A protocol was developed to study the nasal microbiome applying a cpn60 universal target (UT) amplicon as a bacterial phylotyping marker using next generation sequencing. The UT can be used to identify organisms in metagenomic samples to the species level. This study aimed to determine the typical composition of anterior nares microbiota for developing infants over time, and to explore diversity of the infant nasal microflora and the relation to microflora observed within their primary caregivers. Nares swabs were collected from 40 two-week old infants and their primary caregivers over a 1-year period using a single swab in one nare for each subject. These samples were treated to diminish contaminating human DNA, and bacterial template DNA was augmented using a whole-genome linear augmentation procedure. The cpn60 UT target was PCR amplified using degenerate primers and sequenced using high throughput 454/GS FLX pyrosequencing. Reads were quality-filtered and matched to the cpn60 database using an 80% nucleotide identity cutoff. Diversity and metagenomics analyses were performed using mothur v1.27.0 and SIMCA v. 13. Throughout the first year of life, infant nares microflora increased in diversity. Infants showed differential representation of organisms compared with their primary caregivers. The nares microbiota predominantly belong to the phyla Actinobacteria, Firmicutes, and Proteobacteria. Individuals appear to have only one or two predominating genera; however intra-subject variability was noted. High variability between study subjects and within study subjects over time was also observed.

By continually supplying nutrients, a turbidostat presents a steady environment to analyze bacterial growth dynamics. This makes it possible to model microbial community succession in a simple and more realistic way than is possible with batch culture. The problem with current commercial turbidostats are their industrial size and price. With the use of 3D printed parts, printed circuit boards and laser cut pieces, all readily obtained online and in collaboration with laboratories here at McMaster University, I've created a relatively cheap custom turbidostat, ideally suited for longitudinal studies of microbial consortia that can accommodate eight separate experiments simultaneously. I have modified the design to enable microbial growth at 37°C under anoxic conditions, by changing how the growth media and gas is handled. I have also improved sample collection to make it more convenient and flexible. Cell growth dynamics were interrogated separately with one strain of a facultative anaerobe (E. coli) and one strain of an obligate anaerobe (Bacteroides thetaiotaomicron) bacteria. For the individual strains, real-time optical density and dilution rate vs time graphs were created showing that these microbes can be reproducibly cultured, holding steady optical density rates for extended periods of time. Future directions include culturing a complex community without contamination by inoculating the system with microorganisms from an infant stool sample. Community composition and metabolite dynamics could then be analyzed by sampling over time. / Thesis / Master of Science (MSc)

