Our microbiome contains essential bacteria that carry out key metabolic reactions that are necessary for our health but are not encoded for in the human genome. Yet these same beneficial bacteria can make us vulnerable for developing healthcare associated infections (HAI or nosocomial infections).

The CDC estimates there are 1.7 million HAI in the United States each year and almost 100,000 associated deaths. The bacteria causing these infections can be endogenous (part of our existing bacterial flora at admission) or exogenous (acquired during the hospital stay). Some of the problems caused by our existing bacteria (endogenous) of the anterior nares (e.g. S. aureus), skin (e. g. coagulase-negative Staphylococci,) and posterior pharynx (e.g. Klebsiella spp.) include surgical site infections, central-line associated blood stream infections and pneumonia. For example, 80-85% of S. aureus infections in people who are S. aureus colonized are due to the patient's endogenous isolate. The human microbiome of patients is the source of the majority of healthcare associated infections, coming from either endogenous or exogenous sources. The public reporting of HAI has fueled new initiatives to prevent healthcare associated infections. One such healthcare policy initiative is "decolonization", which is applying targeted or non-targeted antimicrobials to the skin and mucosal surfaces. Given the role of our normal flora as a barrier to infection, decolonization regimens could have unintended negative consequences.

IGS scientists are working with Dr. Mary-Claire Roghmann (U. of Maryland School of Medicine) to better understand the microbiome of the anterior nares and upper airways in populations with and without current exposure to the healthcare environment. These comparisons will give us a better understanding of the changes associated with decolonization regimens within individuals in both populations. The long term goal of this research is to use the information gained to develop new ways for better management of the human microbiome during hospital visits. Scientists and medical researchers would like to reduce the risk of healthcare associated infections without the negative consequences that can occur with strategies like decolonization.

IGS scientists are also studying with Dr. Aaron Milstone (Johns Hopkins University, Baltimore) the microbiome of the anterior nares in neonates, in newborn nurseries and neonatal intensive care units (NICUs), where, despite decades of research, S. aureus remains a major threat as the second leading cause of central-line associated bloodstream infection (CLABSI), ventilator-associated pneumonia, and surgical site infections. Rates of invasive S. aureus infections in neonates far exceed the rates of infection in older children and adults. S. aureus can colonize the nasal cavity and colonization is associated with an increased risk of subsequent infection. Intranasal antibiotics and antiseptic agents have been used to decolonize children in an attempt to prevent S. aureus disease. Unfortunately, comprehensive infection control strategies including decolonization have been insufficient at preventing S. aureus disease in hospitalized infants and children. Our long-term goal is to better understand the nasal microbiota and its association with acquisition of S. aureus colonization to inform new strategies for the prevention of S. aureus disease in neonates.

The most important goal in transplantation is to achieve prolonged allograft survival, and eventually alloantigen specific tolerance. Yet, the occurrence of allograft vascular inflammation and fibrosis has not changed in over two decades. In preliminary studies we characterized gut microbiotas and identified single bacterial species that influence myeloid cell responses, lymph node structure, and the outcomes of murine cardiac allografts. The proposed work will use this murine model of cardiac allografting to further identify the functional mechanisms by which proinflammatory and anti-inflammatory microbiota alter adaptive immunity and ultimately allograft survival.

Many aspects of the innate and adaptive immunity are critically regulated by the microbiota. Microbial cells, their metabolites and nucleic acids engage various immune cells, resulting in pro- or anti-inflammatory signals that differ based on chemical structures, cellular receptors, and physiological context. The microbiota not only influences local immunity, but also has distant effects on systemic immunity. Local microbiota stimulation of innate and adaptive immune cells results in those cells or their products to migrate or traffic through lymphatics or blood, and influence diseases. However, the precise causal pathways linking microbiota components to immune cells and downstream effectors in most cases remain to be defined.

Solid organ transplantation has made significant progress over the past 35 years and has become a routine procedure. Cardiac transplantation is a common and successful transplant, with graft survival after one year exceeding 80-90%. Despite advances in all aspects of allografting, the rate of decline of cardiac and other graft function beyond the first year after transplant has not changed in over 20 years. All allografts eventually succumb to chronic vascular, interstitial or epithelial changes. Despite critical improvements in immunosuppressive regimens, immunologic monitoring, and molecular classification of organ pathology, chronic rejection still persists and its primary cause is not understood. Prior work has focused on distal events of fibrosis and inflammation, but not on proximal causes of inflammation and immunity.

