Food-borne infections are a major source of mortality and morbidity. Salmonella causes the highest number of Food-borne bacterial infections in the US. This work contributes towards developing strategies to control Salmonella by (a) defining receptors used by Salmonella to adhere to and invade the host epithelium; (b) developing a host receptor based rapid detection method for the pathogen in food matrix; (C) and defining mechanisms of how probiotics can help alleviate Salmonella-induced cell death in the host epithelium. We developed a cell-cell crosslinking method to discover host-microbe receptors, and discovered three new receptor-ligand interactions. Interaction of Salmonella Ef-Tu with Hsp90 from epithelial cells mediated adhesion, while interaction of Salmonella Ef-Tu with two host proteins that negatively regulate membrane ruffling (myosin phosphatase and alpha catenin) mediated adhesion and invasion. We also showed the role of host ganglioside GM1 in mediating invasion of epithelial cells by Salmonella. Further we exploited pathogen affinity for immobilized gangliosides to concentrate them out of solution and from complex food matrices for detection by qPCR. A sensitivity of 4 CFU/ml (3 hours) in samples without competing microflora was achieved. Samples with competing microflora had a sensitivity of 40,000 CFU/ml. Next we screened several probiotic strains for pathogen exclusion potential and found that Bifidobacterium longum subspp. infantis showed the highest potential for Salmonella enterica subspp. enterica ser. Typhimurium exclusion in a caco-2 cell culture model. B. infantis shared its binding specificity to ganglioside GM1 with S. ser. Typhimurium. Further, B. infantis completely inhibited Salmonella-induced caspase 8 and caspase 9 activity in intestinal epithelial cells. B. infantis also reduced the basal caspase 9 and caspase 3/7 activity in epithelial cells in absence of the pathogen. Western blots and gene expression profiling of epithelial cells revealed that the decreased caspase activation was concomitant with increased phosphorylation of pro-survival protein kinase Akt, increased expression of caspase inhibiting protein cIAP, and decreased expression of genes involved in mitochondrion organization, biogenesis and reactive oxygen species metabolic processes. Hence, B. infantis exerted its protective effects by repression of mitochondrial cell death pathway which was induced in the presence of S. ser. Typhimurium.

Genetic engineering tools are under continuous development. However, hesitation by consumers and governments regarding consumption of genetically modified organism (GMO) affects taking advantage of developments in biotechnology. While being a complicated issue to address, this challenge inspired us to investigate whether it is possible to engineer organisms without altering their wild-type genomes, but with the same customizability level offered by genetic engineering; that is, having the capacity of expressing foreign proteins not codified by the wild-type genome. I used B. subtilis spores as a model organism for this purpose. I took advantage of the sporulation process during which two compartments with differential expression, or different gene expression patterns co-exist, the mother cell and the forespore, and I programmed a single designer plasmid to behave differently in each compartment: the plasmid in the mother cell modifies the spore phenotype, while the plasmid in the forespore undergoes self-digestion. At the end of sporulation, the mother cell lyses and releases the final product — a plasmid-free engineered spore. Following this, I incorporated the forespore-specific "self-digestion" gene circuit into a variety of plasmids with different purposes, including the generation of spores expressing GFP on their protective coats and the artificial induction of sporulation, both of them as a proof-of-concept of genetically intact bioengineered organisms. Production of the different types of genetically intact bioengineered spores resulted in an average of nearly 90% of them free of detectible plasmid or genome alterations. Spores of B. subtilis and other species overall continue to gain attention in the biotechnology sector, with potential applications ranging from biopesticides, probiotics, and vaccines to energy-converting materials, self-healing concrete, and whole-cell biocatalysts. While spores represent a special case of multiple-compartment organisms among bacteria, most eukaryotic organisms possess multiple compartments, structures, or tissues with differential expression, including plants and animals. Therefore, our results in this study could serve as a starting point for new ideas and methods for the genetic modification-free engineering of complex organisms or parts of them.

