Metagenomics is a cultivation-independent approach for obtaining the genomic composition of microbial communities. Microbial communities are ubiquitous in nature. Microbes which are associated with the human body play important roles in human health and disease. These roles span from protecting us against infections from other bacteria, to being the causes of these diseases. A deeper understanding of these communities and how they function inside our bodies allows for advancements in treatments and preventions for these diseases. Recent developments in metagenomics have been driven by the emergence of Next-Generation Sequencing technologies and Third-Generation Sequencing technologies that have enabled cost-effective DNA sequencing and the generation of large volumes of genomic data. These technologies have allowed for the introduction of hybrid DNA assembly techniques to recover the genomes of the constituent microbes. While Next-Generation Sequencing technologies use paired-end sequencing reads from DNA fragments into short reads and have a relatively lower error rate, Third-Generation Sequencing technologies use much longer DNA fragments to generate longer reads, bringing contigs together for larger scaffolds with a higher error rate. Hybrid assemblers leverage both short and long read sequencing technologies and can be a critical step in the advancements of metagenomics, combining these technologies to allow for longer assemblies of DNA with lower error rates. We evaluate the strengths and weaknesses of the hybrid assembly framework using several state-of-the-art assemblers and simulated human microbiome datasets. Our work provides insights into metagenomic assembly and genome recovery, an important step towards a deeper understanding of the microbial communities that influence our well-being.

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MSc Thesis - The effect of wastewater effluent on the gut microbiome of rainbow darter / The microbiome plays an important role in host physiology and can be influenced by species, diet, and environment. Municipal wastewater effluent contains a mixture of chemicals including antibiotics and antimicrobials that may affect the gut microbiome of fish living downstream of these discharges. Thus, this study examines the effect of wastewater treatment plant (WWTP) effluent on the gut microbiome of wild rainbow darter (Etheostoma cearuleum), and examines how the gut microbiome of wild fish changes in the lab. Fish were collected from sites upstream and downstream of 2 major WWTPs along the central Grand River and gut contents were aseptically sampled. After extracting gDNA, nested PCR of the V3-V4 region of the 16S rRNA gene, and Illumina sequencing were performed. The gut microbiome of exposed fish had increased bacterial diversity and was dominated by Proteobacteria, which has been linked to altered health outcomes in mammals. Next, rainbow darters were collected from a reference site on the Grand River. Fish were sampled in the field, after a 14 day lab acclimation, and after a 28 day exposure to environmental stressors (WWTP effluent or triclosan, an antimicrobial found in WWTP effluent). Surprisingly, there were no changes in the microbiome after exposure to environmental stressors. Major changes were observed between the field and laboratory fish suggesting that environment and diet are important factors influencing the gut microbiome. Changes in the gut microbiome continued up to 42 days in the lab, indicating longer acclimation periods may be needed. This study showed that effluents altered the gut microbiome of fish in the field, but not in the laboratory for unknown reasons. Laboratory studies indicated that transitioning to a new environment may require greater than 14 days before achieving a stable microbiome. / Thesis / Master of Science (MSc) / Wastewater is the largest source of pollution affecting Canada’s aquatic ecosystems; effluents contain antibiotics and antimicrobials that can affect fish and other aquatic life. The gut microbiome of fish is influenced by host species, its diet, and the environment, and thus contaminants released via wastewater effluents may alter the gut microbiome of fishes in receiving waters. This study found that the gut microbiota of rainbow darter fish exposed to wastewater effluents in the central Grand River (Waterloo/Kitchener, Ontario) were dominated by Proteobacteria and had increased diversity. Wild fish transitioned to the lab were dominated by Firmicutes and had decreased bacterial diversity in the gut compared to those in the wild. Altogether, these results suggest that wild fish exposed to wastewater effluents had altered gut microbiomes; transitions to new environments and laboratory acclimation periods are important considerations when studying the fish gut microbiome.

