Anthropogenic impacts to the environment are unavoidable currently; however, my research investigates a potential mitigation method for amphibians dealing with poor health outcomes caused by detrimental anthropogenic changes to their wetlands. Environmental stressors such as antibiotics leeching from manure of domesticated farm animals into local wetlands can cause a dysbiosis of the gastrointestinal bacterial flora within tadpoles. Dysbiosis of gastrointestinal bacteria during early tadpole development is associated with a decrease in development rate, decrease in body mass accumulation, and other poor health outcomes. I investigated if increasing the indigestible fiber (prebiotic) content in wood frog tadpole’s alfalfa based diet could return tadpoles with stripped microbiomes (dysbiotic gastrointestinal bacterial community composition) to the same phenotype of healthy control tadpoles. I also did a pilot study to see if diet could help in increasing survival post infection with Ranavirus, and from both studies, I created NGSS aligned curriculum and activities. I found that a 10% corn starch enriched alfalfa diet significantly increased the body mass accumulation and development rate of stripped tadpoles. I found there was an association with metabolism and gut dysbiosis. Unfortunately, the connection in regards to corticosterone release was unclear. There was an association with diet and survival, but it needs to be repeated with a larger sample size.

The microorganisms that live in and on a host (the microbiome) influence host phenotype, health, and behavior in plants and animals. However, the effects of the host and environment on the composition of microbiome communities are unclear. This is especially true in arid and semi-arid environments such as deserts that filter many microorganisms. This study investigated variation in phyllosphere microbiotal bacterial assemblages of prickly pear cactus (Opuntia) across differing spatial scales, ecoregions, and taxa throughout Texas at two timepoints. I expected bacterial assemblages to differ significantly among Texas ecoregions and among species. Results support significant influences on bacterial assemblage composition including ecoregion, taxonomy, and potentially seasonal time of sampling. The dry season timepoint yielded high microbial diversity and abundance across species and ecoregions, with different species and ecoregions harboring unique microbial communities. The rainy season timepoint yielded significantly lower levels of microbial diversity and abundance across species and ecoregions.

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京都大学 / 新制・課程博士 / 博士(理学) / 甲第23749号 / 理博第4839号 / 新制||理||1692(附属図書館) / 京都大学大学院理学研究科生物科学専攻 / (主査)准教授 半谷 吾郎, 教授 湯本 貴和, 教授 古市 剛史 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DFAM

The collection of microbes that inhabits the human gastrointestinal tract is known as intestinal microbiota, and an enormous body of work has shown that their activities contribute to health and disease. Ulcerative colitis (UC), which is a type of inflammatory bowel disease, is considered to arise due to a disruption in the balance between the immune system and microbiota. However, there is little consensus on the mechanism of action and microbes involved in the disease manifestation. In this work, I applied culture-enriched metagenomics (CEMG) to characterize the dynamics of gut microbiota in healthy individuals and UC patients. I showed that CEMG provides a higher resolution to study these microbial communities, and we used this approach to understand microbial colonization after fecal microbiota transplantation (FMT) therapy in UC patient. I showed that sequencing approaches alone did not reveal consistent engraftment across FMT responders. Using CEMG and a collection of bacterial whole-genome sequences, I showed patient-specific microbial strain transfer and a signature of commonly engrafted genes only in patients who responded to FMT. In this work, I also investigated the dynamics of a highly abundant bacteriophage, crAssphage, in an FMT donor and implemented a new method to detect bacteriophage engraftment post-FMT using SNP analysis. Finally, it has been suggested that antibiotic treatment before FMT may increase the efficacy of FMT. However, in this work, I show that while antibiotics alter the microbiome, there was no difference in the composition of the microbiome of antibiotic vs placebo group post-FMT. This is consistent with the randomized controlled trial results that shows pretreatment with antibiotics does not improve FMT outcome. Together, this work demonstrate the importance of in-depth microbiome analysis applied to culture-dependent and -independent sequencing to characterize microbial changes post-FMT. / Dissertation / Doctor of Philosophy (PhD) / Many bacteria reside in the human gut, and they are essential in our health and in disease. It is evident that these bacteria are associated with inflammatory bowel disease, but we do not yet know how and what bacteria are involved in this disease. In this work, I describe a method to study these bacteria from stool that relies on growing them and investigating their DNA. I showed that our approach helped us recover a greater diversity of these bacteria and their genetic content in healthy individuals and patients with inflammatory bowel disease compared to methods that use only DNA based approaches. Using this method, we could better understand why some patients responded to a treatment consisting of transferring stool content from healthy donor to patient. I also investigated a group of viruses that infect bacteria and implemented a new computational method based on DNA sequencing to test whether these viruses transfer to the patient after receiving the fecal therapy. We also found that antibiotic treatment before fecal therapy in patients with inflammatory bowel disease does not improve the patient’s recovery.