Les biofilms bactériens sont des communautés répandues, dans lesquelles la densité cellulaire élevée, la diffusion réduite et la structure hétérogène favorisent les contacts physiques et métaboliques entre les bactéries et induisent de nouveaux comportements par rapport aux microorganismes individuels. [Si] la plupart des connaissances actuelles sur la formation du biofilm et le métabolisme découlent des études réalisées dans les modèles bactériens aérobies pathogènes et environnementaux, […] il existe peu d'informations sur la formation de biofilm dans des anaérobies strictes en général et des anaérobies commensaux associés à l'hôte de la gastro-microbiota intestinal en particulier. […] Nous avons utilisé des tests de biofilm in vitro statiques et dynamiques combinés avec des approches génétiques et transcriptomiques pour étudier les mécanismes moléculaires de la formation de biofilm et du métabolisme dans un prothèses intestinales Bacteroides thetaiotaomicron. Notre écran initial sur 35 souches de B.thetaiotaomicron […] a révélé une capacité généralisée de cette espèce à former un biofilm. Cependant, la production de biofilm semblait réprimée […] dans le VPI 5482 de type sauvage de référence et cela nous a poussé à développer les travaux de thèse selon deux axes principaux : l'identification de conditions induisant la formation de biofilm in vitro dans VPI 5482 et l'identification et la caractérisation de mutants transposons avec augmentation de la capacité du biofilm par rapport à WT VPI 5482. Après avoir testé divers composés qui sont pertinents pour l'environnement intestinal, nous avons montré que les sels biliaires […], induisent la formation de biofilm chez VPI 5482 et nous avons utilisé le RNAseq pour identifier les fonctions impliquées dans la formation de biofilm induite par le sel biliaire. Les résultats de RNAseq montrent que les bactéries dans le biofilm augmentent l'expression des gènes impliqués dans la glycosylation de la surface cellulaire et présentent un modèle d'expression spécifique des opéron de synthèse capsulaire, suggérant que les polysaccharides de surface cellulaire pourraient jouer un rôle clé lors de la formation du biofilm dans B. thetaiotaomicron. Nous avons ensuite utilisé la mutagenèse du transposon pour révéler les facteurs d'adhérence impliqués dans la formation du biofilm en présence de sels biliaires, ce qui a conduit à l'identification d'un opéron non caractéristique (BT3560-2) impliqué putativement dans le transport de micronutriments dépendant de TonB. Même si les cibles des systèmes de transport identifiés par mutagenèse et RNAseq sont […] inconnues, leur caractérisation supplémentaire peut conduire à l'identification des voies réglementaires qui régissent la formation du biofilm. En utilisant la mutagenèse du transposon pour débloquer l'adhérence dans le pauvre biofilm B. thetaiotaomicron VPI 5482, nous avons identifié deux loci - un putatif V Mfa1 pili operon BT3148-7 et un opérat de synthèse de capsule 4 (CPS4) impliqué dans la formation de biofilm indépendamment des sels biliaires. Sur la base de l'homologie structurale avec les pilins mfa1, BT3148 et BT3147 constituent vraisemblablement une structure pilus qui requiert une troncature C-terminale de la pilule BT3147 de la tige prédite pour l'allongement et l'augmentation conséquente de la formation du biofilm. La microscopie électronique continue combinée à la détection d'immunoglole du pilus putatif devrait élucider complètement le mécanisme de formation de biofilm médié par BT3148-7. Le deuxième facteur de biofilm identifié indépendant de la bile est la capsule 4 (CPS4). Comme la suppression du CPS4 à l'état sauvage a conduit à une forte production de biofilm, nous avons émis l'hypothèse que le CPS4 pourrait masquer une adhésine putative. / Bacterial biofilms are widespread communities, in which high cell density, reduced diffusion and heterogeneous structure favor physical and metabolic contacts between bacteria and induce novel behaviors as compared to individual microorganisms. Most of the current knowledge about biofilm formation and metabolism is derived from the studies performed in pathogenic and environmental aerobic bacterial models, by contrast, there is little information on biofilm formation in strict anaerobes in general, and host-associated commensal anaerobes of the gastro-intestinal microbiota in particular. In this project we used static and dynamic in vitro biofilm assays combined with genetic and transcriptomic approaches to study molecular mechanisms of biofilm formation and metabolism in a prominent gut commensal Bacteroides thetaiotaomicron. Our initial screen on 35 B. thetaiotaomicron strains collected from various academic and clinical collections revealed a widespread ability of this species to form biofilm. However, biofilm production seemed repressed or masked in the reference wild type VPI 5482 and this prompted us to develop the thesis works along two main axis: the identification of conditions inducing in vitro biofilm formation in VPI 5482 and the identification and characterization of transposon mutants with increased biofilm capacity compared to WT VPI 5482. Having tested various compounds that are relevant to gut environment, we showed that bile salts – an important environmental clue for gut microbiota, induce biofilm formation in VPI 5482 and we used RNAseq to identify functions involved in bile salt-induced biofilm formation. RNAseq results show that bacteria in biofilm increase the expression of genes involved in glycosylation of cell surface and exhibit a specific expression pattern of capsular synthesis operons, suggesting that cell surface polysaccharides could be playing a key role during biofilm formation in B. thetaiotaomicron. We then used transposon mutagenesis to reveal adhesion factors involved in biofilm formation in presence of bile salts, which led to identification of an uncharacterized operon (BT3560-2) putatively involved in TonB-dependent transport of micronutrients. Even though the targets of the transport systems identified via mutagenesis and RNAseq are yet unknown, their further characterization may lead to identification of regulatory pathways that govern biofilm formation. Using transposon mutagenesis to unlock adhesion in the poor biofilm-former B. thetaiotaomicron VPI 5482, we identified two loci – a putative type V Mfa1 pili operon BT3148-7 and capsule synthesis operon 4 (CPS4) involved in biofilm formation independently of bile salts. Based on structural homology with mfa1 pilins, BT3148 and BT3147 likely constitute a pilus structure that requires C-terminal truncation of the predicted stalk pilin BT3147 for elongation and consequent increase in biofilm formation. Ongoing electron microscopy combined with immunogold detection of the putative pilus should fully elucidate the BT3148-7-mediated biofilm formation mechanism.

