Amy McMillan

Vaginal Microbiome and Epithelial Gene Array in Post-Menopausal Women with Moderate to Severe Dryness

After menopause, many women experience vaginal dryness and atrophy of tissue, often attributed to the loss of estrogen. An understudied aspect of vaginal health in women who experience dryness due to atrophy is the role of the resident microbes. It is known that the microbiota has an important role in healthy vaginal homeostasis, including maintaining the pH balance and excluding pathogens. The objectives of this study were twofold: first to identify the microbiome of post-menopausal women with and without vaginal dryness and symptoms of atrophy; and secondly to examine any differences in epithelial gene expression associated with atrophy. The vaginal microbiome of 32 post-menopausal women was profiled using Illumina sequencing of the V6 region of the 16S rRNA gene. Sixteen subjects were selected for follow-up sampling every two weeks for 10 weeks. In addition, 10 epithelial RNA samples (6 healthy and 4 experiencing vaginal dryness) were acquired for gene expression analysis by Affymetrix Human Gene array. The microbiota abundance profiles were relatively stable over 10 weeks compared to previously published data on premenopausal women. There was an inverse correlation between Lactobacillus ratio and dryness and an increased bacterial diversity in women experiencing moderate to severe vaginal dryness. In healthy participants, Lactobacillus iners and L. crispatus were generally the most abundant, countering the long-held view that lactobacilli are absent or depleted in menopause. Vaginal dryness and atrophy were associated with down-regulation of human genes involved in maintenance of epithelial structure and barrier function, while those associated with inflammation were up-regulated consistent with the adverse clinical presentation.

A multi-platform metabolomics approach identifies highly specific biomarkers of bacterial diversity in the vagina of pregnant and non-pregnant women.

Bacterial vaginosis (BV) increases transmission of HIV, enhances the risk of preterm labour, and is associated with malodour. Clinical diagnosis often relies on microscopy, which may not reflect the microbiota composition accurately. We use an untargeted metabolomics approach, whereby we normalize the weight of samples prior to analysis, to obtained precise measurements of metabolites in vaginal fluid. We identify biomarkers for BV with high sensitivity and specificity (AUC = 0.99) in a cohort of 131 pregnant and non-pregnant Rwandan women, and demonstrate that the vaginal metabolome is strongly associated with bacterial diversity. Metabolites associated with high diversity and clinical BV include 2-hydroxyisovalerate and γ-hydroxybutyrate (GHB), but not succinate, which is produced by both Lactobacillus crispatus and BV-associated anaerobes in vitro. Biomarkers associated with high diversity and clinical BV are independent of pregnancy status, and were validated in a blinded replication cohort from Tanzania (n = 45), where we predicted clinical BV with 91% accuracy. Correlations between the metabolome and microbiota identified Gardnerella vaginalis as a putative producer of GHB, and we demonstrate production by this species in vitro. This work illustrates how changes in community structure alter the chemical composition of the vagina, and identifies highly specific biomarkers for a common condition.

Effect of chemotherapy on the microbiota and metabolome of human milk, a case report

BackgroundHuman milk is an important source of bacteria for the developing infant and has been shown to influence the bacterial composition of the neonatal gut, which in turn can affect disease risk later in life. Human milk is also an important source of nutrients, influencing bacterial composition but also directly affecting the host. While recent studies have emphasized the adverse effects of antibiotic therapy on the infant microbiota, the effects of maternal chemotherapy have not been previously studied. Here we report the effects of drug administration on the microbiota and metabolome of human milk.MethodsMature milk was collected every two weeks over a four month period from a lactating woman undergoing chemotherapy for Hodgkin’s lymphoma. Mature milk was also collected from healthy lactating women for comparison. Microbial profiles were analyzed by 16S sequencing and the metabolome by gas chromatography–mass spectrometry.FindingsChemotherapy caused a significant deviation from a healthy microbial and metabolomic profile, with depletion of genera Bifidobacterium, Eubacterium, Staphylococcus and Cloacibacterium in favor of Acinetobacter, Xanthomonadaceae and Stenotrophomonas. The metabolites docosahexaenoic acid and inositol known for their beneficial effects were also decreased.ConclusionWith milk contents being critical for shaping infant immunity and development, consideration needs to be given to the impact of drugs administered to the mother and the long-term potential consequences for the health of the infant.

