Demonstration Projects

Fifteen demonstration projects were funded to demonstrate hypothesized correlations between the microbiome and human health and disease. These investigator-initiated projects leveraged advances made by the HMP's large scale sequencing efforts, e.g. tool & technology development and protocol standardization, to examine the relationship between changes in the human microbiome and diseases of interest, using 16S rRNA, metagenomic shotgun sequencing or both..

Demonstration project sequence data was submitted to public and controlled access areas of the NCBI sequence read archives, and is available through the HMP Portal, the legacy DACC Data Browser, or under NCBI BioProject 46305.

Project Title Principal Investigator(s) Institution(s)
Evaluation of the cutaneous microbiome in psoriasis Martin J Blaser New York University School of Medicine

Psoriasis, a highly prevalent disease of humans of unknown cause, is a chronic inflammatory disorder primarily involving skin, with distinctive clinical characteristics. With the newly developed tools that facilitate microbiome research, it now is possible to assess whether the cutaneous microbiome plays a role in the pathogenesis of this disorder. Preliminary data from our studies suggest that the cutaneous microbiome in psoriasis is complex and possibly different from normal. To deal with this complexity, we propose to examine the cutaneous microbiome in relation to psoriasis with explorations at several taxonomic and informatic levels. Our overall objective is to examine how changes in the normal cutaneous microbiome contribute to the pathogenesis of psoriasis. Since causality is complex and often difficult to prove, and beyond the scope of this RFP, our overall hypothesis is that there are alterations in the cutaneous microbiome in areas of skin affected by psoriasis in comparison with the range observed in clinically unaffected areas, or in healthy persons. We also hypothesize that the characteristics of the microbiome may affect clinical responses to the immunomodulatory agents used to treat psoriasis. An alternative hypothesis is that effective treatment of psoriasis with systemic immunomodulatory agents will not substantially affect the disordered microbial ecosystem. Such observations would provide evidence for the roles of the microbiota in this disorder. Since an important consideration in microbiome research is the optimal level (e.g. phylum, genus, species, strain, gene) at which to examine a scientific question, and we are not yet certain what are the optimal levels for psoriasis, this also will be examined. Our studies of psoriasis should allow development of both approaches and tools that will have general utility for microbiome research. To test our hypothesis, we propose the following specific aims: 1. To understand the cutaneous microbiome species composition overlaying psoriatic lesions; 2. To investigate differences in metagenome content for psoriatic lesions compared to normal skin; 3. To identify differences in the transcriptional profiles of the microbiome and the host between normal skin and psoriatic lesions using high-throughput sequencing; and 4. To estimate the effects of systemic immunomodulatory therapy for psoriasis on microbiome composition. In total, these studies should help us understand the role of the microbiome in psoriasis pathogenesis.

The Vaginal Microbiome: Disease, Genetics and the Environment Cynthia Nau Cornelissen, Lindon J Eaves, Jerome Frank Strauss, Gregory A Buck Virginia Commonwealth University

