomonas shigelloides,but not toShewanella putrefaciensandVibrio mimicus. When theseA. veronii and P. shigelloidestransconjugants were the donors in matings with the same four isolates, the plasmid now also transferred fromA. veroniitoS. putrefaciens.P. shigelloideswas unable to donate the plasmid andV. mimicuswas unable to acquire it. Finally, when theE. colidonor was addedin vivoto zebrafish through their food, plasmid transfer was observed in the gut but only toAchromobacter sp.,a rare member of the gut microbiome. This work shows that the success of plasmid-mediated antibiotic resistance spread in a gut microbiome depends on the donor-recipient species combinations and therefore their spatial arrangement. It also suggests that rare gut microbiome members should not be ignored as potential reservoirs of multi-drug resistance plasmids from food.ImportanceTo understand how antibiotic resistance plasmids end up in human pathogens it is crucial to learn how, where and when they are transferred and maintained in members of bacterial communities such as the gut microbiome. To gain insight into the network of plasmid-mediated antibiotic resistance sharing in the gut microbiome, we investigated the transferability and maintenance of a multi-drug resistance plasmid among the culturable bacteria of the zebrafish gut. We show that the success of plasmid-mediated antibiotic resistance spread in a gut microbiome can depend on which species are involved, as some are important nodes in the plasmid-host network and others dead-ends. Our findings also suggest that rare gut microbiome members should not be ignored as potential reservoirs of multi-drug resistance plasmids from food.

Objective To determine the presence and identity of extracellular bacteriophage (phage) families, genera and species in the vagina of pregnant women. Design Descriptive, observational cohort study. Setting Sao Paulo, Brazil. Population Pregnant women at 21-24 weeks' gestation. Methods Vaginal samples from 107 women whose vaginal microbiome and pregnancy outcomes were previously determined were analysed for phages by metagenomic sequencing. Main outcome measures Identification of phage families, genera and species. Results Phages were detected in 96 (89.7%) of the samples. Six different phage families were identified: Siphoviridae in 69.2%, Myoviridae in 49.5%, Microviridae in 37.4%, Podoviridae in 20.6%, Herelleviridae in 10.3% and Inviridae in 1.9% of the women. Four different phage families were present in 14 women (13.1%), three families in 20 women (18.7%), two families in 31 women (29.1%) and one family in 31 women (29.1%). The most common phage species detected were Bacillus phages in 48 (43.6%), Escherichia phages in 45 (40.9%), Staphylococcus phages in 40 (36.4%), Gokushovirus in 33 (30.0%) and Lactobacillus phages in 29 (26.4%) women. In a preliminary exploratory analysis, there were no associations between a particular phage family, the number of phage families present in the vagina or any particular phage species and either gestational age at delivery or the bacterial community state type present in the vagina. Conclusions Multiple phages are present in the vagina of most mid-trimester pregnant women. Tweetable abstract Bacteriophages are present in the vagina of most pregnant women.

To investigate associations between bacterial species in the vagina in mid-trimester pregnant women from Brazil.The vaginal microbiome in 613 subjects was identified by analysis of the V1-V3 region of bacterial 16S ribosomal RNA and the relative prevalence of individual bacteria were determined.The bacterial species present in the greatest number of women were Lactobacillus crispatus (306 women), L. iners (298 women) and Gardnerella vaginalis (179 women). When present in the vagina, L. crispatus was the most abundant bacterium more than 85% of the time. In contrast, L. iners and G. vaginalis were most abundant in 63% and 41% of women who were positive for these microorganisms, respectively (p 0.0001 vs. L. crispatus). The proportion of L. crispatus was negatively associated with the proportions of L. iners, L. jensenii, L. gasseri, G. vaginalis, Megasphaera, Atopobium vaginae and Prevotella (p 0.0001). In contrast, the proportion of G. vaginalis was positively associated with levels of Megasphaera, A. vaginae and Prevotella (p 0.0001) while L. iners proportion was unrelated to the proportion of L. jensenii, G. vaginalis, Megasphaera, A. vaginae or Prevotella.The composition of the vaginal microbiota in mid-trimester pregnant women is influenced by the relative concentrations of L. crispatus, L. iners and G. vaginalis.