Necrotic enteritis (NE) is a major enteric disease in commercial poultry and manifests itself in clinical and subclinical forms. Despite years of research, NE is still among the leading diseases with the greatest economic impact on poultry production. Subclinical forms lead to poor performance (reduced feed intake, weight gain and eventually higher feed conversion ratio) but usually occurs with low mortality rates. The use of antibiotic growth promoters (AGPs) is proving to be an effective tool in maintaining gut health and modifying gut microbiota, thus improving broiler performance and reducing NE. Removal of AGPs has led to an increase in NE occurrence, particularly the subclinical forms. The lack of alternative strategies to control NE is mainly due to limited insight into the relationship between NE pathogenesis, the host microbiome and its immune responses. Therefore, key to overcoming NE is to define the cellular and molecular mechanisms that are involved in the progression of the disease, especially with regard to mucosal immune responses and gut microbiome. Also, assessing the impact of these changes on gut cell metabolism and function is of great importance. This information would be a valuable guide to prevent the onset or alleviate the negative impact of NE on bird's health and performance. In a series of experiments conducted for this project, the effect of single or multi-strain probiotics as well as multi-component additives were tested during NE challenge in order to define the cellular and molecular mechanisms that are involved in the progression of the disease. Results of these experiments revealed that challenging broilers with a 'naturally occurring' NE led to differential expression of tight junction (TJ) proteins in the jejunum compared to non-challenged birds. Supplementation of certain additives reduced NE lesion scores, improved performance and increased mRNA abundance of claudin-3, a key epithelial TJ protein. Multi-strain probiotics and multi-component additives (including a symbiotic and a product containing probiotics, prebiotics and essential oils) were more effective than single-strain probiotics or prebiotics. The aforementioned additives were also more effective in modulating immune responses to NE, especially by decreasing the mRNA abundance of IFN-γ and IL-10 in the jejunum. Furthermore, supplementation of these additives led to an increase in the expression of nutrient transporters (SGLT-1) and regulators of energy metabolism (PGC-1α, mTOR and AMPK); thus, improving nutrients absorption and metabolism. Microbial profiling using 16S rRNA sequencing showed that supplementation of each specific additive led to a signature-like microbiome in the ileal scrapings of broilers. However, supplementation of multi-component additives (including a symbiotic and a product containing probiotics, prebiotics and essential oils) modified the ileal microbiome in association with lower NE lesion scores, better performance and modulated immune responses. These additives reduced the relative abundance of bacteria such as ASF356, Bacteroides, Clostridium sensu stricto 1, Faecalibaculum, Lachnospiraceae UCG-001, Muribaculum, Oscillibacter, Parabacteroides, Rikenellaceae RC9 gut group, Ruminococcaceae UCG-014, and Ruminiclostridium 9 and increased the relative abundance of Lactobacillus compared to NE challenged birds. Collectively, these data indicate that during a subclinical naturally occurring NE, the use of multi-strain probiotics or multi-component additives, compared to single-strain probiotics or prebiotics, would be a more promising strategy in alleviating the effect of this enteric disease. / Doctor of Philosophy / Necrotic enteritis, an enteric disease, is one of the major diseases that negatively impacts the poultry industry and producers' profitability. The growing ban on the use of antibiotics that were used to prevent this disease has increased the number of necrotic enteritis outbreaks worldwide. Having a better understanding of the cellular and molecular mechanisms that are involved in the onset of this disease is of crucial importance and could lead to finding more effective ways to control this disease without drugs. The gut is the site of digestion and absorption of nutrients so any damage would lead to poor bird performance. In a series of experiments conducted for this project, several combinations of beneficial bacteria and nutrient sources that help bacterial growth in the gut (prebiotics) improved gut health leading to better performance during the grow-out period (days 0-42) when birds reach market age. These supplements protected the gut lining and reduced damages due to necrotic enteritis with less severe lesions. Barrier function of the gut was also improved by supplementing the diet with combination of beneficial bacteria and nutrients that help their growth in the gut. There are special types of proteins (called tight junctions) that seal up the space between intestinal cells (enterocytes) and prevent pathogens in the gut lumen from entering the body, thus preventing inflammation and disease. This helps the body to use the absorbed nutrients for growth rather than spending energy to fight pathogens, which collectively results in better growth performance. Concurrent supplementation of beneficial bacteria plus nutrients that help their growth balanced the immune responses in the gut by increasing the copy number of cytokines. Cytokines are proteins that orchestrate immune responses that the host mounts against pathogens. Certain cytokines regulate such responses by preventing the immune system from overreacting and mounting unnecessary reactions, thus preserving energy and nutrients for growth while reducing inflammation. Nutrient uptake from the gut lumen is facilitated by nutrient transporter proteins that reside on intestinal cells (enterocytes). Birds concurrently supplemented with beneficial bacteria and nutrients that help their growth in the gut increased the abundance of these proteins, resulting in improved nutrient uptake and performance compared to the control birds. Co-supplementation of beneficial bacteria and nutrient sources that help their growth modified the type and number of bacteria that are present in the gut lumen. The modified bacterial community were able to produce metabolites such as butyrate and propionate, which are beneficial for the health and growth of the intestinal cells, thus improving the bird's health and its performance. Overall, compared to beneficial bacteria alone, co-supplementation of beneficial bacteria with the nutrients that help their growth in the gut significantly reduced intestinal lesions and improved performance of broiler chickens during the production period. Moreover, dietary addition of these supplements improved gut barrier function by regulating the gene expression of tight junction proteins and gut mucosal immune responses as well as modifying the bacterial community of the gut. Therefore, such combination supplements hold promise in controlling necrotic enteritis in poultry and sustain good overall performance that translates into higher profitability to producers.