The vagina is an interactive interface with the environment, and as such is covered by a protective epithelial surface. This surface, in turn, is colonized by bacteria and other microorganisms which, through a variety of mechanisms serve to further protect the host from invasion by pathogens. Alterations in the normal vaginal microflora, particularly those associated with bacterial vaginosis, are thought to contribute to risk of spontaneous pregnancy loss in the second trimester and spontaneous preterm birth. Additionally, alterations in the vaginal microbiome may increase the likelihood of transmission of certain agents including human immunodeficiency virus type 1 (HIV-1). There are physiologic alterations in host condition (e.g., menopause and pregnancy), which are beginning to be investigated as potential selective conditions for change in the "normal" flora, and their impact on disease susceptibility and transmission remains to be more definitively elucidated. The effects of chronically abnormal physiologic states (e.g., diabetes mellitus) on normal vaginal flora have not been well described or studied. Finally, an almost unexplored area of inquiry is the genetic contribution, including race/ethnicity, to the establishment and maintenance of a "normal" vaginal flora, under normal and physiologically altered circumstances. The proposed research will address gaps in our knowledge and shed light on how the vaginal microbiome contributes to adverse obstetrical outcomes and sexually transmitted disease in diverse populations. The aims of the project are intended to answer the following questions: Specific Aim 1. Do the genes of the host contribute to the composition of the vaginal microbiome? We hypothesize that a woman's genetic composition significantly affects the ability of certain commensal, parasitic and pathogenic microbes to colonize and/or infect the genital tract. This aim is divided into 2 subaims, the first of which will compare and quantify the microbial populations inhabiting the vaginas of monozygotic (MZ) and dizygotic (DZ) twins in the Mid Atlantic Twin Registry (MATR). The second subaim will address the question of whether there is a relationship between the microbiomes of the vagina, mouth and GI tract utilizing samples collected from the DZ and MZ twins. Specific Aim 2. What changes in the vaginal microbiome are associated with common physiological perturbations or non-infectious pathological states of the host? We hypothesize that "altered" physiologic (pregnancy, menopause) and pathologic (chronic disease, hysterectomy) conditions, or environmental "exposures" (exogenous hormones, antibiotics, chronic immunosuppressant, smoking; douching) can and often do predictably alter the vaginal microenvironment. These alterations in turn will lead to alterations in microbial populations within the vagina. Changes in the microbial populations may have impacts, positive or more likely negative, on the spontaneous and future well-being of the affected individual. We will characterize the effects of these "altered" physiologic and pathologic conditions, and environmental exposures exert on the composition of the vaginal microbiome, and test the hypothesis that they lead to predictable changes in the vaginal microbiome. The relationship between the molecular analysis of the microbiome and laboratory findings based on Amsel's criteria and the Nugent Score will be evaluated. Specific Aim 3. What changes in the vaginal microbiome are associated with relevant infectious diseases and conditions? We will test the hypothesis that infectious diseases predictably alter the vaginal microbiome, and that these changes have an impact on the disease process. We will also test the hypothesis that the vaginal microbiome has an impact on susceptibility to some relevant infectious diseases. The research to be conducted in this Specific Aim will provide a platform for modeling the impact of physiological, pathological, environmental and ethnic/racial factors, and their interactions, in determining the vaginal microbiome. In addition to providing critical descriptive data, this will be a hypothesis-generating Specific Aim. We will analyze the vaginal microbiomes of: normal women of reproductive age; women in the same age range with common pathological conditions (e.g., vaginosis, vaginitis, viral infections, bacterial STDs); women of three different ethnic/racial groups: European Caucasian, African-American, and Mexican Hispanic. We will address these questions using a combination of high throughput 'nextgen' sequencing technologies, including the Roche 454 FLX and the upgraded Illumina Genome Analyzer II instruments currently installed in the Nucleic Acids Research Facilities at VCU.

Diet, Genetic Factors, and the Gut Microbiome in Crohn's Disease Frederic D Bushman, James D Lewis, Gary D Wu University of Pennsylvania

We propose to investigate the hypothesis that consistent changes in the human gut microbiome are associated with Crohn's disease, a form of inflammatory bowel disease, and that altered microbiota contributes to pathogenesis. Analysis of this problem is greatly complicated by the fact that multiple factors influence the composition of the gut microbiota, including diet, host genotype, and disease state. For example, data from others and us document a drastic impact of diet on the composition of the gut microbiome. No amount of sequencing will yield a useful picture of the role of the microbiota in disease if samples are confounded with uncontrolled variables. Our proposed project will take advantage of our experience with deep sequencing to characterize the composition of the gut microbiome, but also control diet, host genotype, and disease state. Diet will be controlled by analyzing children treated for Crohn's disease by placing them on a standardized elemental diet, and by testing effects of different diets on the gut microbiome composition in adult volunteers. Genotype will be analyzed by large scale SNP genotyping, which is already underway and separately funded team member Hokan Hakonarson is currently genotyping 50 children a week at ~half a million loci each and investigating connections with inflammatory bowel disease. Clinical status will be ascertained in the very large IBD practice in the UPenn/CHOP hospital system. Effects of diet, host genotype, and disease state on the gut microbiome will be summarized in a multivariate model, allowing connections between microbiome and disease to be assessed free of confounding factors.