Objective Does the combined analysis of compounds in vaginal secretions plus vaginal microbiota composition improve the ability to predict risk for spontaneous preterm birth (SPTB) in women with a short cervix. Design Prospective observational study Setting Three hospitals in Brazil Samples Vaginal secretions from 568 women at 21-24 weeks gestation with cervical length measurement. Method Vaginal microbiome composition was determined by analysis of the V1-V3 region of the bacterial 16S ribosomal RNA gene. D- lactic acid and tissue inhibitor of matrix metalloproteinase (TIMP)-1 in vaginal secretions were measured by ELISA. Main outcome measures SPTB in relation to cervical length and dominant vaginal bacteria. Results When Lactobacillus crispatus was the dominant bacterium the concentration of TIMP-1 was lower, and D-lactic acid was higher, than when either L. iners or Gardnerella vaginalis predominated (p 1.5 ng/ml best predicted the absence of L. crispatus dominance. Vaginal levels of TIMP-1 were highest (p = 0.024) and D-lactic acid levels were lowest (p=0.032) in women with the shortest cervical length. The absence of L. crispatus dominance increased the sensitivity for predicting a SPTB from 22.4% to 81.8% or 78.3% in women with a cervical length

Vaginal samples from women with term deliveries were tested for torquetenovirus (TTV) by gene amplification, matrix metalloproteinase (MMP)-8 and D- and L-lactic acid by ELISA, and microbiome composition by analysis of the bacterial 16S ribosomal RNA gene. TTV was detected in 43.2%, 31.5%, and 41.4% of first trimester, third trimester, and postpartum samples, respectively. The viral titer was higher in postpartum than in the first (p = 0.0018) or third (p = 0.0013) trimester. The mean gestational age at delivery was lower in women positive for TTV in their first trimester (p = 0.0358). In the first and third trimester, the MMP-8 level was higher if TTV was also present (p

Background High-fat diet, microbial alterations and lipopolysaccharide (LPS) are thought to cause enteric diabetic neuropathy and intestinal dysmotility. However, the role of the gut microbiota, lipoteichoic acid (LTA) from Gram-positive bacteria and short-chain fatty acids (SCFAs) in the development of diabetic enteric neuropathy and intestinal dysmotility is not well understood. Our aim was to examine the role of the gut microbiota, LTA and SCFAs in the development of diabetic enteric neuropathy and intestinal dysmotility. Methods We fed germ-free (GF) and conventionally raised (CR) mice either a high-fat (HFD) or standard chow diet (SCD) for 8 weeks. We analyzed the microbial community composition in CR mice using 16S rRNA sequencing and damage to myenteric neurons using immunohistochemistry. We also studied the effects of LPS, LTA, and SCFAs on duodenal muscularis externa contractions and myenteric neurons using cultured preparations. Key results High-fat diet ingestion reduced the total number and the number of nitrergic myenteric neurons per ganglion in the duodenum of CR but not in GF-HFD mice. GF mice had fewer neurons per ganglion compared with CR mice. CR mice fed a HFD had increased abundance of Gram-positive bacteria. LTA and LPS did not affect the frequency of duodenal muscularis contractions after 24 hours of cultured but reduced the density of nitrergic myenteric neurons and increased oxidative stress and TNFα production in myenteric ganglia. SCFAs did not affect muscularis contractions or injure myenteric neurons. Conclusions & inferences Gut microbial alterations induced increase in Gram-positive bacterial LTA may contribute to enteric neuropathy.

We characterized the composition and structure of the vaginal microbiota in a cohort of 149 women with genital Chlamydia trachomatis infection at baseline who were followed quarterly for 9 months after antibiotic treatment. At time of diagnosis, the vaginal microbiota was dominated by Lactobacillus iners or a diverse array of bacterial vaginosis–associated bacteria including Gardnerella vaginalis. Interestingly, L. iners–dominated communities were most common after azithromycin treatment (1 g monodose), consistent with the observed relative resistance of L. iners to azithromycin. Lactobacillus iners–dominated communities have been associated with increased risk of C. trachomatis infection, suggesting that the impact of antibiotic treatment on the vaginal microbiota could favor reinfections. These results provide support for the dual need to account for the potential perturbing effect(s) of antibiotic treatment on the vaginal microbiota, and to develop strategies to protect and restore optimal vaginal microbiota., Azithromycin treatment used for Chlamydia trachomatis infection appears to have a long-lasting impact on the vaginal microbiota (up to 9 months), favoring L. iners–dominated communities, a known risk factor for C. trachomatis infection, thus fueling an increased susceptibility to reinfection.