We previously showed in renal transplantation, large and persistent shifts in the composition and complexity of the gut microbiota as a result of immunosuppression and antibiotics. Such shifts in the microbiota are indicative of all organ transplants, including cardiac transplants. We therefore hypothesized that these changes could critically affect graft outcome. Our current studies dissected the interactions between the enteric microbiota and innate and adaptive immunity, in clinically-relevant cardiac transplantation models of acute and chronic rejection. Our results show that both pro-inflammatory and anti-inflammatory microbiota populations, as well as single bacteria, can be defined by their effects on the long-term outcome of the grafts. Mechanistic explorations suggest a differential stimulation of myeloid cells (i.e. macrophages and DC), resulting in changes in LN structure that influence allogeneic immunity. Thus, we hypothesize that the microbiota directly regulates innate immunity, which in turn regulates systemic inflammation and adaptive immunity, thereby determining the occurrence and progression of graft fibrosis, inflammation and graft survival. To investigate this hypothesis, we will take advantage of our expertise in microbiota analysis and in molecular and cellular transplant immunology. The definition of pro-inflammatory and anti-inflammatory microbiota and strains may provide a precise platform to define the most important upstream influences that initiate organ inflammation and scarring and could serve as potent diagnostic markers for allograft management.

The project was funded by the National Institutes of Health (NIH) - National Heart, Lung and Blood Institute (NHLBI): Project information can be found here.

Vaginal discharge, burning, itching, and malodor are the most common vaginal symptoms reported by reproductive-age women resulting in millions of health care visits annually in the United States alone. These symptoms create substantial discomfort, are often recurrent, and negatively impact women’s self- esteem and quality of life. Often, women resort to ill-advised alternative treatments in an attempt to ameliorate their symptoms that only provide a short-lived relief, and in many cases increase symptom severity. These symptoms are associated with numerous serious gynecological and obstetric outcomes, including an increased risk for sexually transmitted infections. The long-term goal of the proposed work is to develop a more complete understanding of the underlying causes of vaginal symptoms so that targeted and effective strategies can be developed to effectively prevent or treat vaginal discharge, discomfort, and malodor. The multi-omics datasets we propose to gather and analyze will allow us to test the central hypothesis that symptoms are emergent properties of the vaginal microbiome that result from the interplay of specific functions, and not simply microbial composition of vaginal microbiomes and host responses elicit these signs and symptoms. To achieve this, we will leverage a unique set of vaginal swabs samples and extensive metadata that were prospectively collected daily by 135 women for 10 weeks. During this study, women experienced either 1) clear episodes of vaginal symptoms, 2) chronic and persistent symptoms or 3) no symptoms. The study design affords a unique opportunity to use ‘omics technologies on samples collected before, during and after episode of vaginal symptoms and compare these to women with chronic or no symptoms, and identify specific predictive biomarkers that will translate to more personalized management of women’s health. The project addresses two specific aims: (1) Determine the composition and function of the vaginal microbiome by characterizing bacterial metagenomes, the host and bacterial transcriptomes and metabolomes, and markers of innate immunity in vaginal samples collected prior to, during and after episodes of vaginal symptoms and compare these to comparable data from asymptomatic women. (2) Develop predictive and causal models of symptom onset using integrative systems biology approaches using a multi- prong modeling strategy that includes lasso regression, ridge regression and elastic net regression combined with robust and modern model selection techniques, and network analyses, we will achieve a predictive and causal model that is a balance of robustness, explanatory power, and size (in terms of the number of predictor variables). Armed with detailed knowledge of changes in genomic factor of the microbiota and the host during the onset and recovery from vaginal symptoms it will be possible to develop more accurate and sensitive diagnostic procedures, new therapeutic strategies, and effective means to ensure vaginal homeostasis.

This research reported is supported by the National Institute of Allergy and Infectious Diseases, of the National Institutes of Health.

Revealing the Role of the Cervico-Vaginal Microbiome in Spontaneous Preterm Birth

