Recent 16S rRNA gene sequencing studies of the duodenal and fecal microbiota have revealed alterations in the abundance of specific bacterial groups in dogs with gastrointestinal (GI) disorders. The aim of this study was to establish a panel of quantitative real-time PCR (qPCR) assays for the evaluation of specific bacterial groups in fecal samples of healthy dogs, dogs with chronic enteropathies (CE), and dogs with acute hemorrhagic diarrhea (AHD). Fecal samples from 242 healthy dogs, 118 dogs with CE, and 57 dogs with AHD were analyzed using qPCR assays targeting Faecalibacterium spp., Turicibacter spp., Bifidobacterium spp., Lactobacillus spp., Streptococcus spp., Ruminococcaceae, C. perfringens, E. coli, gamma-Proteobacteria, Bacteroidetes, and Firmicutes). Differences in bacterial abundance among the three groups were evaluated using a Kruskal-Wallis test followed by a Dunn's post-test. A Bonferroni correction was used to correct for multiple comparisons and an adjusted p<0.05 was considered for statistical significance. Faecalibacterium spp., Turicibacter spp., and Ruminococcaceae were significantly decreased in CE and AHD compared to healthy dogs (p<0.001 for all). Lactobacillus spp. and Streptococcus spp. were significantly increased in dogs with CE (p<0.001 for both) when compared to the healthy dogs. In contrast, Lactobacillus spp. and Streptococcus spp. were significantly decreased in dogs with AHD compared to healthy dogs (p<0.01 and p<0.05, respectively) and also when compared to the dogs with CE (p<0.001 for both). C. perfringens and E. coli were significantly increased in dogs with AHD (p<0.001 and p<0.01, respectively), when compared to healthy dogs. E. coli was also significantly increased in dogs with CE when compared to the healthy dogs (p<0.001). Bacteroidetes were significantly lower in dogs with CE compared to healthy dogs (<0.001). Firmicutes were significantly higher in healthy dogs in comparison to dogs with AHD (p<0.05). Bifidobacterium spp. and gamma-Proteobacteria were not significantly different among all three groups of dogs. In conclusion, the qPCR panel employed here revealed a fecal dysbiosis in dogs with CE and AHD when compared to healthy dogs. These results are similar to recently reported findings using molecular sequencing approaches. Quantification of these bacterial groups by qPCR may be a useful adjunct for the diagnosis or monitoring of gastrointestinal disease in dogs.

Cryptosporidium parvum is an apicomplexan protozoan parasite commonly causing diarrhea, particularly in infants in developing countries. The research challenges faced in the development of therapies against Cryptosporidium slow down the process of drug discovery. However, advancement of knowledge towards the interactions of the intestinal ecosystem and the parasite could provide alternative approaches to tackle the disease. Under this perspective, the primary focus of this work was to study interactions between Cryptosporidium parvum and the intestinal ecosystem in a mouse model. Mice were treated with antibiotics with different activity spectra and the resulted perturbation of the native gut microbiota was identified by microbiome studies. In particular, 16S amplicon sequencing and Whole Genome Sequencing (WGS) were used to determine the bacterial composition and the genetic repertoire of the fecal microbial communities in the mouse gut. Following alteration of the microbial communities of mice by application of antibiotic treatment, Cryptosporidium parasites were propagated in mice with perturbed microbiota and the severity of the infection was quantified. This approach enabled the prediction of the functional capacity of the microbial communities in the mouse gut and led to the identification of bacterial taxa that positively or negatively correlate in abundance with Cryptosporidium proliferation.

