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(1) Introduction: Sulfonates, which can be diet- or host-derived, are a class of compounds detected in the gut, are involved in host–microbiome interactions and have several health effects. Our aim was to develop a method to quantify five of the sulfonates in the intestine and apply it in a simplified human microbiome model. These were taurine, its metabolic precursor cysteate and one of its degradation products isethionate, as well as sulfoquinovose and one of its most relevant degradation products 2,3-dihydroxy-1-propanesulfonate. (2) Methods: An extraction and sample preparation method was developed, without the need for derivatization. To detect and quantify the extracted sulfonates, a multiplexed LC-MS/MS-MRM method was established. (3) Results: The accuracy and precision of the method were within GLP-accepted parameters. To apply this method in a pilot study, we spiked either taurine or sulfoquinovose into an in vitro simplified human microbiota model with and without Bilophila wadsworthia, a known sulfonate utilizer. The results revealed that only the culture with B. wadsworthia was able to degrade taurine, with isethionate as an intermediate. After spiking the communities with sulfoquinovose, the results revealed that the simplified human microbiome model was able to degrade sulfoquinovose to 2,3-dihydroxypropane-1-sulfonate, which was probably catalyzed by Escherichia coli. In the community with B. wadsworthia, the 2,3-dihydroxypropane-1-sulfonate produced was further degraded by B. wadsworthia to sulfide. (4) Conclusions: We successfully developed a method for sulfonate quantification and applied it in a first pilot study.

Prebiotics and probiotics, proposed alternatives to antibiotics in shrimp aquaculture, are reported to improve growth parameters, promote disease resistance, and influence the gut microbial community. This study aimed to investigate the influence of prebiotic- or probiotic-coated feed and/or exposure to the Early Mortality Syndrome-causing strain of Vibrio parahaemolyticus ( VP-EMS) on the mid and hindgut microbiome of Pacific white shrimp (Litopenaeus vannamei). A monoculture probiotic strain of Bacillus subtilis spores: O14VRQ, and a prebiotic product of cultured Saccharomyces cerevisiae cell walls: MOS, were administered to shrimp as feed additives for 14 days, before a pathogen challenge to VP-EMS. Based on previous efforts, animals in this study were fed experimental diets for 14 days to allow ample amount of time for the prebiotic to be metabolized by health-promoting bacteria and for the probiotic spores to germinate. The pathogen challenge consisted of negative disease control (no VP-EMS exposure, commercial feed), positive disease control (VP-EMS exposure, commercial feed) and two treatment groups, probiotic (VP-EMS exposure) and prebiotic (VP-EMS exposure). DNA extraction, 16S rRNA gene amplicon sequencing, polymerase chain reaction (PCR), and sequencing were utilized to create an overview of the mid and hindgut microbial composition. No significant differences in survival were shown between experimental diets following exposure to sublethal levels of VP-EMS. Bioinformatic analyses revealed no distinct shifts in the mid and hindgut microbiome of shrimp across experimental diets and time points. Results of this data revealed that dominant members of the intestinal microbiome, Proteobacteria, Actinobacteriota, Bacteroidota, Verrucomicrobiota, Flavobacteriaceae, Demequinaceae, Vibrionaceae, Shewanellaceae, Rhodobacteriaceae, and Rubritaleaceae were relatively stable across treatments and time points. Sequencing methods such as metagenomics or metatranscriptomics should be utilized for a higher microbiome resolution. Furthermore, the use of quantitative polymerase chain reaction to quantify ingested probiotic spores, prebiotic-associated bacteria, and VP-EMS is recommended. / Master of Science in Life Sciences / Shrimp aquaculture is one of the fastest-growing aquaculture sectors that provides another supply of feed that save wild fish populations. The use of prebiotics and probiotics are reported to improve growth, provide disease protection, influence the gut community, improve the immune system, and serve as substitutes to antibiotics. In this study, the effect of probiotic-, or prebiotic-coated diets and/or exposure to the Early Mortality Syndrome-causing Vibrio parahaemolyticus (VP-EMS) strain on the gut communty of shrimp (Litopenaeus vannamei) was investigated. DNA extraction, 16S rRNA gene amplification, and sequencing were utilized to identify the microbes in the intestines of shrimp. Based on previous studies, animals in this study were fed experimental diets for 14 days to allow enough time for prebiotic-associated bacteria and probiotic spores to multiply within the intestines. Exposure to a sublethal level of VP-EMS did not significantly affect shrimp survival between treatments. Analyses showed no noticeable differences in the intestinal microbial communities between treatments and time points. This research provided initial proof of what microbes occupy the mid and hindgut microbiome. A higher resolution sequencing method is recommended to gain a better understanding of the microbes and their roles in the intestines. The use of quantitative polymerase chain reaction is warranted to evaluate the amount of ingested probiotic spores, prebiotic-associated bacteria, and VP-EMS.