Worldwide, 13 million infants are born preterm each year. In the United States, approximately 1 in 8 live births are preterm. The economic burden from prematurity is enormous costing over 28 billion dollars a year. These costs are not just for immediate neonatal care but also for long term care of these preterm children who are at increased risk for a spectrum of medical and neurobehavioral disorders. Currently, we have no effective strategies to predict or to prevent the majority of preterm births (PTBs). Based on the current model, key events in the pathogenesis of PTB are uterine infection and contractility. Assuming this model to be valid, the only strategies to decrease the risk for PTB are to monitor for increased uterine contractility and/or to treat the presumed underlying infectious process. Unfortunately, clinical trials attempting to treat or prevent uterine contractions and/or to treat infections have failed. Based in part on new findings linking, for example, dysbiosis of microbial communities (microbiota) inhabiting the human gastrointestinal tract to disease states through immune responses at the mucosal barriers, we propose a revised paradigm for the pathogenesis of PTB. Whereas PTB result from microbiota dysbiosis in the cervicovaginal (CV) compartment leading to a local immune response and altered tissue (cervix) homeostasis. The dysregulated immune response, mediated by the CV epithelial barrier, promotes changes in the cervix which leads to PTB. Furthermore, we propose that certain factors (e.g. behavior, social, environmental) known to mildly increase the risk for PTB, actually serve to alter the CV microbiota; thus, contributing to the pathogenesis of PTB. We hypothesize that microbial communities in the CV space will be stably composed of Lactobacillus spp. throughout pregnancy in women who are destined to have a term delivery. However, we propose that microbial communities will be less stable and enter states depleted of Lactobacillus spp. in women destined to have a PTB. Furthermore, specific microbial communities will induce an unfavorable immune response from the CV mucosal barrier; thus, microbial communities will be found to play a mechanistic role in PTB. This new paradigm will dramatically alter clinical care by allowing the identification, through the characterization of the CV microbiota, of the majority of women at risk-months prior to a clinical event- thus, providing opportunities for low-risk, feasible interventions. We have assembled an interdisciplinary team of experts in the field of reproductive medicine, microbiology, and perinatal nursing. We propose to assemble a prospective cohort, enriched with women with a prior PTB, that will allow for 3 longitudinal assessments during the 2nd and early 3rd trimester. At each study time point, we will determine the CV microbiota, the CV host immune response and determine if there are any molecular changes in the cervix consistent with premature cervical remodeling. In addition, we will comprehensively assess if behavioral, social and/or environmental factors modify the CV microbiota and/or its association with PTB.

This research reported is supported by the National Institute of Nursing Research, of the National Institutes of Health

Bacterial vaginosis (BV) is typified by vaginal discharge, discomfort, and malodor, and constitutes the most common vaginal complaint of reproductive-age women, resulting in millions of health care visits annually in the United States alone. The prevalence of BV ranges from 29% in the US to over 50% of women in rural Ugandan villages. The disease is characterized by an elevated pH and vaginal communities with reduced proportions of Lactobacillus sp. and increased proportions of strict anaerobes including species of Atopobium, Gardnerella, Prevotella, and other taxa of the order Clostridiales. A wealth of evidence indicates that BV and high Nugent scores (used in the diagnosis of BV) are independent risk factors for severe reproductive tract and obstetric sequelae, including preterm delivery and low birth weight as well as the acquisition of sexually transmitted infections, including HIV. Despite the high prevalence of this condition and the associated risk for severe adverse outcomes, the etiology of BV remains unknown. Interventions to treat BV focus on the use of antibiotics, but the effectiveness of such treatments is disappointingly low, and relapse is common. Our recent work characterizing the temporal dynamics of the vaginal microbiota demonstrated that vaginal bacterial communities are highly dynamic and undergo rapid fluctuations in and out of BV-like states (9, 31). The frequencies and duration of these fluctuations do not appear to correlate with specific members of the community or behaviors, and the underlying biological causes of these fluctuations remain unknown. What we have learned from environmental and laboratory studies is that bacteriophages are efficient and highly specific predators which have the potential to quickly decimate a population of sensitive bacterial hosts and consequently alter the composition of co-existing microbial communities. One hallmark of BV is rapid changes in the abundance of bacterial populations leading to a vaginal bacterial community responsible for disease outcomes. Thus, in this research we are leveraging a unique combination of scientific expertise to address the hypothesis that bacteriophage infection is a contributing underlying biological causative factor capable of rapidly altering the composition of vaginal bacterial communities, changes that ultimately lead to BV.