The objective of this research was to use Bacillus subtilis-based probiotics alone or combined with various additional antibiotic alternatives (yeast-derived prebiotics and zinc supplementation) to maintain gut health and improve growth performance of antibioticree broilers. Three consecutive studies have been conducted to evaluate dietary effects on intestinal morphology, digestive organ development, microbiota, and growth performance of commercial broilers under different coccidia-challenge circumstances. In the first study, broilers were raised under commercial conditions, without coccidia-challenge (Chapter III). Broilers fed diets supplemented with antibiotics exhibited the highest body weight gain (BWG) from d 15 to 27. Broilers fed diets supplemented with B. subtilis or prebiotics + B. subtilis exhibited higher BWG from d 28 to 41 and higher BWG from d 0 to 41 as compared to birds fed control diets. In the second study, broilers were exposed to a clinical coccidia-challenge (Chapters IV and V). Interaction and main effects of the dietary supplement and coccidial vaccination were studied. Diets supplemented with Prebiotics + B. subtilis facilitated broilers to reach a similar feed conversion ratio (FCR) as to the antibiotic control group from d 0 to 56. Feeding diets supplemented with B. subtilis to coccidial vaccinated broilers reduced microbiota diversity by increasing the proportion of an antibiotic-resistant bacterium, Rikenella microfusus. In the third study, interaction and main effects of the subclinical coccidia-challenge and the dietary supplement were studied (Chapters VI and VII). The dietary anticoccidial supplementation increased feed intake and BWG and decreased FCR of broilers from d 15 to 28. However, dietary B. subtilis supplementation did not improve any growth performance of broilers. The combined use of zinc and B. subtilis lowered the high mortality of broilers fed diet supplemented with B. subtilis or zinc alone. Additionally, the dietary supplementation of zinc lowered the proportion of Clostridium in cecal contents of broilers. In conclusion, different nutrition strategies should be considered when broiler chickens are under different health circumstances. Bacillus subtilis-based probiotics have potential to replace the antibiotics, but not anticoccidial feed additives.

No description available.

Bacteria, especially symbiotic species in the gastrointestinal tract, have lived with human for long time and are involved in many aspects of host physiology. There is growing evidence that microbiota-gut-brain axis plays an important role in modulating the response to stress in both human and animals. Alterations in the gut microbiota can change the central nervous system function through effects on the endocrine, immune and nervous systems. Recent studies suggest that probiotic treatment may help to maintain resistance against the detrimental effects of stress though the microbiota-gut-brain axis. However, how potentially beneficial bacteria interact with specific immune and neural components, to mediate beneficial effects on behavior remain unclear. Using chronic social defeat stress, a model often used in post-traumatic stress disorder research, we found that administration of Lactobacillus rhamnosus JB-1 beginning 48 hours following chronic social defeat led to persistence of fear memory and social deficits. These effects were associated with changes in gene expression related to emotion and memory in the hippocampus. This was in contrast to previous studies showing that probiotic intervention during social defeat prevents stress induced deficits in social behavior. This indicates that timing of L. rhamnosus treatment in relation to stress exposure has important implications for effects of the bacteria on behavior. In relation to the mechanism of action of L. rhamnosus on behavior, we demonstrate through depletion and adoptive transfer experiments that CD4+CD25+ T cells in mice treated with JB-1 were necessary and sufficient for JB-1 induced anxiolytic and antidepressant-like effects. Evidence also suggested that Ly6Chi monocytes may be a downstream target inhibited by Tregs involved in the behavioral effects of the bacteria. We observed that JB-1 could also reduce the number of activated microglia in the hippocampus, and attenuate hypothalamic-pituitary-adrenal axis reactivity with the integrity of vagus nerve. Crucially we demonstrated that JB-1 induced promotion of peripheral Tregs, reduction in microglia activation in the hippocampus, and attenuation of HPA axis reactivity, were all inhibited following vagotomy indicating that vagus nerve integrity is required to maintain immune and endocrine linkages from gut microbes to the brain. These studies demonstrate prerequisites for beneficial probiotic effects on stress related behaviours including a specific time window in relation to stress exposure, the activation of regulatory immune cells, and undisrupted vagal nerve signalling. These findings highlight the inter-systemic communication of the microbiota-gut-brain axis in the stress response, and might help to unveil more therapeutic opportunities in relation to stress-related mood disorders. / Thesis / Doctor of Philosophy (PhD) / Excessive exposure or dysregulated responses to stress in human and animals induces behavioral changes and the development of mood disorders. The Microbiota-gut-brain axis plays an important role in maintenance of homeostasis. However, crosstalk between the different components of microbiota-gut-brain axis, and how specific microbes can modulate these interactions, remains unclear. Thus, we sought to understand the mechanism of inter-systemic communication linking a specific gut microbe to changes in stress response and behavior. We observed immunoregulation by regulatory T cells were essential in Lactobacillus rhamnosus JB-1 induced anxiolytic and antidepressant-like effects. We also found the integrity of vagus nerve was necessary for JB-1 induced promotion of regulatory T cells and decrease in microglial activation and attenuation of hypothalamic-pituitary-adrenal axis that are associated with the anxiolytic effects of the bacteria. We also identified that the temporal relationship between exposures to stress and the bacteria is important as ingestion of JB-1 directly after chronic social defeat lead to persistence of fear memory and social deficits. This work will help us to understand mechanisms underlying the microbiota-gut-brain axis, which may allow for the development of novel microbe based therapeutic intervention against mood disorders.