This thesis presents the first randomised controlled trial (RCT) to investigate whether supplemented probiotic bacteria-"live microorganisms that, when administered in adequate amounts, confer a health benefit on the host" (Sanders, 2008)-affect mood and other psychological outcomes in people presenting with low mood. Seventy-nine participants with at least moderate symptoms of depression were randomised in a double-blind manner to receive either a probiotic preparation containing Lactobacillus helveticus and Bifidobacterium longum or a matched placebo for eight weeks. The RCT phase was followed by an open label extension in which all participants were offered the active study product for a further 8 weeks. Participants were followed up at 6 months post-study. Based on the existing evidence from gut-brain axis research, and on models linking depression with inflammation, immune activation, low vitamin D levels, and the gut microbiota (outlined in Chapters 1 and 2), it was hypothesised that: the overall sample would have elevated levels of inflammatory biomarkers and low levels of vitamin D at baseline, and that this would be associated with scores on psychological and irritable bowel syndrome (IBS) outcome measures; that group differences (active treatment versus placebo) would be observed in scores on psychological outcome measures after eight weeks of probiotic intervention; that group differences would also be observed in blood levels of proinflammatory cytokines, hsCRP, vitamin D and BDNF, and scores on a measure of gut function/IBS, and that levels of these variables may predict or impact on treatment response; and that group differences would be observed on outcome measures at the point of the 6-month follow-up between those who continued to take the probiotic and those who discontinued probiotic use. In total, 58 of the 77 participants who provided baseline blood samples (75%) had at least one marker of inflammation elevated outside the normal reference range at baseline. Baseline vitamin D was approaching the deficient level, displayed a seasonal pattern, and was associated with severity on one measure of cognition. No significant differences were found between the active treatment and placebo groups on any psychological outcome measure, the measure of gut function or in the level of any blood-based biomarker in the randomised phase. Baseline vitamin D level was found to moderate treatment effect on several outcome measures. The results of the open label extension supported the lack of efficacy observed in the randomised phase, and also allowed for the comparison of efficacy over intervention periods of varying durations. The results of the follow-up at 6 months post-trial indicated that, while mean scores on psychological outcome measures remained lower than baseline, there was regression on some outcome measures after the study. When the participants who replied to the 6 month follow-up questionnaire were divided into groups based on their self-reported dominant treatment since the trial (probiotics/nutrition, standard treatment or no treatment) there was no difference in mood or other psychological outcomes among the groups at 6 months. The current trial found no evidence that this probiotic formulation is effective in treating the symptoms of depression or IBS, or in moderating the levels of inflammatory and other biomarkers in a sample recruited with moderate depression. This finding does not support the theory proposed in several narrative reviews which suggests probiotics as a possible intervention for depression and other mental health outcomes, but is supported by the systematic review of human probiotics studies presented in Chapter 3 which found overall limited evidence of probiotic efficacy for psychological outcomes. Future studies in the area should attempt to further broaden this field, in particular by recruiting samples with mild and/or non-chronic depression for interventional studies, or by approaching probiotics as a preventative or adjuvant treatment strategy for depression.

Colorectal cancer (CRC) is one of the most treatable cancers, with a 5-year survival rate of similar to 64%, yet over 50,000 deaths occur yearly in the United States. In 15% of cases, deficiency in mismatch repair leads to null mutations in transforming growth factor beta (TGF-beta) type II receptor, yet genotype alone is not responsible for tumorigenesis. Previous work in mice shows that disruptions in TGF-beta signaling combined with Helicobacter hepaticus cause tumorigenesis, indicating a synergistic effect between genotype and microbial environment. Here, we examine functional shifts in the gut microbiome in CRC using integrated - omics approaches to untangle the role of host genotype, inflammation, and microbial ecology. We profile the gut microbiome of 40 mice with/without deficiency in TGF-beta signaling from a Smad3 (mothers against decapentaplegic homolog-3) knockout and with/without inoculation with H. hepaticus. Clear functional differences in the microbiome tied to specific bacterial species emerge from four pathways related to human colon cancer: lipopolysaccharide (LPS) production, polyamine synthesis, butyrate metabolism, and oxidative phosphorylation (OXPHOS). Specifically, an increase in Mucispirillum schaedleri drives LPS production, which is associated with an inflammatory response. We observe a commensurate decrease in butyrate production from Lachnospiraceae bacterium A4, which could promote tumor formation. H. hepaticus causes an increase in OXPHOS that may increase DNA-damaging free radicals. Finally, multiple bacterial species increase polyamines that are associated with colon cancer, implicating not just diet but also the microbiome in polyamine levels. These insights into cross talk between the microbiome, host genotype, and inflammation could promote the development of diagnostics and therapies for CRC. IMPORTANCE Most research on the gut microbiome in colon cancer focuses on taxonomic changes at the genus level using 16S rRNA gene sequencing. Here, we develop a new methodology to integrate DNA and RNA data sets to examine functional shifts at the species level that are important to tumor development. We uncover several metabolic pathways in the microbiome that, when perturbed by host genetics and H. hepaticus inoculation, contribute to colon cancer. The work presented here lays a foundation for improved bioinformatics methodologies to closely examine the cross talk between specific organisms and the host, important for the development of diagnostics and pre/probiotic treatment.