The numerous benefits associated with natural products isolated from the environmental sources, including soil bacteria, plants and fungi, are long known and well appreciated. Interestingly, the immense number of microorganisms that reside within our bodies and whose cell counts greatly outnumber our own represents a potentially new and practically untapped reservoir of bioactive compounds. With the advent of next generation sequencing we are only now starting to realize the complexity and biological diversity of the human microbiome. With this ever-increasing flow of genomic information, more bioactive potential in these microbes can be identified. For instance, biosynthetic assembly lines responsible for the production of two largest classes of bioactive compounds, polyketides and nonribosomal peptides, can be readily identified within the microbial genomes, providing us with a view of their bioactive profiles. In addition to the identification of biosynthetic assembly lines, the building blocks of polyketide and nonribosomal peptide products can also be accurately predicted, given the well-understood logic of assembly line operations. Nonetheless, the identification of actual products is still lagging behind. The discovery of these bioactive molecules can be achieved, however, by establishing a unique connection between genomes and molecules. Using several concrete examples, this thesis demonstrates how both metabolomic and biochemoinformatic platforms can assist in discovery of bioactive small molecules. More specifically, investigations involving three members of the human microbiome, Streptococcus mutans, Lactobacillus plantarum and Pseudomonas aeruginosa, provide distinct examples of identification of bioactive agents and assessment of their immunomodulatory potential. Interrogating the human microbiome form the angle of small molecules is critical for evaluation of microbial effects on our cells, and ultimately our health. Studying these agents will hopefully reveal interesting principles on how microorganisms speak to human cells and how this communication could lead to therapeutic strategies or downstream mechanistic revelations. / Thesis / Master of Science (MSc) / The numerous benefits associated with natural products isolated from the environmental sources, including soil bacteria, plants and fungi, are long known and well appreciated. Interestingly, the immense number of microorganisms that reside within our bodies and whose cell counts greatly outnumber our own represents a potentially new and practically untapped reservoir of bioactive compounds. With the advent of next generation sequencing we are only now starting to realize the complexity and biological diversity of the human microbiome. With this ever-increasing flow of genomic information, more bioactive potential in these microbes can be identified. For instance, biosynthetic assembly lines responsible for the production of two largest classes of bioactive compounds, polyketides and nonribosomal peptides, can be readily identified within the microbial genomes, providing us with a view of their bioactive profiles. In addition to the identification of biosynthetic assembly lines, the building blocks of polyketide and nonribosomal peptide products can also be accurately predicted, given the well-understood logic of assembly line operations. Nonetheless, the identification of actual products is still lagging behind. The discovery of these bioactive molecules can be achieved, however, by establishing a unique connection between genomes and molecules. Using several concrete examples, this thesis demonstrates how both metabolomic and biochemoinformatic platforms can assist in discovery of bioactive small molecules. More specifically, investigations involving three members of the human microbiome, Streptococcus mutans, Lactobacillus plantarum and Pseudomonas aeruginosa, provide distinct examples of identification of bioactive agents and assessment of their immunomodulatory potential. Interrogating the human microbiome form the angle of small molecules is critical for evaluation of microbial effects on our cells, and ultimately our health. Studying these agents will hopefully reveal interesting principles on how microorganisms speak to human cells and how this communication could lead to therapeutic strategies or downstream mechanistic revelations.

The current findings support the hypothesis that disruption of the microbiome in infancy results in measurable, sex specific changes in behavioural characteristics associated with psychopathology. These early exploratory findings support further study into the mechanisms of potential behavioural effects of disruption of the human microbiome in infancy through exposure to antibiotics or birth by caesarean section. If a causal relationship is determined between early life microbiome disruptors and the development of psychopathology, the relative effect of these disruptors in relation to other developmental processes needs further consideration, given the potential impact these findings may have on the utilization of these intervention in infancy and early life. / Thesis / Master of Science (MSc)

Obsessive-Compulsive Disorder (OCD) is a debilitating, chronic neuropsychiatric disorder estimated to effect approximately 1-2% of the Canadian population. Our understanding of the pathophysiological mechanisms involved in OCD is unclear, as evidenced by the moderate response associated with treatments targeting these putative pathways. As such, there is a need to explore novel mechanisms of disease. Recent research has focused on the gut-brain axis and highlighted the potential role of the gut microbiota in psychiatric conditions. Further, the role of inflammation is also gaining traction in psychiatric research. This thesis investigates the role of these commensal gut bacteria in OCD, by examining stool samples of unmedicated, non-depressed OCD outpatients and healthy community controls. Given that systemic inflammation is a suggested pathway by which gut bacteria effect behaviour, morning levels of C-reactive protein (CRP), interleukin-6 (IL-6) and tumor-necrosis factors-α (TNF-α) were also examined. To our knowledge, this thesis is the first investigation of the gut microbiome in OCD. This thesis describes: (1) a critical review of the literature developing a theoretical basis for a role of microbial dysbiosis in OCD; that (2) three specific genera and species richness/diversity are lower in OCD patients compared to controls; (3) mean CRP, but not IL-6 and TNF-α, is elevated in this sample of OCD patients; and (4) gastrointestinal symptom severity and prevalence of irritable bowel syndrome is higher in OCD. Taken together, this thesis is the first study to provide evidence for microbial dysbiosis in OCD. Although systemic inflammation may not mediate the relationship between reduced diversity and OCD symptomatology, these results provide evidence for mild systemic inflammation. Further gastrointestinal and psychiatric symptom severity are positively correlated, but not specific to patients with IBS. These results suggest the gut microbiome may be a potential pathway of interest for future OCD research, clinical implications are also made. / Thesis / Doctor of Philosophy (PhD)