The human microbiome, the complex and dynamic ecosystem that populates our body, performs essential functions such as aiding digestion and protecting us from harmful pathogens. An increasing number of diseases are found to be associated with a shift of balance - or dysbiosis - of the microbiome. However, we still know little about this delicate balance and how it depends on different microbial functions. In this thesis project, I used metagenomic sequencing data to study the variability of microbes and their functions in different areas of the human body. First, in an attempt to characterize the dysbiosis associated with periodontitis, I examined the microbial community of the oral cavity in presence and absence of this chronic inflammatory disease. Specifically, I catalogued the phylogenetic signatures composed of tetramer nucleotide frequencies and observed that the disease state occupies a much narrower region than the healthy one. This result suggests that upon onset of the disease, through host cell invasion, pathogenic bacteria may find a more consistent environment for their parasitic lifestyle. Motivated by these findings, I sought further evidence of an environment-specific use of metabolic functions in the oral and gut communities. Rather than focusing on the abundance of individual metabolic functions, I evaluated their diversity, i.e., the extent to which these functions are performed by different classes of organisms. My hypothesis was that such diversity may confer increased robustness to taxonomic variability. Using metagenomic sequencing data and NCBI's Protein Clusters database, I characterized the multiplicity of gene families associated with a given metabolic function. I found that different human body sites display different degrees of metabolic functional diversity, as assessed by Shannon entropy. For some well-studied gene functions, such as those involved in glycolytic pathways, I found entropy signatures consistent with the known degree of oxygen availability of different environmental niches. Conversely, in an unsupervised analysis, I identified functions with nontrivial entropy signatures. These results pave the way for a new way to inspect human microbiome activity, and could help understand its functional resilience and suggest ways to shift its balance towards healthy configurations.

There is an increasing interest in how therapeutic drugs could alter the human gut microbiota composition and function. While some knowledge is accumulating on the antimicrobial impact of some psychotropics on isolated strains or the gut microbiota of animal models, information about other classes of psychotropics and representative species from the human gut is poorly investigated. The antimicrobial effect of psychotropic drugs is usually neglected as a confounding factor when investigating gut microbiome biomarkers, knowing that patients are generally put in long-term medication. The purpose of the present study was to investigate (in vitro and ex-vivo) the antimicrobial activity of some oral commonly prescribed psychotropics from different therapeutic classes on colonic microbiota diversity and metabolism and the potential capacity of probiotics to alleviate related dysbiosis. The findings of this study revealed an important in vitro inhibitory activity of psychotropic drugs, which were also expressed as drastic alterations in gut microbiota composition ex-vivo. Indeed, the relative abundances of Firmicutes and Actinobacteria were lowered while the Proteobacteria population was increased. Families of Lachnospiraceae, Lactobacillaceae, and Erysipelotrichaceae were also declined by psychotropics (aripiprazole) treatment. These microbial changes were translated into a decrease of the major SCFA (butyrate, acetate, and propionate) at the metabolic level. The addition of a probiotic combination (Lactobacillus rhamnosus and Bifidobacterium longum) concomitantly with a psychotropic (aripiprazole) had a protective effect by attenuating the decline of microbiota composition and increasing the concentrations of SCFA. These findings provide evidence that psychotropics, through their antimicrobial effect, have the potential to alter the human gut microbiota composition and metabolism, while probiotics can mitigate the related dysbiosis.

Since the invention of high-throughput sequencing, the majority of experiments studying bacterial microbiomes have relied on the PCR amplification of all or part of the gene for the 16S rRNA subunit, which serves as a biomarker for identifying and quantifying the various taxa present in a microbiomic sample. Several computational methods exist for analyzing 16S amplicon based metagenomics, but the most commonly used bioinformatics tools are unable to produce quality genus-level or species-level taxonomic calls and may underestimate the degree to which such calls are possible. In this thesis, I have used 16S sequencing data from mock bacterial communities to evaluate the sensitivity and specificity of several bioinformatics pipelines and genomic reference libraries used for microbiome analyses, with a focus on measuring the accuracy of species-level taxonomic assignments of 16S amplicon reads. With the efficacy of these tools established, I then applied them in the analysis of data from two studies into human microbiomes. I evaluated the metagenomics analysis tools Qiime 2, Mothur, PathoScope 2, and Kraken 2, in conjunction with reference libraries from GreenGenes, Silva, Kraken, and RefSeq, using publicly available mock community data from several sources, comprising 137 samples with varied species richness and evenness, several different amplified regions within the 16S gene, and both DNA spike-ins and cDNA from collections of plated cells. PathoScope 2 and Kraken 2, both tools designed for whole genome metagenomics, outperformed Qiime 2 and Mothur, which are theoretically specialized in 16S analyses. I used PathoScope 2 to analyze longitudinal 16S data from infants in Zambia, exploring the maturation of nasopharyngeal microbiomes in healthy infants, establishing a range of typical healthy taxonomic profiles, and identifying dysbiotic patterns which are associated with the development of severe lower respiratory tract infections in early childhood. I used Qiime 2 to analyze 16S data from human subjects in a controlled dietary intervention study with a focus on dietary carbohydrate quality. I correlated alterations in the gut microbiome with various cardiometabolic risk factors, and identified increases in some butyrate-producing bacteria in response to complex carbohydrates. I also constructed a metatranscriptomics pipeline to analyze paired rRNA-depleted RNAseq data. My evaluation of 16S methods should improve 16S amplicon analyses by advocating for the modernization of computational tools; my analysis of infant nasopharyngeal microbiomes lays groundwork for future predictive models for childhood disease and longitudinal microbiomic studies; my analysis of gut microbes illuminates the mechanisms through which bacteria can mediate cardiovascular health. Taken together, the research I present here represents a significant contribution to 16S metagenomics and its application to epidemiology, clinical nutritional science.