The Role of the Gut Microbiota in Ulcerative Colitis Eugene B Chang, Folker Meyer, Thomas M Schmidt, Mitchell L Sogin, James M Tiedje, Vincent B Young University of Michigan at Ann Arbor

The indigenous microbiota of the gastrointestinal tract is the richest of the human associated microbial communities in terms of absolute biomass and relative diversity. Although the gut microbiota play critical roles in the maintenance of gut homeostasis, evidence is accumulating that disturbances in the gut microbial community ("dysbiosis") underlie a number of gastrointestinal disease states including antibiotic-associated diarrhea and inflammatory bowel disease (IBD). To address our hypothesis we propose to determine the relationship between the gut microbiome and the development of mucosal inflammation in patients with ulcerative colitis. We will coordinate and integrate existing resources for the study of IBD patients, metagenomic analysis, molecular microbial diversity analysis and targeted sequence-aided bacterial culture into a research pipeline for the study of complex host-associated microbial communities. We will characterize the gut microbiota in human patients who have undergone total colectomy with the construction of an ileal pouch anal anastomosis for treatment of ulcerative colitis. These patients will be followed temporally after surgery to monitor for the development of mucosal inflammation in the pouch anastomosis ("pouchitis"). Comparison within patients over time and between patients will be done to associate specific ecologic and functional characteristics of the gut microbiome with the development of pouchitis. To accomplish these goals, we have assembled an experienced, integrated team of researchers from a variety of disciplines. We will study a carefully selected group of patients with ulcerative colitis that will permit us to make inter- and intra-patient comparisons over time. Though the use of high-throughput, cost effective technologies, which members of this team have played a key role in developing, we intend to demonstrate a critical role for the gut microbiome in the development of the dysregulated mucosal immune response that is the hallmark of IBD. This knowledge will lead the way to novel ways to monitor patients with IBD and to the development of novel preventative and therapeutic interventions.

Urethral Microbiome of Adolescent Males Dennis J Fortenberry Indiana University-Purdue University at Indianapolis

The microflora of the anterior male urethra during adolescence is poorly described and no data address the range of "typical" urethral microflora during adolescence as functions of pubertal development or onset of various types of partnered sexual activity. This means that current understanding of the urethral microbiome in adolescent men rests entirely on data drawn from adults, and from epidemiologic and behavioral explanations. Marked ethnic group variation in adolescent sexual behaviors and disparity in conditions such as sexually transmitted infections (STI) justifies exploration of potential ethnic group differences in the male urethral microflora. Some research among adult males suggests important interactions between the male urethral microflora and STI, but this research has not been extended to younger men. Here we propose to characterize the male urethral microbiome in longitudinal urine samples collected from a multi-ethnic cohort of adolescent males. Microbial flora will be assessed as function of pubertal development, sexual activity, and incident STI. Subjects will be Latino (N=24), African-American (N=24), and Euro-American (N=24) males, ages 14 - 17 at enrollment; total N=72). Participants will be recruited from an urban clinic within an area of high prevalence of STI. Socio-demographic data will be collected at enrollment. Daily behavioral and symptomologic data will be collected via cell phone, which will also be used for cohort maintenance and scheduling of sample collections. Urine will be collected at enrollment and up to 36 1-month follow-up samples for characterization of microflora using high-throughput 16S rRNA phylogeny, Q-PCR and metagenomic sequencing. STI testing will be done as well to better understand interactions between the urethral microbiome and infection. Cell phones also will be used to initiate daily sample collection for 10 days following trigger events such as specific sexual acts, antibiotic use, condom failure, or genital symptoms.

The Thrifty Microbiome: The Role of the Gut Microbiota in Obesity in the Amish Alan R Shuldiner, Claire M Fraser University of Maryland Baltimore

Emerging evidence that the gut microbiota may contribute in important ways to human health and disease has led us and others to hypothesize that both symbiotic and pathological relationships between gut microbes and their host may be key contributors to obesity and the metabolic complications of obesity. Our "Thrifty Microbiome Hypothesis" poses that gut microbiota play a key role in human energy homeostasis. Specifically, constituents of the gut microbial community may introduce a survival advantage to its host in times of nutrient scarcity, promoting positive energy balance by increasing efficiency of nutrient absorption and improving metabolic efficiency and energy storage. However, in the presence of excess nutrients, fat accretion and obesity may result, and in genetically predisposed individuals, increased fat mass may result in preferential abdominal obesity, ectopic fat deposition (liver, muscle), and metabolic complications of obesity (insulin resistance, hypertension, hyperlipidemia). Furthermore, in the presence of excess nutrients, a pathological transition of the gut microbial community may occur, causing leakage of bacterial products into the intestinal lymphatics and portal circulation, thereby inducing an inflammatory state, further aggravating metabolic syndrome traits and accelerating atherosclerosis. This pathological transition and the extent to which antimicrobial leakage occurs and causes inflammatory and other maladaptive sequelae of obesity may also be influenced by host factors, including genetics. In the proposed study, we will directly test the Thrifty Mirobiome Hypothesis by performing detailed genomic and functional assessment of gut microbial communities in intensively phenotyped and genotyped human subjects before and after intentional manipulation of the gut microbiome. To address these hypotheses, five specific aims are proposed: (1) enroll three age- and sex-matched groups from the Old Order Amish: (i) 50 obese subjects (BMI > 30 kg/m2) with metabolic syndrome, (ii) 50 obese subjects (BMI > 30 kg/m2) without metabolic syndrome, and (iii) 50 nonobese subjects (BMI < 25 kg/m2) without metabolic syndrome and characterize the architecture of the gut microbiota from the subjects enrolled in this study by high-throughput sequencing of 16S rRNA genes; (2) characterize the gene content (metagenome) to assess the metabolic potential of the gut microbiota in 75 subjects to determine whether particular genes or pathways are correlated with disease phenotype; (3) characterize the transcriptome in 75 subjects to determine whether differences in gene expression in the gut microbiota are correlated with disease phenotype, (4) determine the effect of manipulation of the gut microbiota with antibiotics on energy homeostasis, inflammation markers, and metabolic syndrome traits in 50 obese subjects with metabolic syndrome and (5) study the relationship between gut microbiota and metabolic and cardiovascular disease traits, weight change, and host genomics in 1,000 Amish already characterized for these traits and in whom 500K Affymetrix SNP chips have already been completed. These studies will provide our deepest understanding to date of the role of gut microbes in terms of 'who's there?', 'what are they doing?', and 'how are they influencing host energy homeostasis, obesity and its metabolic complications?

Metagenomic Analysis of the Structure and Function of the Human Gut Microbiota in Crohn's Disease Claire M Fraser University of Maryland Baltimore

Inflammatory bowel diseases (IBD), such as Crohn's disease, are chronic, immunologically mediated disorders that have severe medical consequences. The current hypothesis is that these diseases are due to an overly aggressive immune response to a subset of commensal enteric bacteria. Studies to date on IBD have suggested that the disorder may be caused by a combination of bacteria and host susceptibility; however the etiologies of these diseases remain an enigma. In this application, we propose to develop and demonstrate the ability to profile Crohn's disease at an unprecedented molecular level by elucidation of specific biomarkers (bacterial strains, genes, or proteins) that correlate to disease symptoms. To achieve this goal, we will employ a multidisciplinary approach based on metagenomic and metaproteomic molecular tools to elucidate the composition of the commensal microbiota in monozygotic twins that are either healthy or exhibit Crohn's disease (for concordant, both are diseased; for discordant, one is healthy and one is diseased). The central hypotheses of this proposal are (1) that specific members and/or functional activities of the gastrointestinal (GI) microbiota differ in patients with Crohn's disease as compared to healthy individuals, and (2) that it will be possible to elucidate microbial signatures which correlate with the occurrence and progression of this disease by integration of data obtained from 16S rRNA-based molecular fingerprinting, metagenomics, and metaproteomics approaches. To address these hypotheses, five specific aims are proposed: 1) Obtain data on community gene content (metagenome) in a subset of healthy twins and twins with Crohn's Disease to assess potential differences in the metabolic capabilities of the gut microbiota associated with CD, 2) Obtain data on community protein content (metaproteome) in a subset of healthy twins and twins with Crohn's Disease to assess the state of expressed proteins associated with CD, 3) Prospectively obtain biopsy and fecal samples from a cohort of 80 patients undergoing surgical resections for the treatment of inflammatory bowel according to HMP guidelines, 4) Extend our efforts in molecular fingerprinting, metagenomics, and metaproteomics to a larger population to further explore the relationship between changes in gut microbial community structure and function in CD. These studies will include: (i) completion of the metagenomic and metaproteomic characterization of the gut microbiota from the Swedish monozygotic twins, (ii) evaluation of gut microbiota stability over time, as measured in fecal samples from a group of individuals from the Swedish twin registry using molecular fingerprinting approaches, (iii) molecular fingerprinting and metaproteomic comparison of microbial community composition in intestinal biopsies from multiple CD patient cohorts, and (iv) molecular fingerprinting and metagenomic analysis of the less abundant members of the gut microbiota in healthy individuals and CD patients, and 5) Apply various statistical clustering and classification methods to correlate/associate microbial community composition, gene and protein content with patient metadata, including metabolite profiles and clinical phenotype. The ultimate goal of these efforts is to identify novel biomarkers for non-invasive diagnostics of CD and to eventually identify drug targets (i.e. bacterial strains) for cure or suppression of disease symptoms.

