Despite the association of the human gut microbiome and various diseases, a systematic definition of what constitutes a healthy human gut microbiome has not been established. This is crucial for microbiome research as it provides a basis for evaluating whether a given microbiome sample may deviate from the homeostasis state and is thus prone to the development of chronic diseases. This work aims to propose one such definition by using species/strain-resolved Genome-scale (GEM) models of metabolism. More specifically, we have constructed sample-specific GEMs from 30 healthy subjects using the taxonomic profiling of fecal metagenomic samples. We then computationally simulated these GEMs under a relevant diet (a supplemented typical Western diet) to determine which microbes in each sample contribute to the production of 17 key metabolites curated from literature and reported to be produced and secreted by the gut microbiota of healthy subjects. Beyond this pilot study, we plan to expand our analyses by creating samples-specific GEMs for a large-scale database of all publicly available metagenomic data from healthy subjects (~2,500 samples so far). We will additionally identify a core set of microbial species/strains that are necessary to perform all essential functions of a healthy microbiome. Taken together, this project offers a new paradigm to establish a healthy baseline microbiome definition by identifying generalized and personalized microbial blueprints that could serve as viable markers of health.

Complex microbiomes are part of the food we eat: they are naturally present on the raw material, they merge along the food system, or they can be intentionally inoculated. Whether their presence is desired, such as in case of fermentation or probiotic supplementation, or undesired, in case of pathogenic or spoilage microbes, depends on who they are and what they are doing and therefore several studies investigated the microbiota of specific foods. However, the diversity of food microbiomes remains largely unexplored and similar studies present inconsistencies in methods and results. The study of the food microbiome is relevant also in light of the human microbiome and its multifaceted connection to hosts’ health status. Diet is one of the main factors influencing the human microbiome and many studies investigated how nutrition impacts the endogenous microbial communities both in the gut and in the oral cavity. Nevertheless, they largely overlooked the possibility of direct contribution of food-origin microorganisms. The primary aim of my PhD was the comprehensive characterization of foodborne microbial communities with the ultimate goal of estimating their impact on the human microbiome. This research intended to be humble contribution to the global effort in understanding the microbial sources building these composite ecosystems inhabiting the human body. In order to explore the food microbiome diversity, I selected and collected 583 publicly available food (shotgun) metagenomes and integrated them with 1950 newly sequenced food metagenomes. Through an assembly-based pipeline, I reconstructed >10,000 metagenome-assembled genomes (MAGs) that resulted in 290 previously undescribed taxa and, hence, firstly observed in this work. I characterized the composition of microbial communities in food, proving strong specificity across food categories and types through statistical analysis and machine learning approaches. The uniformly and coherently processed curated metadata, taxonomic profiles and reconstructed genomes are publicly available in a resource called curatedFoodMetagenomicData (cFMD). To investigate the presence of food-associated bacteria among human oral and gut microbiomes, I analyzed 20,000 human metagenomes available in curatedMetagenomicData (cMD) through the same expanded pipeline used for food samples. The overlap between food and human microbiomes showed high variations according to host characteristics and the food prevalent species accounted on average for 3% of relative abundance in adult microbiomes. I recognized 43 bacterial species prevalent in both environments that were investigated at the strain level, showing close genomic similarities of strains found both in food and humans.To our knowledge this was the first attempt to investigate the global food microbiome and to estimate its involvement in human microbiome at a large-scale. Our results showedan expansion of known and yet-to-be-isolated species associated with food microbiomes, their characterization to uncover microbial diversity and provide insights on links with the human microbiome, and the release of a publicly-available resource as cFMD that will support the use of metagenomics in food microbiology and food safety, certificationand quality control applications.

Plants are inhabited by diverse species of bacteria and fungi, which affect plant health and fitness. Endophytes are bacteria or fungi that live within plant host tissues without causing symptoms of disease, and mediate important plant traits in agriculture, such as nutrient acquisition, disease resistance, and abiotic stress tolerance. However, we know little about the general ecology of endophytes, including which factors determine their compositions within plants. Seedborne transmission may represent an important source of bacterial and fungal endophytes, which can significantly impact the plant microbiome and plant traits. However, seeds are also a vehicle for transmission of plant pathogens. Seeds are commonly treated to control against seedborne pathogens, and increasingly bacteria and fungi are inoculated onto seeds to serve as biological control against pathogens. My dissertation explores the theoretical and applied ecology of seedborne endophytes of maize, including their interactions with pathogenic Fusarium fungi, and with seed treatments designed to control Fusarium. In Chapter II, I examine factors that affect the transmission of seedborne fungal endophytes and Fusarium into maize seedlings, including the influence of soil microbiota, and the impact of disinfection and biological control seed treatments. In Chapter III, I determine the long-term effects of seed disinfection and biological control inoculants on maize bacterial and fungal endophytes and Fusarium pathogenicity across three different farms. In Chapter IV, I recruit maize seed growers across the Pacific Northwest and U.S. to participate in a broadscale study of seedborne endophytes. Across submitted seed samples, I find commonly occurring seedborne endophyte taxa, and delineate how maize varieties interact with environmental factors to affect the composition and diversity of seed-associated endophytes in seeds. Throughout these chapters, I explore the potential applications of seedborne endophytes in agriculture, particularly as a source for biological control against Fusarium in maize, and speculate how seed treatments can have significant, lasting impacts on the plant microbiome.