Adhesion of Lactobacillus iners AB-1 to Human Fibronectin: A Key Mediator for Persistence in the Vagina?

Lactobacillus iners is prominent in the human vagina and is able to persist despite development of bacterial vaginosis and treatment with antibiotics. A probable factor in its persistent survival is its ability to be retained in the vaginal epithelia. Genome sequencing of the strain showed an organism deplete of many metabolic pathways, yet equipped with fibronectin (Fn)-binding adhesins. The objective of the present study was to assess the ability of L iners AB-1 to bind immobilized Fn. Results showed that the organism superiorly bound the protein compared to other species of Lactobacillus and known binders such as Staphylococcus aureus. Treatment of L iners cells by protease rendered its binding abilities to Fn nonfunctional. The findings indicate a mechanism of vaginal persistence for a Lactobacillus species, with implications for reproductive health.

A systems biology approach investigating the effect of probiotics on the vaginal microbiome and host responses in a double blind, placebo-controlled clinical trial of post-menopausal women.

A lactobacilli dominated microbiota in most pre and post-menopausal women is an indicator of vaginal health. The objective of this double blinded, placebo-controlled crossover study was to evaluate in 14 post-menopausal women with an intermediate Nugent score, the effect of 3 days of vaginal administration of probiotic L. rhamnosus GR-1 and L. reuteri RC-14 (2.5×109 CFU each) on the microbiota and host response. The probiotic treatment did not result in an improved Nugent score when compared to when placebo. Analysis using 16S rRNA sequencing and metabolomics profiling revealed that the relative abundance of Lactobacillus was increased following probiotic administration as compared to placebo, which was weakly associated with an increase in lactate levels. A decrease in Atopobium was also observed. Analysis of host responses by microarray showed the probiotics had an immune-modulatory response including effects on pattern recognition receptors such as TLR2 while also affecting epithelial barrier function. This is the first study to use an interactomic approach for the study of vaginal probiotic administration in post-menopausal women. It shows that in some cases multifaceted approaches are required to detect the subtle molecular changes induced by the host to instillation of probiotic strains. Trial Registration ClinicalTrials.gov NCT02139839

Metabolic derangements identified through untargeted metabolomics in a cross-sectional study of Nigerian children with severe acute malnutrition

IntroductionSevere acute malnutrition (SAM) is a major cause of child mortality worldwide, however the pathogenesis of SAM remains poorly understood. Recent studies have uncovered an altered gut microbiota composition in children with SAM, suggesting a role for microbes in the pathogenesis of malnutrition.ObjectivesTo elucidate the metabolic consequences of SAM and whether these changes are associated with changes in gut microbiota composition.MethodsWe applied an untargeted multi-platform metabolomics approach [gas chromatography–mass spectrometry (GC-MS) and liquid chromatography–mass spectrometry (LC-MS)] to stool and plasma samples from 47 Nigerian children with SAM and 11 control children. The composition of the stool microbiota was assessed by 16S rRNA gene sequencing.ResultsThe plasma metabolome discriminated children with SAM from controls, while no significant differences were observed in the microbial or small molecule composition of stool. The abundance of 585 features in plasma were significantly altered in malnourished children (Wilcoxon test, FDR corrected P < 0.1), representing approximately 15% of the metabolome. Consistent with previous studies, children with SAM exhibited a marked reduction in amino acids/dipeptides and phospholipids, and an increase in acylcarnitines. We also identified numerous metabolic perturbations which have not been reported previously, including increased disaccharides, truncated fibrinopeptides, angiotensin I, dihydroxybutyrate, lactate, and heme, and decreased bioactive lipids belonging to the eicosanoid and docosanoid family.ConclusionOur findings provide a deeper understanding of the metabolic consequences of malnutrition. Further research is required to determine if specific metabolites may guide improved management, and/or act as novel biomarkers for assessing response to treatment.

