Recent advances have increased the understanding that dysbiosis of the gut microbiome may be a significant contributor to the pathophysiology of ulcerative colitis. Because of this, the use of fecal microbiota transplants (FMT) has become more popular as a potential supplemental treatment option for patients suffering from this disease. Research has shown a possible benefit of FMT in conjunction with varying conventional therapies for patients with mild to moderate disease severity. However, there are scarce publications that have investigated the benefit of FMT in conjunction with a single conventional therapy for patients with moderate to severe disease, specifically. The proposed study is a multicenter, double blind, randomized controlled study of FMT, mercaptopurine (6-MP), and prednisone vs 6-MP and prednisone alone in patients with moderate to severe ulcerative colitis. The study subjects will have a baseline evaluation and the treatment trial will last 8 weeks with follow up throughout the study. Investigators will analyze the primary outcome of clinical remission and secondary outcomes of improvement of fecal calprotectin levels, Inflammatory Bowel Disease Questionnaire (IBDQ) score, C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR) in the treatment vs control groups. The data from this study will help to identify if FMT would be an additional safe, efficacious treatment modality to the current medical management of ulcerative colitis.

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.

Orientador: Katia Denise Saraiva Bresciani / Resumo: A criptosporidiose, uma das principais causas de diarreia infantil, é causada por parasitos do Filo Apicomplexa pertencentes ao gênero Cryptosporidium. A falta de medicamentos eficazes está motivando pesquisas para desenvolver tratamentos alternativos. Para este objetivo, o impacto dos probióticos no curso da criptosporidiose foi investigado. A microbiota intestinal nativa de camundongos imunossuprimidos livres de patógenos específicos foi inicialmente esgotada com antibióticos administrados por via oral. Um produto probiótico comercialmente disponível destinado ao consumo humano foi subsequentemente adicionado à água potável. Os camundongos foram infectados com oocistos de Cryptosporidium parvum. Em média, os camundongos tratados com probiótico desenvolveram uma infecção mais grave. Os probióticos alteraram significativamente a microbiota fecal, mas não foi observada associação direta entre a ingestão de bactérias probióticas e sua abundância na microbiota fecal. Esses resultados sugerem que os probióticos alteram indiretamente o microambiente intestinal ou o epitélio intestinal de maneira a favorecer a proliferação de C. parvum. / Abstract: Cryptosporidiosis, a leading cause of infant diarrhea, is caused by apicomplexan parasites classified in the genus Cryptosporidium. The lack of effective drugs is motivating research to develop alternative treatments. To this aim, the impact of probiotics on the course of cryptosporidiosis was investigated. The native intestinal microbiota of specific pathogen-free immunosuppressed mice was initially depleted with orally administered antibiotics. A commercially available probiotic product intended for human consumption was subsequently added to the drinking water. Mice were infected with Cryptosporidium parvum oocysts. On average, mice treated with probiotic developed a more severe infection. The probiotics significantly altered the fecal microbiota, but no direct association between ingestion of probiotic bacteria and their abundance in fecal microbiota was observed. These results suggest that probiotics indirectly altered the intestinal microenvironment or the intestinal epithelium in a way that favors proliferation of C. parvum. / Doutor

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.

A microbiota intestinal humana desempenha papel essencial no organismo saudável, pois sintetiza vitaminas, influencia no desenvolvimento e maturação do sistema imune da mucosa intestinal, além de exercer importante função protetora, competindo por nutrientes e receptores com bactérias patogênicas. A colonização desta microbiota se inicia na criança recém-nascida e alcança estabilidade em torno do segundo ano de vida, com consequência para a saúde da criança e do adulto. As diferenças na composição da microbiota estão relacionadas a diferentes níveis de contaminação ambiental e de diferentes fatores endógenos. O objetivo do nosso estudo foi analisar a microbiota fecal de crianças com idade entre 2 dias a 1 ano de idade, que vivem em baixas condições socioeconômicas em São Paulo, Brasil. Foram coletadas amostras de fezes de crianças saudáveis, nos seguintes pontos pós-nascimento: 2º e 7º dias, 1 mês, 3 meses, 6 meses e 1 ano de vida. O DNA bacteriano foi extraído diretamente a partir das amostras de fezes e as bibliotecas 16S rRNA foram construídas utilizando 2 iniciadores bactéria-específicos. Os clones foram selecionados aleatoriamente, parcialmente sequenciados e analisados com base em bibliotecas de gene 16S rRNA. Os principais grupos filogenéticos identificados foram Escherichia, Clostridium, Streptococcus e Bacteroides, do 1º ao 30º dia de vida. A partir do 3º mês, Streptococcus e bactérias não cultiváveis, além do gênero Escherichia, ganharam relevância na microbiota. Estes dados, em conjunto com as informações nutricionais, intercorrências clínicas e ambientais, sugerem a influência da contaminação ambiental e interpessoal no aumento da complexidade na composição da microbiota fecal. Essa abordagem molecular permitiu a análise da microbiota fecal do grupo selecionado, encontrando perfil bacteriano diferente do que é descrito nos países desenvolvidos. / The human intestinal microbiota plays essential role in healthy body since it synthesizes vitamins, influences on the development and maturation of the immune system of the intestinal mucosa. Furthermore, it also plays an important protective function competing for nutrients and receptors with pathogenic bacteria. The colonization of this microbiota starts in the newborn child and achieves stability around the second year of life, with consequence for the health of children and adults. The differences in the microbiota composition are related to different levels of environmental contamination and different endogenous factors. The aim of our study was to analyze the fecal microbiota of children ranging from 2º days to 1º year old living in low socioeconomic status in São Paulo, Brazil. We collected fecal samples of healthy children at the following points after birth: 2º e 7º days, 1 month, 3 months, 6 months and one year of life. Bacterial DNA was extracted directly from stool samples, and the 16S rRNA libraries were made using 2 bacterium-specific primers. The clones were randomly selected, and partially sequenced and analyzed based on 16S rRNA libraries. The main phylogenetic groups identified were Escherichia, Clostridium, Streptococcus, Bacteroides ranging from the 1º to 30º days of life. From the third month Streptococcus and uncultured bacteria, and, besides, Escherichia gender gained relevance in the microbiota. These data together with nutritional information, environmental and clinical intercurrents suggest the influence of interpersonal and environmental contamination in the increase of complexity in fecal microbiota composition. This molecular approach allowed the fecal microbiota analysis. This bacterial profile is different from described in developed countries.

