This study investigated the microbial colonization and maternal influences on the neonatal calf gut microbiome. Microbiome samples were collected from dams (n = 6) and calves (n = 6) using sterile flocked swabs. The vaginal, oral, and fecal bacterial communities were examined from the dam and the fecal community of calves was examined from birth to 60 d of age. Microbial communities varied by anatomical location and age of the calf. Metagenomic analysis 16s ribosomal DNA revealed ten phyla associated with microbiomes of the dam and the same ten phyla associated with calf feces at various time points: Bacteroidetes, Firmicutes, Proteobacteria, Actinobacteria, Cyanobacteria, Verrucomicrobia, Spirochaetes, Tenericutes, Fibrobacteres, and Lentisphaerae. Overall, the calf meconium and fecal microbiome is influenced by a combination of the maternal vagina, oral, and fecal microbiomes. Further studies will be needed to identify the transference mechanisms of maternal microbes to offspring and the associated host-microbial interactions. / Master of Science in Life Sciences / This study investigated the microbial colonization and maternal influences on the neonatal calf gut microbiome. Microbiome samples were collected from dams (n = 6) and calves (n = 6) using sterile flocked swabs. The vaginal, oral, and fecal bacterial communities were examined from the dam and the fecal community of calves was examined from birth to 60 d of age. Microbial communities varied by anatomical location and age of the calf. Metagenomic analysis 16s ribosomal DNA revealed ten phyla associated with microbiomes of the dam and the same ten phyla associated with calf feces at various time points: Bacteroidetes, Firmicutes, Proteobacteria, Actinobacteria, Cyanobacteria, Verrucomicrobia, Spirochaetes, Tenericutes, Fibrobacteres, and Lentisphaerae. Overall, the calf meconium and fecal microbiome is influenced by a combination of the maternal vagina, oral, and fecal microbiomes. Further studies will be needed to identify the transference mechanisms of maternal microbes to offspring and the associated host-microbial interactions.

The human gut is colonized by a vast bacterial community that is currently rather well characterized at the species level. Yet, each of these species harbor a tremendous amount of individual genetic variations. Our understanding of the human gut microbiome, its dynamics, composition and impact on host health requires a deeper characterization of its bacteria. The amount of publicly available shotgun sequencing data as well as development of computational tools allowed to reach strain-level resolution in metagenomic analysis. In this thesis, I present systematic approaches to study the strain-level variation using complementary phylogenetic and pangenomic methods aiming to address fundamental questions about microbiome transmission in early life as well as impact of functions encoded by microbiome strains on host health. Across two different cohorts, I used a recently-developed strain-tracking method to assess the impact of delivery conditions on the initial seeding of the infant gut microbiome. While mode of delivery (vaginal or C-section) had a great impact on the amount of mother strains transmitted to the infant, place of delivery (home or hospital) and breastfeeding duration also had an impact on the ongoing development, strain replacement or persistence over the first year of life. In comparison, the father appeared as a stable source of strains independent of the delivery mode. This initial mother seeding, despite being reduced in C-section delivery, can be compensated by Fecal Microbiota Transfer, demonstrating the need of fecal microbiota exposure in seeding during vaginal delivery. In addition, strain dynamics was shown partially explained by differences in the carbohydrates degrading capacities, especially the ability to feed on Human Milk Oligosaccharides. These differences in metabolism between strains were also observed by their respective empirical growth rate that was seen associated with transmission and persistence in the infant gut. To further systematically assess the differences of metabolic capacities between strains and the impact on hosts, I developed a new method to identify gene groups (PanPhlAn Genomic Islands, PGIs) co-present across conspecific strains in metagenomic samples. By applying this method on a large collection of over 10,000 samples, I was able to build a set of 5,315 PGIs. Deeper characterization of these PGIs revealed horizontal gene transfer across species, high variation in carbohydrate metabolism capacities and association with the host lifestyle and health status. Together, these analyses demonstrated the complementary aspects of strain variation andstressed out the need to encompass both strain phylogeny and gene content to fully understand the microbiome at the strain-level.