The host microbiome (mainly bacteria) is essential for host immune function, metabolism, and digestion. Alterations in these microbes, known as dysbiosis, generally results in adverse effects to the host, including diseases. Dysbiosis can be induced from exposures to various anthropogenic contaminants including constituents of municipal wastewater treatment effluents (MWWEs), namely, pharmaceuticals, antibiotics, and excess nutrients. Despite MWWEs being one of the largest dischargers to aquatic ecosystems, impacts of these contaminants on exposed organism microbiomes, especially in aquatic insects, is unclear. In addition, some aquatic contaminants may transfer to riparian habitats through predation on emergent insects that were exposed to contaminants as larvae, and subsequently alter microbiomes of terrestrial predators. Our study evaluated whether MWWEs altered microbiomes of freshwater larval and adult insects and their riparian spider predators using effluent-associated bacteria and stable nitrogen isotopes (δ15N) to confirm effluent exposure. We analyzed microbiome compositions through sequencing of the V3-V4 hypervariable region of the 16S rRNA gene and analyzed food web dynamics with stable carbon (δ13C) and nitrogen (δ15N) isotopes. We found that insects and spiders were enriched in δ15N at one site downstream of wastewater outfalls, indicating exposure to effluents and transfer of nutrients to terrestrial ecosystems. Microbiomes of most larval and adult insects were altered downstream of wastewater outfalls and had lower relative abundances of endosymbiont bacteria, shifts in bacterial diversities, increases in abundances of effluent-associated bacteria, and downregulation of some biosynthesis pathways than those collected at upstream sites. However, spider microbiomes had little evidence of dysbiosis, and were distinct from those of adult insects, despite a close association in their isotopic signatures. Overall, this study provides evidence of biological impacts from MWWEs to exposed insects and suggests that changes in microbial communities of invertebrates may be used as an effective indicator of effluent exposure as part of monitoring frameworks. / Thesis / Master of Science (MSc) / Municipal wastewater treatment facilities release contaminants into aquatic ecosystems that may affect the health of exposed organisms, including their microbiome, which contains bacteria essential for host digestion and immune function. Alterations in microbiomes of contaminant-exposed aquatic insects are poorly characterized and such effects may be transferred to terrestrial ecosystems through emergent insects. This study evaluated microbiomes and food web dynamics of freshwater insects and riparian spiders upstream and downstream of wastewater treatment facilities in the Bow River, AB. Results indicate that microbiomes of some downstream aquatic insects had lower relative abundances of endosymbiont bacteria, shifts in bacterial diversities, and increases in abundances of effluent-associated bacteria than those collected at upstream sites, but no such changes were observed in the spider predators. This study improves our understanding of how freshwater insect microbiomes are altered by municipal wastewater effluents and suggests that directly effluent-exposed organisms are more at risk of dysbiosis.