Promising Prebiotic Candidate Established by Evaluation of Lactitol, Lactulose, Raffinose, and Oligofructose for Maintenance of a Lactobacillus-Dominated Vaginal Microbiota

ABSTRACT Perturbations to the vaginal microbiota can lead to dysbiosis, including bacterial vaginosis (BV), which affects a large portion of the female population. In a healthy state, the vaginal microbiota is characterized by low diversity and colonization by Lactobacillus spp., whereas in BV, these species are displaced by a highly diverse population of bacteria associated with adverse vaginal health outcomes. Since prebiotic ingestion has been a highly effective approach to invigorate lactobacilli for improved intestinal health, we hypothesized that these compounds could stimulate lactobacilli at the expense of BV organisms to maintain vaginal health. Monocultures of commensal Lactobacillus crispatus, Lactobacillus vaginalis, Lactobacillus gasseri, Lactobacillus johnsonii, Lactobacillus jensenii, and Lactobacillus iners, in addition to BV-associated organisms and Candida albicans, were tested for their ability to utilize a representative group of prebiotics consisting of lactitol, lactulose, raffinose, and oligofructose. The disaccharide lactulose was found to most broadly and specifically stimulate vaginal lactobacilli, including the strongly health-associated species L. crispatus, and importantly, not to stimulate BV organisms or C. albicans. Using freshly collected vaginal samples, we showed that exposure to lactulose promoted commensal Lactobacillus growth and dominance and resulted in healthy acidity partially through lactic acid production. This provides support for further testing of lactulose to prevent dysbiosis and potentially to reduce the need for antimicrobial agents in managing vaginal health. IMPORTANCE Bacterial vaginosis (BV) and other dysbioses of the vaginal microbiota significantly affect the quality of life of millions of women. Antimicrobial therapy is often poorly effective, causes side effects, and does not prevent recurrences. We report one of very few studies that have evaluated how prebiotics—compounds that are selectively fermented by beneficial bacteria such as Lactobacillus spp.—can modulate the vaginal microbiota. We also report use of a novel in vitro polymicrobial model to study the impact of prebiotics on the vaginal microbiota. The identification of prebiotic lactulose as enhancing Lactobacillus growth but not that of BV organisms or Candida albicans has direct application for retention of homeostasis and prevention of vaginal dysbiosis and infection.

Pilot assessment of probiotics for pregnant women in Rwanda

Background While the global market for probiotics is soon to reach in excess of US

Post-acquisition filtering of salt cluster artefacts for LC-MS based human metabolomic studies

50 billion, the continent of Africa has been largely ignored, despite these products having the ability to reduce the burden of disease and death. Trial design The present randomised, blinded, placebo-controlled clinical trial was undertaken in Rwanda, a country devoid of well-documented probiotics. The primary outcome aim was to examine receptivity and compliance for orally administered probiotic capsules containing Lactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC-14 in pregnant women and assess any initial side effects or changes to the vaginal microbiome. Methods Pregnant women between the ages of 18 and 55 were recruited from the Nyamata District Hospital in Rwanda and randomly assigned to receive probiotic or placebo capsules for one month. Clinicians were blinded to the treatments. Results The drop-out rate was 21%, with 13 of 18 women in the placebo group and 17 of 20 in the probiotic group completing the study. Only 13 women returned for birthing and additional sample collection. No side effects of either treatment group were reported. Microbiota and metabolomics data showed similar findings to those reported in the literature, with low bacterial diversity and Lactobacillus dominance associated with a healthy vagina, and birthing associated with high diversity. Despite the small sample size and lack of changes in the microbiota, women in the placebo arm were significantly more likely to give birth pre-term. Conclusion Overall women were receptive to the probiotic concept, but the lack of information on such products and logistical and economical challenges pose problems for wider population engagement. Trial registration ClinicalTrials.gov NCT02150655