Epilepsy is one of the most common neurological disorders worldwide. This neurological disorder is characterized by spontaneous recurrent seizures and impacts about 65 million people globally. As there is no cure for epilepsy, the treatment goal for patients is seizure management, and ultimately seizure freedom. The first line of defense in seizure management is anti-epileptic drugs, which aim to restore the excitatory and inhibitory balance in the brain. Unfortunately, about 30% of people with epilepsy are drug resistant, a number which has remained unchanged despite the increasing amount of anti-epileptic drugs. This leads patients to seek alternative treatments, which include surgery, vagus nerve stimulation, or diet alterations such as the ketogenic diet. Due to the invasiveness of surgeries, difficulty to maintain specialty diets, or lack of effectiveness of these treatments in some patients, additional therapies are needed. The gut-brain axis is a bidirectional communication network connecting the central and enteric nervous systems. Part of this network includes communication via the gut microbiota. The gut microbiota consists of all the microorganisms living in the gut, including bacteria, viruses, and fungi. It is involved in aiding nutrient absorption, promoting the maturation of immune cells and functions, and protection against pathogens. There is growing interest in the role of the gut microbiome in human health and disease. Studies have shown that patients with epilepsy have altered gut microbiomes compared to healthy controls, and that gut microbiome alteration can impact seizure frequencies. These exciting findings have ignited research on the potential therapeutic role of the gut microbiome in epilepsy. Although studies have explored the impact of alterations in the gut microbiome on seizure activity, they have not studied how anti-epileptic drugs may contribute to this relationship. Thus, this dissertation explores the role of the commonly prescribed anti-epileptic drug topiramate on the gut microbiome. Fecal samples of mice treated with topiramate were analyzed using 16S ribosomal RNA gene sequencing. Analysis revealed that topiramate ingestion increased the probiotic bacteria Lactobacillus johnsonii in the gut microbiome. In addition, cotreatment of topiramate and Lactobacillus johnsonii reduced seizure susceptibility in a pentylenetetrazol-kindling seizure model. Moreover, cotreatment increased the butyrate producing family Lachnospiraceae and subsequently increased the neuroprotective SCFA, butyrate in the gut microbiome. Importantly, cotreatment also resulted in an increased GABA/glutamate ratio in the cortex of mice that underwent pentylenetetrazol-kindling. These results are the first to demonstrate that the anti-seizure effect of topiramate may be facilitated by the modulation of the gut microbiota via increasing butyrate and altering the GABA/glutamate ratio in the cortex. Lastly, this work highlights the potential for probiotics as an adjuvant therapy in seizure management. / Doctor of Philosophy / Epilepsy is one of the most common neurological disorders worldwide. This neurological disorder is characterized by spontaneous recurrent seizures and impacts about 65 million people globally. As there is no cure for epilepsy, the treatment goal for patients is seizure management, and ultimately seizure freedom. The first line of defense in seizure management is anti-epileptic drugs, which aim to restore the excitatory and inhibitory balance in the brain. Unfortunately, about 30% of people with epilepsy are drug resistant, a number which has remained unchanged despite the increasing amount of anti-epileptic drugs. Due to this unmet need, epilepsy patients utilize alternative treatments, which include surgery, vagus nerve stimulation, or diet modifications such as the ketogenic diet. Due to the invasiveness of surgeries, difficulty to maintain specialty diets, or lack of effectiveness of these treatments in some patients, additional therapies are needed. The gut microbiota consists of all the microorganisms living in the gut, including bacteria, viruses, and fungi, which can be both harmful and helpful. In healthy individuals, the gut microbiota coexists in a balance that prevents diseases and helps the host, however, disruptions in this balance can lead to susceptibility to several diseases. As a result, researchers are increasingly interested in the role of the gut microbiota in human health and disease. In epilepsy, the gut microbiome is altered compared to healthy individuals, and gut microbiome alterations can impact seizure activity. This has led researchers to investigate the potential therapeutic role of the gut microbiome in epilepsy. Although studies have explored the impact of alterations in the gut microbiome on seizure activity, they have not studied how anti-epileptic drugs may contribute to this relationship. Thus, this dissertation explores the role of the commonly prescribed anti-epileptic drug topiramate on the gut microbiome. The results demonstrate that topiramate increases probiotic bacteria in the gut microbiome of mice. Moreover, this probiotic bacterium facilitates topiramate in reducing the susceptibility to seizures in a mouse model by resulting in a beneficial gut microbiome and restoring excitatory and inhibitory balance to the brain. These results are the first to demonstrate that the anti-seizure effect of topiramate may be facilitated by the gut microbiome. Lastly, this work highlights the potential for probiotics as an adjuvant therapy in seizure management.