The skin microbiome is a critical component of human health, however, little is understood about the daily dynamics of skin microbiome community assembly and the skin’s potential to acquire microorganisms from the external environment. I performed a series of microbial transfers using three skin habitat types (dry, moist, sebaceous) on human subject volunteers. Microbial communities were transferred to recipient skin using a sterile swab 1) from other skin sites on the same individual, 2) from other skin sites on a different individual, 3) and from two environmental donor sources (plant leaf surfaces and farm soil). With these experiments I was able to test for the presence of initial transfer effects and for the persistence of those effects over the time period sampled (2-, 4-, 8-, and 24-hours post-transfer). The sebaceous skin community was associated with the strongest initial effect of transfer and persistence on the moist recipient skin site, and to a lesser extent the dry skin site. The soil donor community when transferred to dry skin resulted in the strongest initial transfer effect and was persistent over 8- and even 24-hours post-transfer. These experiments are the first in scope and scale to directly demonstrate that dispersal from other human or environmental microbial communities are plausible drivers of community dynamics in the skin microbiome.

The gut and the brain are in constant communication through pathways that include the immune system, the nervous system, neurotransmitters, and hormones. Modifications in the gut, especially the gut microbiome, have the potential to cause changes in the brain resulting in behavioral and cognitive changes. A healthy and diverse microbiome, which may be achieved by a high fiber diet or probiotic or prebiotic treatments, is associated with improvements in cognition. Gut dysbiosis and a decrease in diversity of the microbiota, which may be caused by a western diet or antibiotic treatments, is associated with cognitive decline and decreased memory. There are many possible pathways through which these changes in the gut act to change cognition, including the immune system, the expression of brain derived neurotropic factor, metabolites such as short chain fatty acids, gut hormones, and neurotransmitters. If researchers can decipher which pathways are involved in modifying cognition, they may be able to identify treatments that can help improve memory and specifically decrease age-related cognitive decline.

abstract: Introduction: Cystic fibrosis (CF) is the most common life-shortening autosomal recessive genetic disease affecting Caucasians. The disease is characterized by a dysfunctional cystic fibrosis transmembrane regulator (CFTR) protein and aberrant mucus accumulation that subsequently alters the physicochemical environment in numerous organ systems. These mucosal perturbations have been associated with inflammation and microbial dysbiosis, most notably in the lungs and gastrointestinal (GI) tract. Genistein, a soy isoflavone and dietary polyphenol, has been shown to modulate CFTR function in cell cultures and murine models, as well exert sex-dependent improvement of survival rates in a CF mouse model. However, it is unknown whether dietary genistein affects gut microbiome diversity and community structure in cystic fibrosis. This study sought to examine associations between dietary genistein treatment and gut microbiome diversity and community structure in a murine model of CF. Methods: Twenty-four male and female mice homozygous for the DF508 CFTR gene mutation were maintained on one of three diet regimens for a 45-day period (n=11, standard chow; n=7, Colyte-treated water and standard chow; n=6, 600 mg dietary genistein per kg body weight). One fecal pellet was collected per mouse post-treatment, and microbial genomic DNA was extracted from the fecal samples, quantified, amplified, and sequenced on the Illumina MiSeq platform. QIIME 2 was used to conduct alpha- and beta-diversity analyses on all samples. Results: Measures of alpha-diversity were significantly decreased in the dietary genistein group as compared to either standard chow or Colyte groups. Measures of beta-diversity showed that community structure differed significantly between dietary treatment groups; these differences were further illustrated by distinct clustering of taxa as shown by principal coordinates analysis plots. Conclusion: This 3-arm parallel experimental study showed that dietary genistein treatment was associated with decreased microbial diversity and differences in microbial community structure in DF508 mice. / Dissertation/Thesis / Masters Thesis Nutrition 2019