Abundant lactobacilli in the human vagina are thought to protect against invasion by non-indigenous bacteria, including sexually transmitted infections caused by Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (GC). The means by which this happens are not well understood. It could be that these exclusionary mechanisms are properties of the vaginal microbiome, features of the host immune system and physiology, or some combination of both. The goal of this project is to employ a systems biology approach to identify biomarkers of the vaginal and penile microbiome, the host and the pathogens that are associated with increased or decreased risks of infection by CT, GC or both. Project 3 of this research program will rely on samples collected by the Clinical Core C from STING networks of sex partners who have been exposed to and possibly infected by CT, GC, or both. In these networks we expect that about 20-40% of the participants will have been exposed to, but not infected by these pathogens. This will give us the unique opportunity to assess the role of the microbiome in preventing or facilitating infections by CT and GC. Our overarching hypothesis is that when pathogen transmission does not occur the genetic traits of the infecting pathogen(s) may be insufficient to overcome the host response or the exclusionary mechanisms of the microbiome environment; or that features of the microbiome are protective or induce a protective mucosal environment. In this project, we will build on these findings and use modern ‘omic technologies to identify specific functional features of the vaginal and penile microbiota associated with susceptibility and resistance to infection and co-infection and the importance of host and pathogen genetic variation in this infection process, which will be done in collaboration with Projects 1 & 2. We will achieve these goals by addressing three integrated specific aims: Aim 1. Characterize the genomic variations in CT/GC in participants of the STING networks of sex partners; Aim 2. Use ‘omic approaches and system biology analysis characterize the molecular interactions between the host, the pathogens and the genital microbiota in discordant and concordant couples for CT/GC infections; Aim 3. Validate and explore mechanistic explanations for how the microbiota prevent or facilitate infection by CT/GC using an in vitro three-dimensional model of endocervical epithelial cells. Our long-term goal is to leverage the information generated in this project to develop improved diagnostic methods, identify novel targets for new drug development and develop targeted and effective curative or preventive therapies, and ultimately, promote health, reduce risk to unintended adverse sequelae of STI and improve the quality of life for men and women who are at risk of STIs.

The "Eco-Pathogenomics of Sexually Transmitted Infections" (EPSTI), project builds upon the research started under the "Eco-Pathogenomics of Chlamydial Reproductive Tract Infection"(EPCRTI) project and examines the triangular relationship between human genetic variation, sexually transmitted infections (STIs) and co-infections caused by Chlamydia trachomatis andNeisseria gonorrhoeae, and microbiota composition among partners with distinct infection outcomes. Sexually transmitted infections pose a major health challenge in the United States where Chlamydia trachomatis (CT) genital infections, with an estimated 2.8M cases yearly, are the most frequently reported bacterial infectious disease. Likewise there are an estimated 820,000 cases of Neisseria gonorrhoeae (GC) each year. The sequelae of infections and co-infections caused by these two pathogens are insidious and account for the majority of the 750,000 annual cases of pelvic inflammatory disease (PID) in the United States, a precursor to life-threatening ectopic pregnancy and tubal factor infertility (TFI) in women. Thus, research to prevent, control and treat these STIs will provide broad health and economic benefits.

In the battleground of an infection site, both the host cells and the microbes employ complex signaling mechanisms and weaponry to destabilize, neutralize or kill the other. Identifying and understanding these biomarkers of infection and disease are the main research goals of this Cooperative Research Center (CRC). The anticipated impact will be to reduce the incidence of sexually transmitted infections and diseases (STIs & STDs) in humans worldwide. We believe that: a) the genetic variance of the infected host, the genetic diversity of the infecting pathogen(s), and the composition and function of the resident microbiota directly impact the evolution of STIs and could be biomarkers of disease severity or protection from STIs; b) genes, RNAs and proteins that are expressed or produced by the host, the pathogens, and/or the genital microbiota in response to one another are biomarkers of a specific type of STI or STD. The aim of the CRC is therefore to identify host, pathogen and/or microbiota biomarkers of STIs that may reveal mechanisms of pathogenesis and therapeutic or diagnostic targets that can be exploited for the development of translational curative or prophylactic interventions that have a direct impact in public health.

To test these hypotheses and realize these objectives, this CRC, EPSTI, will build on data acquired by the parent CRC, EPCRTI, a Chlamydia-centric program that laid the methodological and conceptual foundations of EPSTI. Within EPSTI, STING (STI Network Groups), consisting of multiple networks of sexual partners, will be leveraged to examine the triangular relationship between human genetic variation, CT, GC infections and co-infections, and microbiota composition among partners with distinct infection outcomes. The experimental approach will adopt a systems biology strategy focused on the identification of biomarkers of genital/reproductive infection and disease and are eminently amenable to translational applications in clinical and public health. These are expected to include predictive diagnosis for individuals at greatest risk of STI and STD based on their biological and microbiome characteristics, development of sensitive and specific point-of-care diagnostic tests and highly specific targets for vaccine development.

This Sexually Transmitted Infections Cooperative Research Center (STI CRC) is funded through grant U19 AI08044 from the National Institutes of Health, National Institute for Allergies and Infectious Disease.