Irritable Bowel Syndrome (IBS) is a gut-brain-axis disorder with a prevalence of 5.8% in Canada and 3.8% globally. Despite evidence suggesting complex interactions among neural, immune, and epithelial cells, influenced by factors such as diet, microbiota, and stress, the pathophysiology of IBS remains incompletely understood. Key knowledge gaps include the irregular nature of symptoms and the specific bacterial taxa responsible for symptom generation. Therefore, this thesis aims to perform an integrated analysis of a longitudinal study to investigate the relationship between IBS symptom dynamics, gut microbiota composition, and metabolite profiles, to determine whether temporal changes in microbiota predict or drive IBS symptoms. Clustering analysis identified distinct patterns in symptom occurrence and progression, categorizing samples into clusters that captured changes in both gut and mood symptoms, and distinguishing between symptom flares and remission. Subjects with constipation-predominant IBS exhibited consistently higher levels of symptoms while diarrhea-predominant IBS subjects showed varying symptom levels among abnormal stool weeks and normal stool weeks. Examining parallel changes in symptom scores and microbiota beta diversity over time, we found a significant correlation between the two in only 25% of IBS subjects in our cohort. This suggests a potential link between microbiota composition and symptom variability in specific subsets of patients, highlighting the heterogeneity in the microbiota-symptom relationship. Integrated microbiota-metabolite analysis revealed signatures linked to IBS subtypes, with bacteria such as Lachnoclostridium and Olsenella ,and metabolites like chenodeoxycholic acid among others identified as key nodes in network analysis. In conclusion, this study offers comprehensive insights into the episodic nature and heterogeneity of IBS, revealing dynamic symptom patterns and persistent burdens across subtypes. The findings highlight the complex relationship between microbiota composition changes and symptom variation, as well as the microbiota-metabolite axis in IBS patients. / Thesis / Doctor of Philosophy (Medical Science)

Development of type 2 diabetes (T2D) is preceded by prediabetes, which is a metabolically "atypical" state associated with chronic low-grade inflammation, overweight and obesity, lack of exercise, and detrimental changes to the gut microbiome. Dietary intake and exercise are modifiable lifestyle factors for reducing T2D risk; however, several questions remain unanswered related to the efficacy and role of prebiotics and exercise, and their respective influences on gut microbiome composition, intestinal permeability, insulin sensitivity and metabolic flexibility. Sedentary to recreationally active overweight and obese adults 40-75 years old at-risk for T2D were recruited (n=22) and randomized to either supplementation with inulin, a prebiotic dietary fiber, (10g/d) or maltodextrin while consuming a controlled diet for six weeks. At baseline and week 6, participants completed a stool collection, a 4-sugar probe test, an intravenous glucose tolerance test (IVGTT), and high-fat meal challenge with skeletal muscle biopsies to evaluate changes in the gut microbiome composition, intestinal permeability, insulin sensitivity and metabolic flexibility, respectively. There were no baseline group differences (all p>0.05). Following the intervention, Bifidobacteria operational taxonomic units increased in the intervention group ([placebo: Δ 9.5 ± 27.2 vs inulin: 96.3 ± 35.5][p=0.03]). There were no other group differences over time in any other outcome variables with the exception of changes in metabolic flexibility. Secondarily, a systematic review of literature was conducted to determine the influence of exercise engagement on gut microbiome composition. Overall, exercise interventions appeared to diversify taxa within the Firmicutes phylum, and