A specific pathogen free (SPF) barrier colony of breeding marmosets (Callithrix jacchus) was established at the Barshop Institute for Longevity and Aging Studies. Rodent and other animal models maintained as SPF barrier colonies have demonstrated improved health and lengthened lifespans enhancing the quality and repeatability of aging research. The marmosets were screened for two viruses and several bacterial pathogens prior to establishing the new SPF colony. Twelve founding animals successfully established a breeding colony with increased reproductive success, improved health parameters, and increased median lifespan when compared to a conventionally housed, open colony. The improved health and longevity of marmosets from the SPF barrier colony suggests that such management can be used to produce a unique resource for future studies of aging processes in a nonhuman primate model.

Epithelial cells lining the vagina are major components of genital tract immunity. The influence of the vaginal microbiome on properties of host epithelial cells is largely unexplored. We evaluated whether differences in the most abundant lactobacilli species or bacterial genera in the vagina of first trimester pregnant women were associated with variations in the extent of stress and autophagy in vaginal epithelial cells. Vaginal swabs from 154 first trimester pregnant women were analyzed for bacterial composition by amplification and sequencing of the V1-V3 region of bacterial 16S rRNA genes. Vaginal epithelial cells were lysed and autophagy quantitated by measurement of p62. Intracellular levels of the inducible 70kDa heat shock protein (hsp70), an indicator of cell stress and an autophagy inhibitor, were determined. When Lactobacillus crispatus was the most abundant member of the vaginal microbiota, epithelial p62 and hsp70 levels were lowest as compared to when other bacterial taxa were most abundant. The highest concentrations of p62 and hsp70 were associated with Streptococcus and Bifidobacterium abundance. The p62 level associated with Gardnerella abundance was lower than that observed when lactobacilli other than L. crispatus were most abundant. In conclusion, in the first trimester of pregnancy the abundance of different bacterial taxa is associated with variations in autophagy and magnitude of the stress response in vaginal epithelial cells.

Pregnancy permanently alters maternal anatomy, physiology and immunity. We evaluated if the vaginal microbiome differed between women with a first or subsequent conception. Relative abundance of bacteria in the vaginal microbiome in first trimester pregnant women, 52 with their first known conception, 26 with a prior spontaneous or induced abortion but no deliveries and 77 with at least one prior birth, was determined by classifying DNA sequences from the V1-V3 region of bacterial 16 S rRNA genes. Lactobacillus crispatus was the numerically most abundant bacterium in 76.4% of women with a first conception, 50.0% with only a prior spontaneous or scheduled abortion and 22.2% with a prior birth (p ≤ 0.01). L. iners was the most abundant bacterium in 3.8% of women with a first conception as compared to 19.2% (p = 0.03) and 20.8% (p = 0.03) in those with a prior abortion or birth, respectively. Gardnerella as the most abundant bacterial genus increased from 3.8% in women with a first conception to 15.4% and 14.3% in those with a prior abortion or birth, respectively (p > 0.05). L. iners dominance was also associated with a history of spontaneous abortion (p ≤ 0.02). The composition of the vaginal microbiome and its influence on pregnancy outcome varies with pregnancy history.

Lactobacillus crispatus and Lactobacillus iners are common inhabitants of the healthy human vagina. These two species are closely related and are thought to perform similar ecological functions in the vaginal environment. Temporal data on the vaginal microbiome have shown that nontransient instances of cooccurrence are uncommon, while transitions from an L. iners -dominated community to one dominated by L. crispatus , and vice versa, occur often. This suggests that there is substantial overlap in the fundamental niches of these species. Given this apparent niche overlap, it is unclear how they have been maintained as common inhabitants of the human vagina. In this study, we characterized and compared the genomes of L. iners and L. crispatus to gain insight into possible mechanisms driving the maintenance of this species diversity. Our results highlight differences in the genomes of these two species that may facilitate the partitioning of their shared niche space. Many of the identified differences may impact the protective benefits provided to the host by these two species. IMPORTANCE The microbial communities that inhabit the human vagina play a critical role in the maintenance of vaginal health through the production of lactic acid and lowering the environmental pH. This precludes the growth of nonindigenous organisms and protects against infectious disease. The two most common types of vaginal communities are dominated by either Lactobacillus iners or Lactobacillus crispatus , while some communities alternate between the two over time. We combined ecological theory with state-of-the-art genome analyses to characterize how these two species might partition their shared niche space in the vagina. We show that the genomes of L. iners and L. crispatus differ in many respects, several of which may drive differences in their competitive abilities in the vagina. Our results provide insight into factors that drive the complicated temporal dynamics of the vaginal microbiome and demonstrate how closely related microbial species partition shared fundamental niche space.