The microflora inhabiting the human gastrointestinal tract can be considered an essential 'metabolic organ', in a symbiotic relationship with its host. Due to the low cultivability and inappropriate sampling methodology the microflora is poorly explored and ill-defined. Preliminary, molecular-based research at the University of Waikato revealed the presence of 16S rRNA gene sequences originating from novel Mollicute-like species inhabiting the human GI tract. A ~830bp 'consensus' sequence representing these novel Mollicute-like sequences was classified within the Mollicute Genus Anaeroplasma the type species of which is Anaeroplasma abactoclasticum. It also displayed near exact matches with 16S rRNA sequences obtained from the human GI tract and matches of high similarity to those from the mouse GI tract in the NCBI database. This thesis describes an attempt to design and create primers that would amplify and characterize full-length versions of these Mollicute-like sequences from samples obtained from the mucosal surface of the human gastrointestinal tract. Primers sets targeted extended 5' and 3' versions of these novel 'known' sequences and were designed from sequence matches found in the preliminary work and other related sequences from the NCBI database. The attempt to amplify a full-length version of these novel Mollicute-like sequences was proven to be unsuccessful. No sequences were classified within the Genus Anaeroplasma, although 81% of amplicons from the 5' extending primer sets were classified within the same division as the Mollicutes, the Firmicutes, only 6% of the sequenced amplicons from the 3' extending primer set belonged to this division. Phylograms containing these 'relevant' sequences and the 'consensus' sequence grouped the 'consensus' sequence separately, indicating a lower relatedness than would have been seen if any of the amplicons contained the 'consensus' sequence.

Humans harbour a diverse suite of microorganisms in and on their bodies. These microorganisms collectively amount to 10 times more cells than human cells in the body, and their combined genomes have more than 100 times more genes than the human genome does. Despite our understanding of the composition, diversity, and abundance of microorganisms of the human body, it is surprising how little we know about the structure and function of the human microbiome. Here, I use network structure to describe interactions among human-associated microbiota and the human body by exploring differences in structure of human microbiomes across five regions of the body and the robustness of these networks to perturbations. My results show that positive interactions among microbiota are extremely important in structuring microbiome networks and those structural aspects of microbiome networks play a major role in their response to perturbations.

Metabolic syndrome is a disease affecting 25% of North America’s population causing strain on the medical systems. With diet and exercise, genetics, environment, and the gut microbiota all being targeted as potential causes of the disease, there is a lack of consensus on the exact aetiology and pathophysiology. With improved methods in bioinformatic sequencing of faecal bacterial DNA in recent years, our research indicates that a dysbiosis in the gut microbiome is both necessary and sufficient in causing immunological changes in the host in order for the development of metabolic syndrome, T1Ds, and T2Ds. Specifically, our data indicates these shifts in microbiota occur prior to the onset of disease, and produce the disease regardless of diet and genetics. The findings in the current study indicate that future research towards manipulating the gut microbiota to prevent disease, as well as using the faecal bacteria as a screening tool should be pursued.

The cpn60 gene is a DNA barcode for bacteria. Recently, the PCR primers that have been used extensively to amplify the cpn60 Universal Target (UT) region of bacteria were redesigned to improve their utility for fungal taxa. Additional novel primers were designed to amplify other regions of the cpn60 gene, specifically from fungal genomes. Design of the redesigned and novel primers was based on 61 nucleotide full-length cpn60 reference sequences available in 2012, including Ascomycota (51), Basidiomycota (5), Chytridiomycota (2), Glomeromycota (1), and Oomycota (2). The research described here investigated the utility of these primers for detecting and identifying fungal taxa and for profiling mixed communities of bacteria and fungi. The redesigned primers were used to discover cpn60 UT sequences for Ascomycota (1), Basidiomycota (2), and Chytridiomycota (1). The novel primers were used to discover new cpn60 sequence data for Ascomycota (3), Basidiomycota (1), and Zygomycota (1). To be adopted for use in studies of microbial communities that are predominantly bacterial, the redesigned cpn60 UT primers must perform at least as well as the original primers for bacterial profiling. Bacterial profiles, created using the original and redesigned primers and two DNA template samples created by pooling DNA extracts from vaginal swabs from individual women, were compared. These included comparisons of diversity indices, rarefaction curve analysis and Operational Taxonomic Unit abundances. Diversity indices and rarefaction curve analysis for bacterial profiles with original and redesigned primers were similar. OTU abundance estimates with the original and redesigned primers were compared at higher and lower taxonomic levels. The overall patterns produced were similar. For one template only, the phylum Bacteroidetes had a greater apparent abundance with the original primers than with the redesigned primers. The greater apparent abundance of Bacteroidetes taxa was balanced by a lesser apparent abundance of taxa that were not assigned to a phylum. These differences may reflect differences in the performance of the two primer sets. At lower taxonomic level, most OTU were represented with apparently equal abundances with redesigned and original primers in same template. Very few OTU were represented with different proportional abundances with redesigned and original primers. Different OTU having same reference cpn60 UT sequence as best hit were sometimes represented by different proportional abundance with same primer in same template that made the analysis difficult. On the whole, the redesigned cpn60 UT primers behaved at least as good as the original cpn60 UT primers. The overall results showed that the redesigned and novel primers used in this study had substantial utility for the identification of fungal samples and mixed microbial communities.