specifically in several taxa associated with butyrate production and gut barrier function. Due to unclear risk of bias in all studies and low quality of evidence, additional research is needed using well- designed trials. In summary, the respective influences of prebiotics and exercise on human gut microbiome composition and their subsequent effects on metabolic function and disease risk are not well understood. / PHD / Type 2 diabetes (T2D) is common in the United States. Prediabetes occurs before T2D, and goes frequently undiagnosed, yet lifestyle changes (e.g. dietary changes and exercise engagement) may prevent or delay the development of T2D. Gut bacteria is a newer area of research that may have an important role in disease prevention. Several dietary supplements, such as pre- and probiotics, and their influence on gut bacteria have been studied, but the effectiveness of the prebiotic inulin for delaying or preventing T2D is unknown. Additionally the effects of exercise on gut bacteria and its role for T2D prevention is still not well understood. To address these questions, sedentary to recreationally active overweight and obese adults 40-75 years old at increased risk for T2D were recruited (n=22) and randomly assigned to either supplementation with inulin (10g/d) or maltodextrin and all consumed a six week standardized diet. At baseline and week 6, all participants completed a stool collection, a 4-sugar probe test, a high-fat challenge (HFC), and intravenous glucose tolerance test (IVGTT), and changes were evaluated in the gut microbiome, intestinal permeability, and indicators of metabolism, respectively. At week 6 Bifidobacteria, which is associated with improved gut health, increased in the inulin group ([placebo: Δ 9.5 ± 27.2 vs inulin: 96.3 ± 35.5][p=0.03]). There were no other differences over time for any other measurements with the exception muscle metabolism meal response. A systematic review of currently available research was also conducted to determine the influence of exercise engagement on gut microbiome composition. Overall, exercise engagement appeared to increase bacteria that is associated with better gut health. These findings are preliminary, and most evidence is from animal studies. Therefore, more research is needed to confirm these changes in humans. In summary, the roles of prebiotics and exercise on gut bacteria and human health are not well understood.

There is a rising global prevalence of type 2 diabetes (T2D), which poses both health and economic burdens. Effective research on T2D and clinical interventions can decrease the impact of these burdens and fill knowledge gaps in their efficacy, safety, and long-term outcomes. This comprehensive literature-based review examines the role of the gut microbiome in T2D and treatments, such as metformin and scutellaria radix-coptidis rhizoma (SC) herb couple, for gut microbiome (GM) modulation and improvement in T2D. For the purposes of this thesis, the GM is defined as a healthy nondiabetic GM, whereas dysbiosis, a deviation from a healthy GM maybe associated with the pathogenesis of T2D in terms of glucose metabolism, insulin sensitivity and inflammatory pathways and dysbiosis has been observed in individuals with a diagnosis of T2D. Microbial metabolites, such as short chain fatty acids and bile acids, and inflammatory processes have been associated with diabetes-related outcomes. Metformin, a traditional first line antidiabetic agent, as well as the traditional Chinese medicine herb couple, SC, modulate the composition of the GM in alleviating T2D. Based on the current body of knowledge, we propose to further investigate the effect of metformin and SC on the GM in T2D alleviation and measure the composition of the GM before and after an interventional clinical trial period. Investigating the effects of these study drugs and their relation to the GM may provide new insights on the pathogenesis and management of T2D.