The upper respiratory tract (URT), including the nasal and oral cavities, is a reservoir for pathogenic and commensal microbial species, collectively known as the microbiota. Microbial colonization of the URT occurs right after birth, and URT microbial composition has been linked to development of respiratory infections, allergy, and asthma, though few direct mechanisms have been uncovered. Thus, I set out to establish animal models for characterizing the URT microbiota, and its role in infections. I found that nasal washes, a predominant method for measuring URT bacterial colonization, were insufficient for completely extracting the URT microbiota. The age and source of mice greatly affected the composition of the microbiota, which could be transferred to germ-free mice via cohousing. I also established that mice colonized with the Altered Schaedler’s Flora in the gut microbiota have no cultivable URT microbiota. To test the function of the URT microbiota, I colonized mice with Streptococcus pneumoniae, the leading cause of bacterial pneumonia worldwide. I show that the presence of a nasal microbiota increases permissiveness to pneumococcal infection in murine models. Addition of a single URT isolate, Actinomyces naeslundii, increased pneumococcal adherence to human respiratory epithelial cells in vitro and increased pneumococcal dissemination in vivo in a sialidase-dependent manner. The microbiota affects expression of several host genes throughout the respiratory tract involved in pneumococcal pathogenesis. Together, this work establishes new models for assessing the URT microbiota, and highlights the contribution of the URT microbiota to pneumococcal pathogenesis and identifies druggable targets to prevent and treat infections. / Dissertation / Doctor of Philosophy (PhD) / Bacteria living in the gut have been shown to benefit our health, but the role of bacteria living in our respiratory tract is relatively unknown. I describe the methods for characterizing the bacteria in the nose of a mouse as a model of the human nose. I found that pockets of the mouse nose are colonized by different bacteria. I also characterized a mouse model that had bacteria in the gut without nasal bacteria. I used this mouse model to understand infections with Streptococcus pneumoniae, the worldwide leading cause of bacterial pneumonia. The mice without nasal bacteria were protected from infections, which was due to a nasal bacteria helping S. pneumoniae escape from the nasal tissue. This work established new models for understanding how bacteria affect respiratory health, and identified new targets for protecting against infections.

Background: The introduction of solid foods is an important dietary event during infancy and is associated with a time of dramatic shifts in gut microbial composition. The influence of solid food introduction on gut bacterial dynamics remains understudied. Methods: 15 healthy, full-term, vaginally born, and breast-fed infants of the Baby, Food and Mi sub-study of the Baby & Mi Study were investigated. Caregivers were asked to collect daily stool samples and food diaries for 17 days, commencing three days prior to the introduction of solids. Additional stool samples were available up to one year as part of the Baby and Mi study. The exposure of interest, nutritional patterns, was analyzed using food composition output from ESHA’s Food Processor. The number of food items and food groups introduced were used to calculate dietary diversity scores. The outcome of interest, gut bacterial dynamics, was analyzed using RStudio. Results: The mean (SD) age at the introduction of solid foods is 5.5 (0.66) months (n = 15). Over the study period, the proportion of estimated energy intake from solid foods was low (7.5%; SD 6.74%) (n = 14). Alpha diversity increased over time and was highest at 1 year. The gut microbial community influenced by dominant bacterial taxa changed with increasing age. With introduction of solids, individual community composition changed, though to a varying extent. Shannon alpha diversity was directly associated with calories from carbohydrates, particularly daily fiber intake. The infant’s dietary diversity score was directly associated with alpha diversity and was also positively associated with the degree of change occurring in this time period. Conclusion: Fiber intake and the dietary diversity scores had the closest relationships to the gut microbiome’s alpha diversity and community structure in infants at the time of solid food introduction. / Thesis / Master of Science (MSc) / The introduction of solid foods is an important life-event during infancy. This is also when the gut microbiome is developing to its mature state. Since nutrition is an important factor influencing the microbiome, investigating the dietary choices at the introduction to solid foods is the aim of the following study. Here, daily stool samples and food diary entries were collected for 15 healthy, breast-fed infants. It is important to measure the diversity of the bacteria in the gut of an individual (alpha) and between people (beta), as well as bacteria present. Carbohydrates drive the change in alpha diversity, especially fiber. Feeding infants a diet with many different foods shows increased alpha diversity and change in the microbiome immediately after introduction. Interestingly, the infant gut microbiome reacts to fiber in a manner comparable to the adult gut microbiome, i.e. increased bacterial diversity, which is associated with better health outcomes in adults.