Effect of Crohn's Disease Risk Alleles on Enteric Microbiota Ellen Li Washington University

Abnormal host-microbial interactions are implicated in the pathogenesis of inflammatory Bowel disease (IBD). Crohn's disease (CD) risk alleles, such as the NOD2 and ATG16L1, have been associated with defective host containment of commensal bacteria. We hypothesize that these CD risk alleles contribute to compositional changes in intestinal microbiota. These changes in microbiota might reduce the proportion of protective commensal organisms and increase the numbers of proinflammatory organisms that incite intestinal inflammation. We propose to take a metagenomic approach towards testing this hypothesis in three Specific Aims: 1.) Analyze the effect of CD-risk alleles on mucosal associated microbiota in GI tissues collected from ileal CD, non-ileal IBD and non-IBD patients; 2. ) Develop and test novel genetic probes for pro-inflammatory commensal organisms; 3.) Expand prospective collection of GI tissues in four major IBD centers. During the UH2 pilot year, we will focus our study on 755 existing GI tissues from 364 subjects already collected at three institutions. High throughput 16S rRNA sequence analysis of the combined ileal tissues and high throughput genotyping for all highly reproducible CD risk alleles (~30, including NOD2 and ATG16L1) will be performed at Washington University. Quantitative PCR will be performed to quantify specific microbial subgroups. Multivariate analysis of the metagenomic data and the genotyping data will include potentially confounding variables such as age, disease state, medications and smoking. The adherent invasive E. coli (AIEC) are a novel group of bacterial strains that have been implicated as candidate "pathogenic" organisms that incite intestinal inflammation. Because there is limited genome information on AIEC strains, we will produce genome sequences of AIEC strains isolated from the GI tissues. During the UH3 phase of the project, prospective collection of GI tissues from 700-800 ileal CD, non-ileal IBD and non-IBD patients a year will be conducted at four major IBD centers: Washington University, University of North Carolina, Cleveland Clinic, and Mount Sinai Medical Center. Shotgun sequencing will be performed on selected fecal samples linked to the ileal tissues to identify additional, or auxiliary, or synergistic pathogenic factors or other functional changes in the microbiome that are contributed by uncultivatable organisms. Genetic probes for pathogenic commensal organisms developed by mining the bacterial genome data and shotgun sequencing data will be used to test the hypothesis that increased numbers of these organisms are associated with dysbiosis and/or CD risk alleles. Our combined expertise in multiple disciplines across multiple institutions, our demonstrated ability to collect a large number of well-phenotyped samples with longitudinal clinical information that will be linked to host response profiling and morphologic studies, and our consortium's capacity for high-throughput sequencing will be used to investigate how alterations in the human microbiome relate to CD risk alleles and CD pathogenesis.