The evolution of pest resistance to Cry proteins threatens the long-term use of Bt crops. Busseola fusca developed resistance to Bt maize in South Africa but the mechanism of resistance is not well understood. According to the gut microbiota theory, extensive cell lysis caused by Cry proteins provide gut microbes access to the more favourable environment of the hemocoel where they germinate and reproduce, causing septicemia and death of the host. This theory brought on questions about the role of gut microbes in the efficacy of Bt maize against target lepidopteran pests. The aim of this study was to determine whether microbes present in the mid-gut of B. fusca influence the efficacy of Cry 1Ab proteins. Larvae were collected from 30 different geographical locations, dissected to excise the midgut and mid-gut content which was separated according to morphological types. The morphological types were used to test the antibiotic susceptibility of the bacteria and proved that ciprofloxacin, ampicillin and doxycycline were the most effective bacteriostatic and bactericidal antibiotics. These three antibiotics were exposed to the morphological types at different concentrations to visualise the possible deleterious effects of the antibiotics on the bacteria. This visualisation was performed by observing the growth curve of the bacteria in the presence of the combination of antibiotics. The antibiotics concentration of 500 μg/ml showed the highest efficacy compared to the other concentrations tested. An antibiotic concentration of 500 μg/ml of ciprofloxacin, ampicillin and doxycycline was incorporated into an artificial diet for the larvae to feed on for 7 days. This method was used to rid the larvae of gut microbes before allowing them to feed on Bt maize (MON810) plant material expressing Cry proteins. The results suggests that by placing antibiotic reared larvae on a Bt plant, the absence of the mid-gut microbes contributed to larvae survival on Bt maize. This observation will contribute to understanding the role of gut microbes on the efficacy of Cry proteins. / MSc (Environmental Sciences), North-West University, Potchefstroom Campus, 2015

The evolution of pest resistance to Cry proteins threatens the long-term use of Bt crops. Busseola fusca developed resistance to Bt maize in South Africa but the mechanism of resistance is not well understood. According to the gut microbiota theory, extensive cell lysis caused by Cry proteins provide gut microbes access to the more favourable environment of the hemocoel where they germinate and reproduce, causing septicemia and death of the host. This theory brought on questions about the role of gut microbes in the efficacy of Bt maize against target lepidopteran pests. The aim of this study was to determine whether microbes present in the mid-gut of B. fusca influence the efficacy of Cry 1Ab proteins. Larvae were collected from 30 different geographical locations, dissected to excise the midgut and mid-gut content which was separated according to morphological types. The morphological types were used to test the antibiotic susceptibility of the bacteria and proved that ciprofloxacin, ampicillin and doxycycline were the most effective bacteriostatic and bactericidal antibiotics. These three antibiotics were exposed to the morphological types at different concentrations to visualise the possible deleterious effects of the antibiotics on the bacteria. This visualisation was performed by observing the growth curve of the bacteria in the presence of the combination of antibiotics. The antibiotics concentration of 500 μg/ml showed the highest efficacy compared to the other concentrations tested. An antibiotic concentration of 500 μg/ml of ciprofloxacin, ampicillin and doxycycline was incorporated into an artificial diet for the larvae to feed on for 7 days. This method was used to rid the larvae of gut microbes before allowing them to feed on Bt maize (MON810) plant material expressing Cry proteins. The results suggests that by placing antibiotic reared larvae on a Bt plant, the absence of the mid-gut microbes contributed to larvae survival on Bt maize. This observation will contribute to understanding the role of gut microbes on the efficacy of Cry proteins. / MSc (Environmental Sciences), North-West University, Potchefstroom Campus, 2015