A Thesis submitted to The University of Arizona College of Medicine - Phoenix in partial fulfillment of the requirements for the Degree of Doctor of Medicine. / The human microbiome is the collection of microorganisms in the body that exist in a mutualistic relationship with the host. Recent studies indicate that perturbations in the microbiome may be implicated in a number of diseases, including cancer. More specifically, changes in the gut and vaginal microbiomes may be associated with a variety of gynecologic cancers, including cervical cancer, uterine cancer, and ovarian cancer. Current research and gaps in knowledge regarding the association between the gut and vaginal microbiomes and the development, progression, and treatment of gynecologic cancers are reviewed here. In addition, the potential use of probiotics to manage symptoms of these gynecologic cancers is discussed. A better understanding of how the microbiome composition is altered at these sites and its interaction with the host may aid in prevention, optimization of current therapies, development of new therapeutic agents and/or dosing regimens, and possibly limit the side effects associated with cancer treatment.

It is known that the birth process and initial life exposures, such as feeding, may have an important impact on the acquisition of bacterial communities throughout the human body, including the gut. Preterm infants usually have special dietary needs and undergo increased oxidative stress related to intensive care, which can ultimately impair their gastrointestinal microbial colonization and microbial diversity in the bowel. Using molecular techniques, we analyzed the fecal microbiome of 20 preterm infants and tested the association between bacterial communities and feeding type, as well as levels of F2-isoprostanes. We found that feeding influences the fecal microbiome of preterm infants, however more research is needed to clarify the role of human milk fortifiers in this process. We also observed preliminary evidence of an association between microbial composition and oxidative stress, indicating that future studies in this area should be conducted. / February 2017

The rumen microbiome is a dynamic environment consisting of bacteria, protist, and fungi responsible for fiber degradation. Advances in molecular techniques have enabled description of bacterial microbiome via pyrosequencing. Cattle consuming low-quality forage are often supplemented with protein to increase forage intake and digestion, but effect on the rumen bacterial community is unknown. Thus, increasing post-extraction algal residue (PEAR) and cottonseed meal (CSM) supplementation was provided to steers consuming oat straw to observe the rumen microbiome within the liquid and solid fraction. Weighted UniFrac analysis indicated different fraction-associated communities with greater similarity across treatments in the solid fraction. Bacteroidetes was the predominant phyla detected in all samples (>65%). Within Bacteroidetes, Prevotella was the most abundant genus. In the liquid fraction, Lachnospiraceae, Ruminococcaceae, and Clostridiaceae increased with PEAR provision (P < 0.05). Similar proportions of bacteria between unsupplemented control and CSM supplemented steers indicate factors other than N supply may impact ruminal bacteria populations. A second experiment evaluated the effect of supplemental CSM or dried distillers’ grain (DDG) for Brahman steers consuming rice straw. Total digestible OM intake and total tract OM digestion increased linearly with additional CSM and DDG provision (P < 0.01). Provision of CSM increased ruminal ammonia linearly (P < 0.01), but DDG supplementation resulted in a quadratic response (P = 0.02). Overall, protein supplementation may increase LQF utilization via changes in the rumen microbiome.

Master of Science / Department of Diagnostic Medicine and Pathology / Megan Niederwerder / Evidence has shown that the gastrointestinal microbiome plays an important role in response to infectious disease. Porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2) are two of the most important pathogens affecting the swine industry worldwide. Co-infections are common on a global scale, resulting in pork production losses through reducing weight gain and causing respiratory disease in growing pigs. Our initial microbiome work demonstrated that the fecal microbiome was associated with clinical outcome of pigs 70 days post-infection (dpi). However, it remained uncertain if microbiome characteristics could predispose response to viral challenge. The purpose of this study was to determine if microbiome characteristics present at the time of viral challenge were associated with outcome after co-infection. Using the Lawrence Livermore Microbial Detection Array, we profiled the microbiome in feces on 0 dpi from pigs identified as having high or low growth rates after co-infection. High growth rate pigs had less severe interstitial pneumonia, reduced PRRSV replication, and a significant increase in average daily weight gain throughout the study. At the level of the fecal microbiome, high growth rate pigs had increased microbial diversity on both a family and species level. Shifts in the microbiome composition of the high growth rate pigs included reduced Methanobacteriaceae species, increased Ruminococcaceae species, and increased Streptococcaceae species when compared to low growth rate pigs. Our results indicate that both microbiome diversity and composition prior to virus exposure may play a role in the subsequent response of pigs to PRRSV/PCV2 co-infection. We followed this study by investigating the microbiome characteristics that are present after co-infection and the role of the microbiome in subclinical infections. Microbiome analysis at 3 and 6 weeks post-infection showed no significant difference between high and low growth rate pigs. The results from both exploring the impact that the initial microbiome has on outcome as well as examining the trends in the microbiome during the post-infection period demonstrate that microbiome pre-infection composition may play a larger role in the outcome of subclinical disease in pigs than microbiome composition during viremia or after viral clearance.