Molecular profiling of blood for several purposes, 1) To identify prostate cancer biomarkers, 2) to identify commonalities between asthma and mood disorders, and 3) to identify mRNAs that may be involved in psychobiotic changes to behaviour. / Every cell of the body has the opportunity to secrete molecules into the blood. These molecules: proteins, RNAs, and DNAs, can be secreted freely, or within extracellular vesicles (EV). The complement of specific molecules secreted by cells can vary in accordance with changes to their immediate environment, such as disease in a particular organ. Cells of the immune system which circulate in the blood may also change the rates at which they produce these molecules in response to a disease or unusual event occurring somewhere within the body. The full complement of proteins, RNAs, or DNAs from all sources within the blood can therefore be measured to garner information about disease states and communication between every tissue of the body. In this body of work, we leveraged this to address three separate challenges within medical science. First, we utilized blood as a source of biomarkers for disease and disease severity; isolating EVs from the blood of prostate cancer patients and healthy subjects and characterized their proteins with mass spectrometry to identify potential biomarkers for prostate cancer and its stages. Next, we explored the ability of blood to identify commonalities between distinct but often comorbid diseases; here we utilized publicly available datasets to identify transcripts or gene sets potentially facilitating the relationship between PTSD, MDD, and asthma. Finally, we utilized differential gene and gene sets expression to gain mechanistic insight into microbiota-gut-brain axis; investigating the hippocampus and blood of mice fed one of two psychobiotic bacteria: Lactobacillus rhamnosus JB1, Lactobacillus reuteri 6475. The analysis identified several mRNA expression differences potentially responsible for the mood-altering characteristics of these psychobiotic bacteria. This body of work illustrates the utility of blood omics data for addressing many problems within medical science, and highlights the large scale of information stored within the blood. / Thesis / Doctor of Philosophy (Medical Science) / Every cell of the body has the opportunity to secrete molecules into the blood. These molecules: proteins, ribonucleic acids (RNAs), and deoxyribonucleic acids (DNAs), can be secreted freely, or within small membrane compartments called extracellular vesicles (EV). Specific molecules are secreted more or less by cells depending on changes to their immediate environment, such as disease in a particular organ. We leveraged this to the benefit of medical science in three separate scenarios: 1) using the molecular contents of EVs to determine when someone has prostate cancer, and at what stage; 2) examining RNAs of the blood to determine why so many with asthma also have depression or PTSD; 3) measuring RNAs in the blood and hippocampus of mice to better understand how certain bacteria in the gut can alleviate depression. This work illustrates the utility of blood in tackling many challenging problems within medical science.

Accumulation of triglyceride (TG) in enterocytes of the small intestine follows two specific absorption pathways: the monoacylglycerol acyltransferase pathway (MGAT) and glycerol acyltransferase pathway (GPAT), the latter (GPAT) is usually found in the small intestine. In this study, we investigated the effects of Lactobacillus rhamnosus MRS6AN, an isolate from “amabere amaruranu” a Kenyan traditional cultured milk, on triglyceride accumulation and expression of GPAT3, I-FABP, MTP and NPC1L1 in Caco-2 cell enterocyte model. Intracellular triglyceride level (TG) of Caco2 cells was significantly reduced by live L. rhamnosus (LB) compared with other bacterial products. MTP expression in Caco2 cells was minimally reduced in live L. rhamnosus (LB) treated Caco2 cells. However, the expression level GPAT3, I-FABP and NPC1L1 was reduced in Caco2 cells treated with live bacteria. Data from this study suggests that Lactobacillus rhamnosus MRS6AN may reduce lipid uptake and accumulation perhaps via modulation of GPAT3 pathway.

Evidence suggests that the gut-brain axis can influence stress-related behaviour, mood and neuropsychological disorders, including anxiety. Stress exposure can increase anxiety-related symptoms such as muscle tension & worrying. Medical treatment has low success and a range of side effects on anxiety. This review aimed to see if probiotics can reduce anxiety symptoms in humans. Where relevant articles on people with anxiety disorders are lacking, the review evaluates articles addressing healthy participants in stressful situations such as exams or public speeches via anxiety questionnaires. I hypothesized that probiotics could be an effective anxiolytic treatment in combination with therapy. Most articles demonstrated reduced subjective and objective results in anxiety and stress measurements after a daily intake of probioticstrains. Findings demonstrate potential anxiolytic benefits with a daily probiotic intake. However, future research on participants with an anxiety disorder is needed to conclude the hypothesis.