Metagenomic study of the human skin microbiome associated with acne Huiying Li University of California Los Angeles

The human microbiota contributes to our normal postnatal development and plays a significant role in defining our physiology. To understand the role of microbiota in human health and disease, we propose to study the skin microbiome in pilosebaceous units (hair follicles) and its association with acne. Acne is one of the most common skin diseases. Although its etiology still needs to be defined, a bacterial factor has been suggested in the development of the disease. In fact, antibiotic therapy targeting Propionibacterium acnes has been a mainstay treatment for more than 30 years. Our preliminary study shows that the microcomedone, a specialized skin compartment where acne arises, has a tractable microbiome, with a single dominant species, P. acnes. This system offers a unique advantage allowing in-depth analysis of a human microbiome at the subspecies level by DNA sequencing, whereas the microbial communities at many other body sites are just too complicated to deconvolute even at the species level. In our preliminary study, we find that P. acnes strains in acne patients appear to be clonal, with distinct nucleotide signatures of 16S rDNA sequences, whereas P. acnes strains from normal individuals are much more diverse and scatter through many lineages. This indicates that the microbiome associated with acne offers promise for understanding the correlation between the composition of the microbiome and human health and disease. In this project, we plan to investigate in three directions the microbiome associated with acne. First, we plan to investigate the strain diversity of P. acnes in a disease cohort and normal cohort and examine whether certain strains of P. acnes are correlated with disease. Second, we plan to investigate the non-P. acnes microbes in microcomedones and disease lesions and examine whether they correlate with acne pathogenesis. These microbes include small eukaryotes, bacteria, viruses and bacteriophages. Third, we will examine the interactions between the microbes and the host by transcriptional profiling of both the microbiota and the host. By the end of the first year of this project, we plan to complete the sequencing of 10,000 16S rDNA clones and 48 full genomes of P. acnes strains. We believe our analysis of the microbiome of the pilosebaceous units will shed light on the microbial composition of the skin biota and its relation to acne.

Foregut microbiome in development of esophageal adenocarcinoma Karen E Nelson, Zhiheng Pei New York University School of Medicine

Esophageal adenocarcinoma (EA), the type of cancer linked to heartburn due to gastroesophageal reflux diseases (GERD), has increased six fold in the past 30 years, which cannot be explained by the usual environmental or host factors. EA is the end result of a sequence of GERD-related diseases, preceded by reflux esophagitis (RE) and Barrett's esophagus (BE). Our preliminary study in elderly male veterans found two types of microbiotas in the esophagus. Patients who carry the type II microbiota are >15 fold likely to have esophagitis and BE than those harboring the type I microbiota. In a small-scale study, we also found that 3 of 3 cases of EA harbored the type II biota. The findings have opened a new approach to understanding the recent surge in the incidence of EA. Our long-term goal is to identify the cause of GERD sequence. The hypothesis to be tested is that changes in the foregut microbiome are associated with EA and its precursors, RE and BE in GERD sequence. We will examine whether the finding in elderly male subjects also applies to younger as well as female subjects. We will conduct a case control study to demonstrate the microbiome-disease association in every stage of GERD sequence as well as analyze the trend in changes in the microbiome along disease progression toward EA, by two specific aims. Aim 1 is to conduct a comprehensive population survey of the foregut microbiome and demonstrate its association with GERD sequence. Furthermore, spatial relationship between the esophageal microbiota and upstream (mouth) and downstream (stomach) foregut microbiotas as well as temporal stability of the microbiome-disease association will also be examined. Aim 2 is to define the distal esophageal metagenome and demonstrate its association with GERD sequence. Detailed analyses will include pathway-disease and gene-disease associations. Archaea, fungi and viruses, if identified, also will be correlated with the diseases. A significant association between the foregut microbiome and GERD sequence, if demonstrated, will be the first step for eventually testing whether an abnormal microbiome is required for the development of the sequence of phenotypic changes toward EA. If EA and its precursors represent a microecological disease, treating the cause of GERD might become possible, for example, by normalizing the microbiota through use of antibiotics, probiotics, or prebiotics. Causative therapy of GERD could prevent its progression and reverse the current trend of increasing incidence of EA.