Microbial communities in the human vagina exhibit symbiotic relationships with the host and play critical roles in maintaining health and preventing disease. These roles include protection against colonization and disease by pathogens and regulation of local immune responses. Several different kinds of vaginal communities occur in reproductive age women and are associated with health; the majority of these community types are dominated by one of several Lactobacillus species (L. iners, L. crispatus, L. gasseri, and L. jensenii), each of which is believed to provide key ecosystem services. Lactic acid production is a particularly important trait of these species, one consequence of which is acidification of the vaginal habitat. Lactobacillus species and strains vary in their production of lactic acid, as well as in their associations with vaginal health- and disease-associated states. Other members of these Lactobacillus-dominated communities presumably play supporting and/or modulating roles in community-associated beneficial services. In contrast, a distinct set of vaginal communities is characterized by low-Lactobacillus abundance and high species diversity, including the presence of Gardnerella vaginalis and other strict and facultative anaerobic bacteria; these low-Lactobacillus, high diversity communities are sometimes associated with adverse health states, such as bacterial vaginosis, and increased risks of HIV acquisition, sexually-transmitted infections, vaginal candidiasis, and premature birth. These adverse states are believed to be associated with either overt or subclinical local inflammation, suggesting a potentially important role for host responses in these outcomes.

Vaginal community composition is temporally dynamic as the community composition shifts from one type to another (e.g., Lactobacillus-dominant versus Lactobacillus-impoverished), but this dynamic behavior differs among individual women. Importantly, the relationships between community composition and function (ecosystem services), potential functional changes during these periods of shift, and the drivers responsible for these shifts are all poorly understood. Some have hypothesized that the frequency and the duration of shifts to a low-Lactobacillus state correlate with risk of adverse health and that combinations of microbial species and strains (bacterial, viral and fungal), genes and/or gene expression, metabolites and host immune factors in the vagina may explain the frequency of changes and duration of the alternative community states. As in any robust ecosystem, the interactions among these features and factors are likely to play fundamental roles in determining stability and function. Given variation in composition, communities presumably will also differ in the number and kinds of interactions among community members and the host. Work on this critical ecosystem so far has focused on individual features and failed to take a holistic, ‘systems’ approach with measurements of many features, both microbial and host-based, and an integrative strategy. As a result, we have an insufficient understanding about the relationships among community species and strain composition, ecological functions, stability of the vaginal microbiota, and risk for disease or adverse gestational outcome. (http://vmrc4health.org)

This project is funded by a grant from the Bill and Melinda Gates Foundation.

The vaginal microbiota is thought to play a major role in preventing Sexually Transmitted Infections (STIs) through ecological competition, lactic acid and target-specific bacteriocins production by Lactobacillus species. A condition known as bacterial vaginosis (BV) characterized by the depletion of Lactobacillus spp. and an overabundance of anaerobic bacteria has been shown to be associated with acquisition of STIs including HSV-2 and HIV-1. Chlamydia trachomatis (Ct) is an obligate intracellular bacterial pathogen associated with cervicitis, pelvic inflammatory disease, and subsequent tubal factor infertility and ectopic pregnancy. It is the most common bacterial STI worldwide, including in the United States. Epidemiological data suggest that the incidence of this disease has increased recently despite efforts to prevent it, which motivates the search for vaccine candidates and updates to current recommendations for Ct screening. The long-term goal of this research is to leverage biological and epidemiological data to adapt strategies for Ct control by integrating these data types. Our hypothesis is that the composition of the vaginal microbiota, and potentially its disruption, could lead to an increased susceptibility to Ct infection, independent of the effect immunogenetics may have on this relationship. If such a role could be highlighted, a change to the treatment and prevention guidelines for Ct and STI in general would be warranted. However, modification to the current recommendations for Ct screening, prevention and treatment would need to be supported by information on the natural history of Ct infection. Therefore, the objective of this project is to study the role of the vaginal microbiota in Ct acquisition and clinical presentation in a unique and already established cohort of young women prospectively followed in France. A multidisciplinary international research network has been assembled to answer these questions by focusing on two specific aims: 1) Characterize the vaginal microbiota composition, abundance and dynamics over 2 years, and the frequency of a set of immunogenetic biomarkers in a cohort of 18-24 years old women; 2) Model the interactions between the vaginal microbiota, immunogenetic factors and C. trachomatis infection using a cohort of 18-24 years old women prospectively followed. In the first aim, we will characterize the vaginal microbiota (composition and abundance) of samples from 300 women prospectively followed at four time points. We will then evaluate under the second aim the independent effects of vaginal microbiota composition and immunogenetic biomarkers on susceptibility to Ct infection as well as presence of symptoms, in order to build host-pathogen-microbiota interaction models. This research is novel in that the role of the vaginal microbiota composition in the susceptibility to Ct infection has never been studied in a large-scale prospective cohort. Its innovative potential lies in the use of novel molecular methods that allow us to refine our approach to “health” and “disease” by integrating individual predisposition to STIs, thus leading the way for personalized medicine.