A microbiota intestinal humana desempenha papel essencial no organismo saudável, pois sintetiza vitaminas, influencia no desenvolvimento e maturação do sistema imune da mucosa intestinal, além de exercer importante função protetora, competindo por nutrientes e receptores com bactérias patogênicas. A colonização desta microbiota se inicia na criança recém-nascida e alcança estabilidade em torno do segundo ano de vida, com consequência para a saúde da criança e do adulto. As diferenças na composição da microbiota estão relacionadas a diferentes níveis de contaminação ambiental e de diferentes fatores endógenos. O objetivo do nosso estudo foi analisar a microbiota fecal de crianças com idade entre 2 dias a 1 ano de idade, que vivem em baixas condições socioeconômicas em São Paulo, Brasil. Foram coletadas amostras de fezes de crianças saudáveis, nos seguintes pontos pós-nascimento: 2º e 7º dias, 1 mês, 3 meses, 6 meses e 1 ano de vida. O DNA bacteriano foi extraído diretamente a partir das amostras de fezes e as bibliotecas 16S rRNA foram construídas utilizando 2 iniciadores bactéria-específicos. Os clones foram selecionados aleatoriamente, parcialmente sequenciados e analisados com base em bibliotecas de gene 16S rRNA. Os principais grupos filogenéticos identificados foram Escherichia, Clostridium, Streptococcus e Bacteroides, do 1º ao 30º dia de vida. A partir do 3º mês, Streptococcus e bactérias não cultiváveis, além do gênero Escherichia, ganharam relevância na microbiota. Estes dados, em conjunto com as informações nutricionais, intercorrências clínicas e ambientais, sugerem a influência da contaminação ambiental e interpessoal no aumento da complexidade na composição da microbiota fecal. Essa abordagem molecular permitiu a análise da microbiota fecal do grupo selecionado, encontrando perfil bacteriano diferente do que é descrito nos países desenvolvidos. / The human intestinal microbiota plays essential role in healthy body since it synthesizes vitamins, influences on the development and maturation of the immune system of the intestinal mucosa. Furthermore, it also plays an important protective function competing for nutrients and receptors with pathogenic bacteria. The colonization of this microbiota starts in the newborn child and achieves stability around the second year of life, with consequence for the health of children and adults. The differences in the microbiota composition are related to different levels of environmental contamination and different endogenous factors. The aim of our study was to analyze the fecal microbiota of children ranging from 2º days to 1º year old living in low socioeconomic status in São Paulo, Brazil. We collected fecal samples of healthy children at the following points after birth: 2º e 7º days, 1 month, 3 months, 6 months and one year of life. Bacterial DNA was extracted directly from stool samples, and the 16S rRNA libraries were made using 2 bacterium-specific primers. The clones were randomly selected, and partially sequenced and analyzed based on 16S rRNA libraries. The main phylogenetic groups identified were Escherichia, Clostridium, Streptococcus, Bacteroides ranging from the 1º to 30º days of life. From the third month Streptococcus and uncultured bacteria, and, besides, Escherichia gender gained relevance in the microbiota. These data together with nutritional information, environmental and clinical intercurrents suggest the influence of interpersonal and environmental contamination in the increase of complexity in fecal microbiota composition. This molecular approach allowed the fecal microbiota analysis. This bacterial profile is different from described in developed countries.

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.

Major depressive disorder (MDD) is a highly heterogeneous disorder, with some patients displaying immune activation and altered intestinal microbiota composition when compared to healthy controls. In recent years, the transfer of fecal microbiota pooled from several MDD patients has been used to model depression in recipient rodents. However, we have previously observed the induction of donor-specific phenotypes in mice receiving microbiota from individual irritable bowel syndrome and generalized anxiety disorder patients. Therefore, we assessed the efficacy of fecal microbiota transplant (FMT) using individual versus pooled MDD patient microbiota to induce depressive-like behaviour in recipient rodents. We observed that pooling microbiota from several patients abrogated microbial features unique to individual donors. Mice that received pooled microbiota displayed different behavioural and immune phenotypes when compared to mice that received individual patient microbiota. Two individual MDD microbiota donors, patients MDD1 and MDD5, altered the behaviour of recipient mice when compared to controls. We identified several microbial species that may underlie the anxiety- and depressive-like behaviours observed in MDD1 and MDD5 mice. Additionally, altered expression of neural and immune genes was observed along the gut-brain axis of mice colonized with MDD1 microbiota. As microglia activation may play a role in our model, we developed a protocol for the isolation and phenotyping of adult mouse microglia that will facilitate future research efforts. Overall, our results demonstrate the heterogeneity of the microbial underpinnings of MDD and support the use of individual patient microbiota in future FMT experiments. / Thesis / Master of Science (MSc)

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.