Probiotic bacteria are increasingly prevalent in food and nutritional products today. These remarkable microorganisms are capable of imparting exceptional health benefits on their host, including prevention of infection by pathogens and stimulation of immune system function. Their most common mode of delivery is through dairy products (e.g. yogurt), which are also one of their preferred habitats. The interactions between probiotic bacteria and dairy systems have been studied, but are still not well discerned. There is a need for better understanding of these associations, as well as those surrounding the mode of bacterial transfer from the food product to the human gastrointestinal tract. Discoveries into the optimal means of probiotic transport to the body may lead to great advancements in both the design of probiotic foods and their exploitation in the support of human health. Much of the previous research on probiotic bacteria has explored their possible means of adherence in the intestine, as well their strengths in the promotion of human health. Studies relating to their interaction with dairy products are lacking, however, thus this work aims to elucidate some of these aspects. The primary endeavor of this thesis was to develop a technique to quantify the binding affinity of probiotic lactic acid bacteria for milk phospholipids. An additional objective was to exploit these bacteria, as well as dairy ingredients rich in bioactive molecules, in the creation of a highly nutritious food product. In these experiments, a collection of methods were used in progression in order to arrive at a novel protocol to assess binding with excellent reproducibility and simplicity. These included various membrane blotting techniques, as well as thin-layer chromatography. Essentially, phospholipids from both animal-derived standards and milk extracts were applied to a surface (e.g. PVDF membrane), and bacteria were incubated with them to allow binding reactions. The lactic acid bacteria selected for the final assays consisted of four strains of Lactobacillus, including L. reuteri (SD2112 and T-1), L. acidophilus, and L. casei (LC-10). Their adhesion to phospholipids was detected by either colorimetric or fluorescent labeling systems. To illustrate this, the final method developed was a procedure in which bacteria fluorescently stained with acridine orange were allowed to bind to dots of PVDF membrane coated with phospholipids. The results of this study showed that lactic acid bacteria undeniably exhibit selective binding affinity for phospholipids as opposed to other lipids such as triglycerides. The bacteria demonstrated significantly greater binding for a phospholipid extract from milk as opposed to individual phospholipid standards from other sources (p<0.05). Nonetheless, adhesion to all phospholipids was substantially greater than that to triglycerides. These findings, as well as the development of this method, should prove valuable in future research regarding the associations of probiotics with dairy systems. An additional purpose of this thesis was to design a dairy-based food product containing ingredient sources rich in milk bioactives. A gel-type product was created using primarily colostrum, buttermilk powder, and whey protein isolate, as well as selected strains of Lactobacillus. With the inclusion of immunoglobulin-rich colostrum, the product was analyzed alongside fluid milk and colostrum in order to quantify and compare these bioactive molecules. An enzyme-linked immunosorbent assay (ELISA) was used to complete this, and the results revealed concentrations that would be expected by the literature. Specifically, immunoglobulin G (IgG) was quantified by interpolation from a bovine IgG standard regression curve. The results showed that the concentration of IgG in the gel was nearly twice that of colostrum, and almost eight-fold higher than that of milk. This indicates that use of bioactive-rich substances, such as colostrum, in a food product may serve as a means of delivering more concentrated doses of bioactives than their respective ingredients. The research completed in this thesis is significant in that it contributes a valuable method to the elucidation of bacterial binding interactions with milk components, and also demonstrates the successful application of dairy ingredients to an innovative food product high in beneficial compounds. The insight provided by these studies could encourage further work in improving the understanding of probiotic delivery and advancing the development of bioactive-rich food products.

Puberty and adolescence are periods of brain-driven physiological development that display increased incidences of depression development. Adolescents display significant alterations to their stress response signaling, sleep patterns, and metabolism when compared to pre-pubescents. Increased exposure to stress, sleep disturbances, and impaired energy acquisition is typical during puberty and adolescence and similarly increases the likelihood of developing depression. A promising avenue of limiting the deleterious effects of stress and sleep disruption on pubertal and adolescent depressive behaviour is the use of treatments that blunt underlying metabolic impairments associated with depression. Treatments that directly or indirectly increase availability of the glucose metabolite L-lactate are associated with depression reduction. The investigations included in this dissertation evaluate the usability of L-lactate treatments in reducing depression development in pubertal CD-1 male and female mice. This work first examines a previously proposed oral lactate solution, its effect on energy substrate concentration and drowsiness, and its efficacy as a safe oral L-lactate treatment (Chapter 2). Subsequent research aimed to identify a pubertal model of depression that would allow future testing of L-lactate antidepressant treatment. Pubertal male and female mice exposed to chronic sleep disruption were evaluated for stress reactivity and depressive behavior and were identified as a model for antidepressant testing (Chapter 3). In the final study, we evaluated the effects of chronic sleep disruption on the expression of energy metabolites like L-lactate and glucose within the brain, its effect on neurotransmitters associated with depression, and changes to sleep architecture in relation to depression behaviour. Sleep disrupted and depressed animal models were administered L-lactate producing probiotics and were evaluated for improvements to energy substrate concentration, neurotransmitter expression, sleep recovery, and depression reduction (Chapter 4). The present thesis provides groundwork for the use of L-lactate therapies in depressed pubertal and adolescent groups and provides initial evaluations of probiotic intervention as a prevention strategy for juvenile depression.