Η εντερική χλωρίδα θα μπορούσε να περιγραφεί ως ένα μικροβιακό όργανο, μείζονος σημασίας για την ομοιόσταση του οργανισμού, εξαιτίας της συμμετοχής του σε πολλαπλές και διαφορετικές λειτουργιές όπως η πέψη, η απορρόφηση των θρεπτικών συστατικών, η αποβολή των άχρηστων ουσιών και η φυσική ανοσία. Η σύστασή της έχει παρατηρηθεί ότι διαφέρει από άνθρωπο σε άνθρωπο. Ενώ οι κυριότεροι παράγοντες που διαμορφώνουν το τελικό μικροβιακό περιεχόμενο περιλαμβάνουν την ηλικία, το περιβάλλον, τις διαιτητικές συνήθειες, το γενετικό υπόβαθρο, την καταγωγή, την χρήση αντιβιοτικών, πρεβιοτικών ή προβιοτικών, την έκθεση σε ποικιλλία μικροβίων και τις χειρουργικές επεμβάσεις. Η ανθρώπινη εντερική χλωρίδα αποτελείται κατά κύριο λόγο από μόλις τέσσερα φύλα βακτηρίων, τα Bacteroidetes (23%), τα Firmicutes (64%), τα Actinobacteria (3%), και τα Proteobacteria (8%). Τα τελευταία χρόνια αρκετό είναι το ερευνητικό ενδιαφέρον στην μελέτη της σχέσης μεταξύ της σύνθεσης της εντερικής χλωρίδας και της παχυσαρκίας. Η έρευνα των πιθανών μηχανισμών αλληλεπίδρασης του μικροβιακού περιεχομένου και του ξενιστή εκτελείσεται τόσο με την χρήση πειραματοζώων όσο και σε ανθρώπινο επίπεδο. Έχει αποδειχθεί ότι σε παχύσαρκα άτομα υπάρχει χαμηλότερο ποσοστό Bacteroidetes και μεγαλύτερο Firmicutes, σε σύγκριση με άτομα κανονικού βάρους. Αν και έχουν επίσης διατυπωθεί αντίθετα αποτελέσματα. Οι διαιτητικές συνήθεις είναι ένας από τους κύριους παράγοντες που επηρεάζουν την σύσταση της εντερικής χλωρίδας. Παράλληλα, είναι γνωστό ότι το μοντέλο της οικογένειας και το γονεϊκό πρότυπο είναι καθοριστικής σημασίας στην διαμόρφωση διαιτητικών και διατροφικών επιλογών. Στην παρούσα μελέτη στόχος ήταν να ερευνήσουμε την επιρροή του οικογενειακού περιβάλλοντος, της μεσογειακής διατροφής και της παχυσαρκίας στην σύσταση της εντερικής χλωρίδας. Στην έρευνα συμμετείχαν 35 άτομα, από 12 οικογένειες, ηλικίας από 18 ετών έως και 77 ετών. Όλοι είναι μέλη οικογενειών που κατοικούν στην περιοχή της Πάτρας, και αφού πληροφορήθηκαν εκτενώς για τον σκοπό και την μεθεδολογιά της, συνένεσαν εθελοντικά. Μετρήθηκαν τα ανθρωπομετρικά χαρακτηριστικά κάθε εθελοντή, και υπολογίστηκε ο δείκτης μάζας σώματος (ΔΜΣ), καταγράφηκαν, μέσω ερωτηματολογίων, δημογραφικά, κοινωνικο-οικονομικά στοιχεία και οι διαιτητικές-διατροφικές τους συνήθειες. Ακόμα έγινε συλλογή κοπράνων, από όπου απομονώθηκε DNA και αναλύθηκε με την χρήση Real Time PCR. Μετά την στατιστική επεξεργασία, δεν επιβεβαιώθηκε η αρχική εκτίμηση για παρόμοια βακτηριακή κατανομή μεταξύ των μελών της κάθε οικογένειας και τελικά δεν σχετίστηκε στατιστικά σημαντικά η υιοθέτηση της Μεσογειακής διατροφής (MedDiet score) με την σύσταση της εντερικής χλωρίδας. / The intestinal flora is a microbial organ of major importance for the homeostasis of human organism, because of its participation in multiple and diverse functions such as digestion, absorption of nutrients, elimination of waste products and immunity. Gut flora is affected by various factors, such as the age, environment, dietary habits, host’s genotype, origin, using of antibiotics, prebiotics or probiotics, exposure to a variety of microbes and surgeries. Human intestinal flora consists primarily of four bacterial genders: the Bacteroidetes (23%), the Firmicutes (64%), the Actinobacteria (3%), and the Proteobacteria (8%). In the last years, many scientists study the correlation between the gut microbiota and obesity. Although, there are conflicting results, there is evidence that obese people have lower percentage of Bacteroides and higher percentage of Firmicutes, when compared with normal weight. It is known that dietary habits affect the composition of gut flora. Furthermore, it has also been proved that type of family and/or parenting model are crucial factors in shaping dietary and nutritional choices. The objective of this study was to investigate the correlation between the Mediterranean diet, obesity and family’s environment with gut microbiota. The survey involved 35 people, from 12 different families, aged from 18 years to 77 years. We measured anthropometric characteristics of each volunteer, calculated body mass index (BMI) and then demographic, socio-economic data and dietary-nutritional habits were recorded through questionnaires. We collected stool sample from every partitipant, DNA was isolated and analyzed using Real-Time PCR. Statistical analysis did not confirm the initial estimate for similar bacterial distribution among the members of each family, and eventually the adherence to Mediterranean Diet (MedDiet score) was not significally related with changes of the intestinal flora.

Univ., Diss--Genf.