Mangrove forests provide a suite of critical ecosystem services ranging from local to global scales.Soil conditions and associated micro-organisms play a fundamental role in maintaining these services, that include nutrient cycling, carbon sequestration and plant growth-promoting properties. Despite its importance, the microbial abundance and function of mangrove soil has received little effort in current research. On the ecotone between the land and the sea, mangrove soils are subject to high variability of geomorphological and ecological conditions that imply a strong distinction of ecological niches. These conditions can diversify the structure and function of the prevalent microbiome. To identify the structure of bacterial communities of mangrove soils, 16S rRNA gene sequencing techniques were applied on samples from arid Avicennia marina forests across different spatial scales: (i) at different depths of the soil profile(surface/subsurface); (ii) between two distinct zones within a given forest (seaward/landward); (iii) among forests with varying local hydrodynamic conditions (exposed/sheltered); and (iiii) among forests in different geographic regions (Saudi Arabia and Australia). This study found that the bacterial soil community varied more within each forest than between forests in different countries. Notably, differences between countries became more apparent at the finest taxonomic resolution (ASVs). Bacterial communities from the landward zone of the mangrove forest are more conserved across countries than those from the dynamic seaward zone. Theoretically assigned functions showed high levels of sulfate respiration and chemoheterotrophy as major metabolic pathways. Differences across local factors in the functional traits reflect the within forest variability. The distinct microbial assemblages from the landward zone were associated with high salinity and phosphorus, and nitrogen and larger grain size were associated with the seaward samples. Understanding patterns of microbial communities in mangrove soils is important to predict changes and mitigate anthropogenic pressures on the ecosystem.

Diet and health are becoming an increasingly important topic in all aspects of society, politics, and everyday life. A growing concern is obesity, which is marked by a large number of circulating fatty acids. This increased number of fatty acids may alter a body's systems such as immune responses. This makes it even more important to scientifically study the effects of one's diet on several factors such as disease, inflammation, and the gut microbiota. Several studies and hypotheses have been performed and proposed, respectively, to find the underlying implications of a high fat diet. Some studies explore the similarity between fatty acids and bacterial antigens, and their resulting stimulation of similar cascades. Others explore the effects of diet on the gut microbiome which has many implications in the context of disease. It has been shown that the microbiota share a relationship with the host that is usually beneficial, but alterations may cause it to become harmful to the host. This manuscript aims to explore these studies and analyze their results, as well as the many connections between them. It also aims to connect these studies with those that explore the far-reaching results of diet, such as the effects it may have on one's offspring. These effects include disease susceptibility and alterations in the gut microbiome in the offspring.

The human gut microbiome is composed of millions of microbial genes, performing a variety of functions contributing to the host’s homeostasis. The disturbance of normal gut microbiome composition and function is associated with diseases. Dietary components including food additives, i.e., sweeteners, play a pivotal role in shaping the human gut microbiome. Despite many studies pointing out the association between sweeteners consumption and health issues, the mechanisms are still unclear and whether sweeteners can directly change the gut microbiome remains largely unknown. In this study, we investigated the responses of the human gut microbiome to 20 common sweeteners, using an approach combining high-throughput in-vitro microbiome culturing and metaproteomics, which provided both taxonomic and functional profile. Sweeteners that belonged to sugar alcohols and glycosides were revealed to induce larger changes in the microbiome metaproteome, as compared with other non-caloric artificial sweeteners (NAS). Changes in taxa abundance were found to be associated with all tested sweeteners at genus level. Clustering analysis based on functional profiling categorized sweeteners into two major clusters, including one cluster comprising 6 sugar alcohols which induced greater functional responses including reduced transport and metabolism of lipid and amino acids, and promoted translation, ribosomal structure and biogenesis, as compared with the other cluster comprising NAS. Taxon-specific functional analyses showed that microbial enzymes from Lachonospiraceae, Faecalibacterium, Eubacterium, Coprococcus and Roseburia hominis were the major contributors to altered butyrate-producing pathways by sweeteners. This study provides a comprehensive profiling of sweeteners-induced gut microbiome changes, and may serve as a basis to understand sweeteners-relevant health issues from a microbiome point of view.