The Microbial Ecology of Bacterial Vaginosis: A Fine Scale Resolution Metagenomic Jacques Ravel University of Maryland Baltimore

The vaginal microbiota play an important protective role in maintaining the health of women. Disruption of the mutualistic relationship that exists between bacterial communities in the vagina and their hosts can lead to bacterial vaginosis (BV), a condition in which lactic acid producing bacteria are supplanted by a diverse array of strictly anaerobic bacteria. BV has been shown to be an independent risk factor for adverse outcomes including preterm delivery and low infant birth weight, acquisition of sexually transmitted infections and HIV, and development of pelvic inflammatory disease. National surveys indicate the prevalence of BV among U.S. women is 29.2%, and yet, despite considerable effort, the etiology of BV remains unknown. Moreover, there are no broadly effective therapies for the treatment of BV, and reoccurrence is common. In the proposed research we will test the overarching hypothesis that vaginal microbial community dynamics and activities are indicators of risk to BV. To do this, we propose to conduct a high resolution prospective study in which samples collected daily from 200 reproductive-age women over two menstrual cycles are used to capture molecular events that take place before, during, and after the spontaneous remission of BV episodes. We will use modern genomic technologies to obtain the data needed to correlate shifts in vaginal microbial community composition and function, metabolomes, and epidemiological and behavioral metadata with the occurrence of BV to better define the syndrome itself and identify patterns that are predictive of BV. The five specific aims of the research are: (1) Evaluate the association between the dynamics of vaginal microbial communities and risk to BV by characterizing the community composition of vaginal specimens archived from a vaginal douching cessation study in which 39 women self-collected vaginal swabs twice-weekly for 16 weeks; (2) Enroll 200 women in a prospective study in which self-collected vaginal swab samples and secretions are collected daily along with data on the occurrence of BV, vaginal pH, and information on time varying habits and practices; (3) Determine the gene content (metagenome) of vaginal microbial communities to assess the metabolic potential of representative vaginal communities in women before, during, and after the spontaneous remission of BV; (4) Characterize suites of expressed genes (metatranscriptome) in communities representative of vaginal community types in healthy women, as well as before, during, and after the spontaneous remission of BV; and (5) Apply model-based statistical clustering and classification approaches to associate the microbial community composition and function, with metadata and clinical diagnoses of BV. The large body of information generated will facilitate understanding of vaginal microbial community dynamics, the etiology of BV, and drive the development of better diagnostic tools for BV. Furthermore, the information will enable a more personalized and effective treatment of BV and ultimately, prevent adverse sequelae associated with this highly prevalent disruption of the vaginal microbiome.

Skin Microbiome in Disease States: Atopic Dermatitis and Immunodeficiency Julia Segre U.S. National Human Genome Research Inst

No information available at this time.

The human virome in children and its relationship to febrile illness Gregory A Storch Washington University

Definition of the human microbiome is an important scientific priority. This study will expand the scope of the investigation to include viruses, which account for a substantial proportion of infectious disease morbidity and mortality, especially in children. The long-term goal of this project is to describe the human virome in children and to investigate its relevance to febrile illnesses in children. The project will also seek to understand the relationship of the immune system to the composition of the virome. Thus, the project's specific aims are 1) To elucidate the spectrum of viruses that can be detected using non-biased, high throughput sequencing on samples of blood, respiratory, and gastrointestinal secretions from healthy children and to use this information as a basis for understanding the role of viruses in acute febrile illnesses without an obvious source, and 2) to investigate the effect of various forms of immunosuppression on the spectrum of viruses detected in children, and to use this information as a basis for understanding the role of viruses in acute febrile illnesses occurring in these children. Our preliminary studies show that diverse viruses can be detected in children having undiagnosed fever. To carry out the specific aims, well children will be enrolled prior to having elective surgery, and febrile otherwise well children will be enrolled from the Emergency Department at St. Louis Children's Hospital. Immunocompromised children will be recruited from hematopoietic stem cell and solid organ transplant clinics, the HIV/AIDS clinic, and the rheumatology/immunology clinic from the same hospital. Children with fever will have samples obtained at the time of the febrile illness and at 1 and 6-month follow-up visits. Selected samples from each study group will be analyzed at the Genome Center at Washington University (GCWU) using next generation 454 high throughput sequencing to detect and sequence all viral sequences present. We anticipate detecting and sequencing a broad range of viruses, including previously unrecognized agents. A variety of techniques will be used to investigate the significance of viruses detected. Virus-specific PCR assays will be used to determine the frequency and extent of viruses detected by sequencing, using the full range of samples collected. Host response to the detected viruses will be investigated using serologic analysis, cytokine profiling, and microarrays to characterize host gene expression. These studies will take advantage of follow-up samples to compare the acute response with the response in the convalescent period. This study will draw upon the expertise and technological assets of one of the world's most powerful sequencing centers to provide the research community with a comprehensive sequence database of the viruses that are present in children, which can be used to improve our understanding of the causes of febrile illnesses in young children, many of which are currently undiagnosed.

The Neonatal Microbiome and Necrotizing Enterocolitis Phillip I Tarr Washington University

Necrotizing enterocolitis (NEC) is a devastating disorder that affects approximately 10% of premature infants. Its mortality remains high (15-30%), and its cause remains unknown. About 80% of cases occur within 35 days of birth among hospitalized newborns of low birth weight. Probiotics diminish the incidence and severity of NEC, and NEC does not occur antepartum. NEC affects a readily identifiable at-risk group, has a tightly defined interval before its onset, occurs in an organ system that is intimately associated with a microbial population in flux, has a plausible association with the intestinal microbiota, and cohorts at risk have rarely been studied in large numbers, or prospectively. This disorder, therefore, provides a unique opportunity to explore the role of the human enteric microbiome in a devastating disease. Moreover, NEC epidemiology and age-incidence present an ability to enroll and study cohorts that are highly likely to provide valuable pathophysiologic and microbiologic insights. In this project, we will identify and quantify the microbial components of stool and its products before and at the onset of NEC. In doing so, we will test the overarching hypothesis that NEC is a direct or indirect consequence of the enteric biomass, its products, or both. We will use multicenter cohorts of premature infants at high risk of developing NEC, extend our research on this disease currently sponsored by the Washington University Institute of Clinical and Translational Sciences, and continue our longstanding collaborations with the Genome Center at Washington University and the Washington University Digestive Diseases Research Core Center (Informatics Core). The Aims of this proposal are to (1) conduct a case cohort study in which we compare clinical data and biological specimens from cases and well-matched controls; (2) determine if the kind and density of intestinal biomass, its gene content, and transcriptional activity are associated with, and potential determinants of, NEC; and (3) determine if host risk alleles for intestinal inflammation play a role in the development of NEC. These efforts will be accomplished using subjects from three collaborating neonatal intensive care units (NICUs), focusing on the critical, instructive, and understudied pre-NEC stage of illness, and formulating a data repository that will be a resource for investigators worldwide who wish to focus their efforts on NEC, its precipitants, and its prevention and cure.

The Human Microbiome in Pediatric Abdominal Pain and Intestinal Inflammation James Versalovic Baylor College of Medicine

This proposal will explore the nature of the human intestinal microbiome in healthy children and children with gastrointestinal (GI) disorders. The overall goal is to obtain a robust knowledge-base of the intestinal microbiome in a set of GI disorders that represent a broad spectrum of important disease phenotypes in pediatric gastroenterology. In addition to the detailed clinical assessment of healthy children and children with irritable bowel syndrome, constipation, and inflammatory bowel disease (Crohn disease), multiple strategies will be deployed to navigate and understand the nature of the intestinal microbiome in childhood. These strategies will include Sanger sequencing and pyrosequencing-based strategies to understand the detailed composition of microbes in healthy and disease groups. Whole genome shotgun sequencing will be used as an exploratory strategy to explore metagenomes in patients in a comprehensive manner. Microarray-based hybridization with the PhyloChip, denaturing HPLC, quantitative real-time PCR, and bacterial fluorescence in situ hybridization probes will be applied as complementary strategies to gain an understanding of the intestinal microbiome from various perspectives in molecular microbiology. The first hypothesis is that healthy children have a core, identifiable microbiome. The second hypothesis is that disease-specific signatures in the human microbiome are present, and these microbial signatures may be correlated with pediatric gastrointestinal disease phenotypes. This proposal will explore the nature of core and variable human microbiomes in pre-adolescent healthy children and children with GI disorders. Finally, spatial architecture of intestinal microbes and human factors will be studied in order to examine higher-order alterations in microbial communities in different disease states and the relative contributions of human immune response genes.

Member Organizations