For millions of years, the nutrient exchange symbiosis between corals and their endosymbiotic algae has formed the foundation of the ecological success of coral reefs. Yet, in recent decades anthropogenic climate change is increasingly destabilizing this symbiosis, and thus the reefs that rely on it. High-temperature anomalies have caused mass mortality of corals due to repeated coral bleaching, the expulsion or digestion of symbionts by the host during stress. Hence, in-depth knowledge of the cellular processes of bleaching is required to conceive strategies to maintain the ecological functioning of coral reefs. In this thesis, we investigated the role of symbiotic nutrient cycling in the bleaching response of corals. For this, we examined the mechanisms that underlie the functioning of the symbiosis in a stable state and how heat stress affects these metabolic interactions during coral bleaching. Our findings reveal that the functioning of the coral – algae symbiosis depends on the resource competition between host and symbionts. In a stable state, symbiotic competition for ammonium limits nitrogen availability for the algal symbiont, thereby ensuring symbiotic carbon translocation and recycling. During heat stress, however, increased metabolic energy demand shifts host metabolism from amino acid synthesis to degradation. The resulting net release of ammonium by the host, coupled with the stimulated activity of associated nitrogen-fixing microbes, substantially increases nitrogen availability for algal symbionts. Subsequently, stimulated algal growth causes selfish retention of carbon, thereby further reducing energy availability for the host. This positive feedback loop disturbs symbiotic nutrient recycling, eventually causing the collapse of carbon translocation by the symbiont. Hence, heat stress causes shifts in metabolic interactions, which directly and indirectly destabilizes the symbiosis, and ultimately undermines the ecological benefits of hosting algal symbionts for corals. In summary, this thesis shows that integrating symbiotic nutrient cycling into our conceptual understanding of coral bleaching is likely to improve our ability to predict coral bleaching in light of environmental conditions and may ultimately help to conceive new strategies to preserve coral reef functioning.

No description available.

Crohn’s disease (CD) is an inflammatory disease of the gastrointestinal tract attributed to an aberrant immune response to environmental and microbial triggers. Individuals with CD exhibit an enrichment of pro-inflammatory strains of Adherent-Invasive E. coli (AIEC) and often report a relapse of symptoms following a period of acute psychological stress. Despite a known immunosuppressive role, the mechanism by which stress contributes toward the development and progression of intestinal inflammation remains unknown. Here, we use a well characterized model of restraint stress to investigate the influence of psychological stress on host protection against a CD-associated strain of AIEC. We found that stress results in profound intestinal dysbiosis, allowing for a complete dominance of Enterobacteriaceae. Interestingly, while stress alone drives a state of low-grade inflammation and loss of barrier integrity in the gut, in the presence of a pathobiont strain of AIEC, stress drives a substantially heightened inflammatory response which exacerbated the resultant loss of barrier integrity. Moreover, we have found stress induces an augmented nutritional immune response, providing AIEC a competitive niche against commensal bacteria lacking alternative methods of iron uptake. Further, we see that stress-induced glucocorticoids mediate broad apoptosis of the CD45+CD90+ lymphocytic population in the gut. The loss of this population prevents an appropriate IL-22 mediated response to dysbiosis. Accordingly, blocking glucocorticoid signalling or exogenous administration of IL-22 prevents the stress-induced expansion of AIEC. This work underscores the complex nature of psychological stress such that the combination of iron limitation and glucocorticoid mediated immune attrition are simultaneously required for the stress-induced expansion of AIEC. These findings present novel insight into the mechanistic consequences of glucocorticoid signalling on impaired immune function and the provision of an inflammatory environment, resulting in a distinct impact on CD susceptibility. As such, deeper insight regarding the complex underpinnings of CD will assist in efforts to design representative models and will strengthen the discovery of targeted therapeutics. / Thesis / Master of Science (MSc) / Crohn’s disease (CD) is an inflammatory disease of the gastrointestinal tract resulting from an exaggerated immune response. CD patients often report a relapse of symptoms following a period of psychological stress and are at an increased likelihood of having pro-inflammatory strains of E. coli within their gut. Here, we use a model of restraint stress to investigate how psychological stress modulates the abundance of bacterial species associated with CD. We found stress results in the limitation of essential nutrients, allowing for an outgrowth of E. coli. Further, stress hormones lead to the loss of a protective immune response in which E. coli expansion can be prevented by blocking these hormones or restoring immune signalling. Together, we conclude that stress leads to immune cell death and creates an iron limited environment that favours E. coli expansion. Such work begins to uncover the functional consequence of stress and its’ role in disease progression.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第22499号 / 農博第2403号 / 新制||農||1077(附属図書館) / 学位論文||R2||N5279(農学部図書室) / 京都大学大学院農学研究科応用生物科学専攻 / (主査)教授 佐藤 健司, 教授 菅原 達也, 准教授 豊原 治彦 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM