Clostridium difficile infection (CDI) is a disease associated with the wide-spread use of antibiotics and causes 450,000 infections and almost 30,000 deaths in the United States annually. Recurrence is a major problem, with approximately 1/3rd of patients relapsing after antibiotic treatment for CDI. Fecal Microbiota Transplantation (FMT) has emerged as a novel therapy for recurrent CDI, but the majority of the literature to date is made up of uncontrolled case series, so FMT’s true efficacy compared with standard antibiotic regimens remains unknown. Only a few randomized control trials (RCTs) have been published, and these have studied small numbers of patients and exhibited marked methodological heterogeneity. As such, there is uncertainty about the appropriate indications for FMT with respect to recurrent CDI, as well as the best methodology for the procedure, which has been carried our using various fecal preparations and modes of delivery. In particular, questions remain about if FMT should be recommended for patients with a first CDI recurrence, and if minimally invasive methods of performing FMT such as administration of enteric coated capsules are more efficacious than standard antibiotic treatments. We propose a double blind, placebo controlled, RCT that will be run as two parallel RCTs, where Trial 1 will enroll patients experiencing a first CDI recurrence, and Trial 2 will enroll patients experiencing a second or later CDI recurrence. The treatment arms in each trial will receive FMT in the form of orally administered frozen capsules, while the control arms will receive standard antibiotic treatments based on the number of recurrences they have experienced. If shown to be efficacious in a large RCT, oral capsulized FMT alone as treatment for recurrent CDI has the potential to increase access to FMT, decrease unnecessary antibiotic use, and substantially reduce morbidity and mortality attributable to CDI.

Fecal microbiota transplantation (FMT) is the process by which fecal suspension from a healthy individual is transferred into the gastrointestinal tract of another individual in an attempt to cure certain diseases. This transplantation process has been accredited as being a potential remedy for a growing number of diseases that have been associated with gut microbial imbalances. Interest in FMT has largely been driven by the science community’s increasing interest in the gut microbiome and its role in potentially regulating a multitude of different functions and processes within the human body. One disease that has been found to respond exceptionally well to FMT treatments is Clostridium difficile infection (CDI). However, while FMT has demonstrated high cure rates for CDI, this transplantation process is no panacea. In fact, the results from FMT treatments on other diseases, such as Inflammatory Bowel Disease (IBD), have not been as impressive as CDI’s. This review will examine the existing literature surrounding FMT usage on IBD and will propose a series of experiments and studies needed to truly test the safety and efficacy of FMT for IBD patients. This review will also reference current literature documenting FMT treatments for CDI as a comparative tool for investigating if this form of bacteriotherapy is indeed a viable therapeutic option for treating IBD.

Thesis advisor: Catherine Y. Read / Background. Clostridium difficile infections (CDI) account for 20-30% of healthcare-acquired infections, resulting in serious patient and economic burdens. CDI incidence has grown rapidly due to overuse of antibiotics and an aging population, posing a significant public health threat. Fecal microbiota transplantation (FMT) using donor stool has demonstrated clinical efficacy rates up to 94% and long-term restoration of a healthy intestinal microbiome. Challenges with donor screening, lack of research about optimal stool donor characteristics and intestinal microbiome composition, and a poorly fit screening model, create barriers to the availability of FMT. Purpose. This study aimed to generate essential information about FMT donor characteristics predictive of passing the screening and donor intestinal microbiome compositions associated with FMT clinical efficacy. The primary aims were to 1) identify previously unstudied characteristics of prospective FMT donors that are predictive of passing a stool bank’s screening process; and 2) determine whether donor intestinal microbial diversity is related to FMT clinical efficacy in preventing recurrent CDI. Methods. This study was conducted as a secondary analysis on a cohort of previously screened donors (n=770). Aim 1 was tested through a logistic regression of donor characteristics (gender, age, body mass index, frequency of bowel movements, diet, tobacco and alcohol use, and seasonality) with screening outcomes. Aim 2 was tested through a simple regression evaluating donor intestinal microbial diversity and rates of FMT clinical efficacy. Results. One donor characteristic in the logistic regression, frequency of bowel movements (p = 0.018), was significantly predictive of whether a donor passed the screening. Specifically, donors who had fewer than two bowel movements per day were more likely to pass. All other characteristics were not predictive. Similarly, the linear regression evaluating alpha diversity and FMT clinical efficacy was not significantly predictive of clinical efficacy (p = 0.140). Conclusion. Findings were used to support recommendations for improving prospective donor screening that nurses and other clinicians can implement to decrease challenging logistics, reduce costs and barriers, and potentially increase FMT clinical efficacy. / Thesis (PhD) — Boston College, 2019. / Submitted to: Boston College. Connell School of Nursing. / Discipline: Nursing.

Immunotherapy has emerged as one of the four “standard” cancer therapies, alongside surgery, chemotherapy, and radiotherapy. Immune checkpoint inhibitor (ICI) therapy is an immunotherapy that blocks inhibitory immune checkpoint interactions, allowing T cells and other immune cells to kill tumor cells. In the tumor microenvironment, there is often overexpression of immune checkpoint proteins, whose binding interaction with cytotoxic T cells and other immune cells results in the dampening of the antitumor response. Programmed cell death protein 1 (PD-1) and T-lymphocyte-associated protein 4 (CTLA-4) are the two most targeted immune checkpoint proteins. Antibodies against PD-1 and CTLA-4, as well as other checkpoint proteins, are approved for clinical use as well as in clinical trials. While ICIs have changed the treatment landscape for many cancers, particularly those with significant immunogenicity, only 20-40% of patients respond to ICI therapy. Many factors are behind the lack of response and resistance, and significant efforts are aimed at improving the response to ICI therapy. One major area is modulating the gut microbiome, as it is well-established that microbial dysbiosis is associated with various human diseases. The concept is that by modulating the microbiome, we might be able to return it to a composition more similar to that seen in healthy individuals or provide microorganisms beneficial to clinical response. In the case of ICI therapy, it is proposed that there is a connection between certain microbial species and the immune system via metabolites and other signaling effects. The microbiome can be manipulated through many methods, including fecal microbiota transplantation (FMT), transferring bacterial isolates or consortia, probiotics, antibiotics, and soluble dietary fiber. For clinical insights, it is important to consider how the pre-treatment microbiome of patients may affect their response to ICI therapy, as well as how their microbiomes can be manipulated to enhance their response. Initial clinical trials have been promising, but this is an emerging field with additional work to be done. Particularly, a better understanding of the microorganisms involved in the response to ICI therapy and the mechanism by which they communicate with the immune system is essential. Future studies will need to be much larger to reduce noise between studies and to allow for emerging computational techniques to be applied.

The human gut microbiome is home to many hundreds of different microbes which play a crucial role in human physiology. For most of them, little is known about how their genetic diversity translates into functional traits and how they interact with their host, which is to some extent due to the lack of isolate genomes. Cultivation-free metagenomic approaches yield extensive amounts of bacterial genetic data, and recently developed algorithms allow strain-level resolution and reconstruction of bacterial genomes from metagenomes, yet bacterial within-species diversity and transmission dynamics after fecal microbiota transplantation remain largely unexplored over cohorts and using these technological advances. To investigate bacterial within-species diversity I first undertook large-scale exploratory studies to characterize the population-level genomic makeup of the two key human gut microbes Eubacterium rectale and Akkermansia muciniphila , leveraging many hundreds of bacterial draft genomes reconstructed from short-read shotgun metagenomics datasets from all around the planet. For E. rectale , I extended previous observations about clustering of subspecies with geography, which suggested isolation by distance and the putative ancestral loss of four distinct motility operons, rendering a subspecies specifically found in Europe immotile. For A. muciniphila, I found that there are several closely related but undescribed Akkermansia spp. in the human gut that are all likely human-specific but are differentially associated with host body mass index, showcasing metabolic differences and distinct co-abundance patterns with putative cognate phages . For both species, I discovered distinct subspecies-level genetic variation in structural polysaccharide synthesis operons. Next, utilizing a complementary strain-resolved approach to track strains between individuals, I undertook a fecal microbiota transplantation (FMT) meta-analysis integrating 24 distinct clinical metagenomic datasets. I found that patients with an infectious disease or those who underwent antibiotic treatment displayed increased donor strain uptake and that some bacterial clades engraft more consistently than others. Furthermore, I developed a machine-learning framework that allows optimizing microbial parameters - such as bacterial richness - in the recipient after FMT based on donor microbiome features, representing first steps towards making a rational donor choice. Taken together, in my work I extended the strain-level understanding of human gut commensals and showcased that genomes from metagenomes can be suitable to conduct large-scale bacterial population genetics studies on other understudied human gut commensals. I further confirmed that strain-resolved metagenomics allows tracking of strains and thus inference of strain engraftment characteristics in an FMT meta-analysis, revealing important differences in engraftment over cohorts and species and paving the way towards better designed FMTs. I believe that my work is an important contribution to the field of microbiome research, showcasing the power of shotgun metagenomics, modern algorithms and large-scale data analysis to reveal previously unattainable insights about the human gut microbiome.

Le microbiote intestinal équin joue un rôle important dans le maintien de la santé de l'hôte. Le microbiote intestinal est composé de nombreux micro-organismes tels que les bactéries, les virus, les champignons et les archées. Cependant, la majorité de ces cellules microbiennes sont bactériennes, et par conséquent, de nombreuses études, y compris la présente, se concentrent sur l'exploration des communautés bactériennes dans l'intestin. Un déséquilibre du microbiote intestinal, appelé dysbiose, a été observé dans plusieurs conditions, telles que la colite, après l’administration d'antibiotiques ou la modification du régime alimentaire. La restauration du microbiote peut être effectuée par la transplantation de microbiote fécal (FMT). Des études utilisant les recommandations actuelles pour la FMT ont montré une récupération clinique chez les chevaux souffrant de diarrhée, mais le microbiote reste largement inchangé après la FMT et aucune étude randomisée avec contrôle placébo n'a été réalisée. Les hypothèses de ce projet étaient que le traitement avec une FMT concentrée corrigera la dysbiose plus rapidement qu’une FMT conventionnelle et le véhicule, et que le microbiote intestinal des chevaux traités avec une FMT concentrée ressemblera au microbiote intestinal du cheval donneur. L'objectif de ce projet était de développer un protocole pour améliorer la FMT chez les chevaux, en augmentant la concentration de bactéries présentes dans les selles du donneur par centrifugation, et de le tester chez les chevaux atteints de dysbiose intestinale induite par les antibiotiques. L'antibiotique triméthoprime sulfadiazine (TMS) a été administré à neuf chevaux pour induire une dysbiose intestinale. Les chevaux ont été séparés en trois groupes: les chevaux recevant une FMT concentrée (cFMT, n = 3); les chevaux recevant la FMT fraîche (fFMT), selon les recommandations actuelles (n = 3); et les chevaux recevant un véhicule (VEH) avec 10% de glycérol dans une solution saline à 0,9% (n=3). Des échantillons fécaux ont été prélevés avant et après l'administration du TMS, ainsi qu'avant, pendant et après la transplantation. Le séquençage a été réalisé à l'aide de la plateforme Illumina MiSeq et les données analysées à l'aide du logiciel Mothur. Tel qu’attendu, l'antibiotique TMS a significativement diminué la richesse microbienne chez tous les chevaux. De manière inattendue, la composition des suspensions fécales des donneurs cFMT et fFMT était significativement différente de la composition de base des receveurs cFMT et fFMT, respectivement. La composition du microbiote des chevaux ayant reçu une transplantation fécale (concentrée ou non) était significativement différente après la transplantation, alors que ce n’était pas le cas chez les chevaux ayant reçu le véhicule. En outre, l’abondance relative de Escherichia était significativement plus élevée dans les suspensions fécales du donneur cFMT par rapport aux suspensions fécales du donneur fFMT. Les principales limites de ce projet sont la petite taille des groupes et l'exposition des selles des donneurs à l'oxygène et à la congélation-décongélation. En outre, le modèle de dysbiose peut ne pas être optimal pour tester l'efficacité de la FMT, et des études réalisant la FMT chez les chevaux souffrant de diarrhée sont nécessaire. Cette étude a contribué à la recherche de nouvelles approches pour améliorer la FMT chez les chevaux. Le faible effet mesuré avec les deux protocoles de FMT et l’augmentation de Escherichia démontre que les protocoles actuels doivent être optimisés avant de pouvoir recommander la FMT pour traiter et prévenir la dysbiose chez les chevaux. / The equine gut microbiota plays an important role in maintaining the health of the host. The gut microbiota is composed of many microorganisms such as bacteria, viruses, fungi, and archaea. However, the majority of these microbial cells are bacterial cells, and consequently, many studies, including the present one, focus on exploring bacterial communities in the gut. An imbalance of the gut microbiota, termed dysbiosis, has been observed in several conditions such as colitis, colic, after antibiotic administration, or diet modification. Restoration of the gut to a healthy state can be performed through fecal microbiota transplantation (FMT). Studies using current recommendations for FMT have shown clinical recovery in horses with diarrhea, but the microbiota remains largely unchanged after FMT and no controlled studies have been performed. The hypotheses of this project were that treatment with concentrated FMT will correct dysbiosis faster than conventional FMT and the vehicle, and that the gut microbiota of horses treated with concentrated FMT will resemble the gut microbiota of the donor. The objective of this project was to develop an improved protocol for FMT in horses, by increasing the concentration of bacteria found in the donor stool using centrifugation, and to test it in horses with antibiotic-induced intestinal dysbiosis. The antibiotic trimethoprim sulfadiazine (TMS) was administered to nine horses to induce intestinal dysbiosis. Horses were separated into three groups: horses receiving concentrated FMT (cFMT) (n=3); horses receiving fresh FMT (fFMT), as per current recommendations (n=3); horses receiving a vehicle (VEH) with 10% glycerol in 0.9% saline (n=3). Fecal samples were collected before and after antibiotic administration, as well as before, during, and after transplantation. Sequencing was performed using the Illumina MiSeq platform and data analysed using the software Mothur. As expected, the antibiotic TMS significantly decreased the richness in all horses (P < 0.05). Unexpectedly, the membership of the cFMT and fFMT donor fecal suspensions was significantly different from cFMT and fFMT recipients’ baseline membership, respectively. The membership of the cFMT and fFMT recipient horses was significantly different after transplantation, while the vehicle recipients were not. In addition, the Escherichia genus was found in significantly higher relative abundances in the cFMT donor fecal suspensions when compared to the fFMT donor fecal suspensions. The main limitations of this study are the small sample size and exposure of cFMT donor stool to oxygen and freeze-thawing. In addition, the dysbiosis model may not be optimal to test the efficacy of FMT, and studies performing FMT in horses with diarrhea are warranted. This study contributed to the search for novel approaches to improve FMT in horses. The weak effect of both FMT protocols on the gut microbiota and the increase in Escherichia suggest that further clinical studies are needed before FMT can be recommended to treat and prevent dysbiosis in horses.