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Various disorders closely linked to gut dysbiosis have been associated with poor dietary patterns. Dietary prebiotic fibers play an essential role in modulating the gut microbiome by enhancing the abundance of beneficial microorganisms and improving the production of short-chain fatty acids. Arabinoxylan (AX) is a major component of most dietary fibers and has been shown to exhibit potential prebiotic properties and modulate gut microbiome composition. This study aimed to investigate the in vitro impact of bioengineered wheat arabinoxylan on depression-inversely linked gut microbes and human gut microbiome diversity and metabolism. This study demonstrates the ability of bioengineered AX to stimulate the growth of depression-inversely linked gut bacterial species (Faecalibacterium prausnitzii and Lacticaseibacillus rhamnosus LGG). On the microbiome composition, the bioengineered AX induced an increased abundance of beneficial bacterial taxa (Bacteroides, Bifidobacterium, Anaerofustis, and Eubacterium) compared to the control and native AX. These effects on microbes translated into significant metabolic activity and produced primary SCFAs (acetate, butyrate, and propionate). The findings from this study suggest that bioengineered wheat arabinoxylan could be considered a promising strategy for fostering next-generation prebiotics targeting depression-inversely linked gut microbes and also supports the structure-function relationship between AX and the human gut microbiome.

<p dir="ltr">Dietary fibers have been observed to modulate the gut microbiome in ways that prevent and moderate human diseases and confer health benefits onto their human host. How dietary fibers do this is through their structure; gut microbes are equipped with a variety of differ- ent carbohydrate-active enzymes (CAZymes) that allow some to hydrolyze glycosidic bonds, thereby utilizing the dietary fiber. The more complex the dietary fiber, the more diverse the maintained gut microbiota may be, as specialist species may be required for complete hydrol- ysis. Therefore, increasing structural complexity of dietary fibers may increase gut microbial diversity and help prevent diseases. To understand if structural features impact the gut mi- crobiome, a set of resistant glucans varying in structures, including mixed-linkage -glucans, resistant maltodextrins (similar to type IV resistant starch) and polydextroses, which are comprised entirely of glucose, were used as substrates in an in vitro sequential batch fermen- tation using fecal microbiota form three healthy donors as inocula. I measured metabolic outputs, growth curves, and community structures by 16S rRNA amplicon sequencing, which I analyzed for through alpha and beta diversity differences and taxa that overrepresented and increased in each treatment. My results show that, depending on the donor and the resistant glucan, structure does significantly impact the concentrations of short-chain fatty acids (SCFAs) and other metabolites that are produced. Resistant glucan structure also impacts alpha and beta diversity to a degree and linear discriminant analysis (by LEfSe) results also support that specific species have preference towards substrates as well. Next, resistant glucans were supplemented into a high-fat diet, and compared these diets to a low- fat diet (LFD), high-fat diet with cellulose (HFD), and high-fat without cellulose (HWC) in a mouse study using C57BL/6J mice over 4 weeks. Increasing microbial diversity will not only increase diversity in the gut microbiome, but it will also provide protective effects in behavior such as helping to prevent anxiety. I measured weight, metabolic outputs, 16S community structure, changes in alpha and beta diversity, and differential abundances of OTUs and taxa by discriminant analysis Effect Size (LEfSe) and Metastats, and anxiety behaviors using open field and light/dark box tests. Microbial community structure was significantly different in treatment groups from controls. Anxiety for mice in tapioca dextrin 01 (TD01), tapioca dextrin 03 (TD03), and resistant maltodextrin (RMF) treatment groups were gen- erally increased, suggesting that the chemical structure of these resistant dextrins may alter the gut microbiome in ways that may influence behavior.</p><p dir="ltr">My overall results support the hypothesis that the fine structural features of dietary fibers do significantly impact the gut microbiome by selecting for specific microbiota, and may even impact cognition and behavior.</p>