Premature birth and its complications are the largest contributors to infant death in the United States and globally. A short cervical length and the depletion of Lactobacillus species are known risk factors for preterm birth. However, in many resource-poor areas of the world, the technology to test for their occurrence is unavailable, and pregnant women with these risk factors are neither identified nor treated. In this study, we used path analysis to gain an unprecedented understanding of interactions between vaginal microbiome composition, the concentrations of various compounds in vaginal secretions, and cervical length. We identified low-cost point-of-care measures that might be used to identify pregnant women at risk for preterm birth. The use of these measures coupled with appropriate preventative or treatment strategies could reduce the incidence of preterm births in poor areas of the world that lack access to more sophisticated diagnostic methods., In many impoverished regions of the world, it may not be possible to assess two major risk factors for preterm birth: a short cervical length and the depletion of vaginal lactobacilli. We determined whether measuring specific compounds in vaginal fluid might be a simple, noninvasive, and cost-effective way to predict the bacteria that dominate the vaginal microbiome and indicate the presence of a shortened cervix (

The vaginal microbiota is believed to protect women against Chlamydia trachomatis, the etiologic agent of the most prevalent sexually transmitted infection (STI) in developed countries. The mechanism underlying this protection has remained elusive. Here, we reveal the comprehensive strategy by which the cervicovaginal microbiota modulates host functions to protect against chlamydial infection, thereby providing a novel conceptual mechanistic understanding. Major implications of this work are that (i) the impact of the vaginal microbiota on the epithelium should be considered in future studies of chlamydial infection and other STIs and (ii) a fundamental understanding of the cervicovaginal microbiota’s role in protection against STIs may enable the development of novel microbiome-based therapeutic strategies to protect women from infection and improve vaginal and cervical health., The mechanism(s) by which Lactobacillus-dominated cervicovaginal microbiota provide a barrier to Chlamydia trachomatis infection remain(s) unknown. Here we evaluate the impact of different Lactobacillus spp. identified via culture-independent metataxonomic analysis of C. trachomatis-infected women on C. trachomatis infection in a three-dimensional (3D) cervical epithelium model. Lactobacillus spp. that specifically produce d(−) lactic acid were associated with long-term protection against C. trachomatis infection, consistent with reduced protection associated with Lactobacillus iners, which does not produce this isoform, and with decreased epithelial cell proliferation, consistent with the observed prolonged protective effect. Transcriptomic analysis revealed that epigenetic modifications involving histone deacetylase-controlled pathways are integral to the cross talk between host and microbiota. These results highlight a fundamental mechanism whereby the cervicovaginal microbiota modulates host functions to protect against C. trachomatis infection.

BACKGROUND Histone deacetylase (HDAC) influences the acetylation status of histones at gene promotor loci, providing an epigenetic mechanism that regulates gene expression. METHODS We determined if variations in the composition of the vaginal microbiome in pregnant women were associated with alterations in the level of HDAC1 in vaginal epithelial cells and whether this influenced the concentration of compounds present in vaginal fluid. Vaginal epithelial cells were obtained from 150 women in their first trimester of pregnancy, lysed and assayed for HDAC1 by ELISA. Composition of the vaginal microbiome was determined by classification of sequences amplified from the V1-V3 region of bacterial ribosomal 16S rRNA genes. Vaginal secretions were assayed for total protein, matrix metalloproteinase (MMP)-8, the 70kDa heat shock protein (hsp70) and the D- and L-lactic acid isomers. RESULTS Lactobacilli were numerically dominant in 119 (79.3%) of the women, with Lactobacillus crispatus being the most prevalent (45.3% of women). Gardnerella was the most prevalent non-Lactobacillus species (10.7% of women). The median HDAC1 level in epithelial cells was 6.1 ng/mL when lactobacilli predominated vs. 20.5 ng/mL when non-lactobacilli were dominant (P=0.0039). Levels were lowest when L. crispatus was dominant (3.8 ng/mL) and highest with Streptococcus dominance (38.1 ng/mL). The concentration of HDAC1 was negatively correlated with the D-lactic acid level (P=0.0183) and positively correlated with concentrations of MMP-8 and hsp70 (P

BACKGROUND The Red Queen hypothesis is an evolutionary theory that describes the reciprocal coevolution of competing species. We sought to study whether introduction of the 7- and 13-valent pneumococcal conjugate vaccines (PCV7 and PCV13, respectively) altered pneumococcal serotype dynamics among children with invasive pneumococcal disease (IPD) as predicted by the Red Queen hypothesis. METHODS This study examined pneumococcal isolates (n = 641) obtained from children