The emerging evidence on the interconnectedness between the gut microbiome and host metabolism has led to a paradigm shift in the study of metabolic diseases such as obesity and type 2 diabetes with implications on both underlying pathophysiology and potential treatment. Mounting preclinical and clinical evidence of gut microbiota shifts, increased intestinal permeability in metabolic disease, and the critical positioning of the intestinal barrier at the interface between environment and internal milieu have led to the rekindling of the “leaky gut” concept. Although increased circulation of surrogate markers and directly measurable intestinal permeability have been linked to increased systemic inflammation in metabolic disease, mechanistic models behind this phenomenon are underdeveloped. Given repeated observations of microorganisms in several tissues with congruent phylogenetic findings, we review current evidence on these unanticipated niches, focusing specifically on the interaction between gut permeability and intestinal as well as extra-intestinal bacteria and their joint contributions to systemic inflammation and metabolism. We further address limitations of current studies and suggest strategies drawing on standard techniques for permeability measurement, recent advancements in microbial culture independent techniques and computational methodologies to robustly develop these concepts, which may be of considerable value for the development of prevention and treatment strategies.

Introduction: La sclérose en plaques (SEP) est une maladie inflammatoire et démyélinisante du système nerveux central (SNC). Les lymphocytes T pro-inflammatoires CD4 TH1/TH17 sont considérés pathogéniques en SEP et dans son modèle animal, l'encéphalomyélite auto-immune expérimentale (EAE). La restriction alimentaire en méthionine (MR) est associée à un effet anti-inflammatoire en périphérie. Cependant, l’impact de la disponibilité de la méthionine sur la fonction des lymphocytes T et sur la neuroinflammation centrale médiée par les lymphocytes T dans la SEP et l'EAE n’est pas connu. Il a été récemment découvert que le métabolisme de la méthionine est induit dans les lymphocytes T murins activés in vitro et que la restriction en méthionine affecte les fonctions effectrices et la prolifération des lymphocytes TH17. Nous formulons donc l’hypothèse que la manipulation du métabolisme des lymphocytes T via la restriction diététique en méthionine représente une nouvelle voie thérapeutique pour contrôler les maladies neuroinflammatoires telles que la SEP. Méthode: Des femelles C57BL/6 sont exposées à une diète contrôle ou réduite en méthionine puis immunisées au MOG35-55 pour induire une EAE active. Un suivi clinique et des expériences de cytométrie en flux permettent de caractériser le profil et l’activation immunitaire. Le prélèvement d'échantillons fécaux et le séquençage de l'ARNr 16S permettent d’évaluer l'influence de la diète sur la composition du microbiome intestinal. Résultats: La MR est associée à un délai significatif de l'apparition des déficits neurologiques chez les femelles C57BL/6 immunisées au MOG. Ceci est associé à une réduction du nombre de cellules immunitaires et de lymphocytes T pro-inflammatoires dans la rate au 7e jour post-induction (présymptomatique) et dans le SNC aux jours 10-13 (début) et 15-16 (pic). Nos résultats préliminaires suggèrent que le microbiome intestinal des souris sous la MR est différent de celui des souris sous la MC et est enrichi de bactéries ayant des effets bénéfiques en inflammation. Conclusion: Nos résultats suggèrent un impact bénéfique de la MR sur l'évolution clinique et les processus neuroinflammatoires dans un modèle animal de SEP. / Introduction: Multiple Sclerosis (MS) is an inflammatory and demyelinating disease of the central nervous system (CNS). Pro-inflammatory CD4 TH1/TH17 are considered pathogenic in MS and its animal model, experimental autoimmune encephalomyelitis (EAE). Dietary methionine restriction (MR) is associated with an anti-inflammatory impact in the periphery. However, little is known about how methionine availability can affect the function of T lymphocytes and impact T-lymphocytes mediated central neuroinflammation in MS and EAE. It was recently discovered that methionine pathway is upregulated in activated murine T lymphocytes in vitro and that methionine restriction affects the effector functions and proliferation of TH17 lymphocytes. We therefore hypothesize that the manipulation of T lymphocyte metabolism via the restriction of dietary methionine intake represents a new therapeutic avenue for controlling neuroinflammatory diseases such as MS. Method: Active MOG35-55-EAE is induced in C57BL/6 female mice exposed to low methionine vs. control diet. Clinical evaluation and flow cytometry studies are used to characterize immune cells phenotype and activation. Fecal samples are collected and 16S rRNA sequencing is used to assess the influence of the diet on the composition of the intestinal microbiome. Results: Dietary MR is associated with a significantly delayed onset of neurological deficits in active EAE (female mice). This is paralleled by a lower number of immune cells and pro-inflammatory T lymphocytes in the spleens at day 7 post-induction (presymptomatic) and in the CNS at day 10-13 (onset) and 15-16 (peak). Our preliminary results suggest that the intestinal microbiome of mice under dietary MR is different from that of mice under control diet and is enriched for bacteria with beneficial effects on inflammation. Conclusion: Our results suggest a beneficial impact of MR on clinical course and neuroinflammation in an animal model of MS.

This thesis investigates how air pollution, both natural and anthropogenic, affects changes in the proximal small intestine and ileum microbiota profile, as well as intestinal barrier integrity, histological changes, and inflammation. APO-E KO mice on a high fat diet were randomly selected to be exposed by whole body inhalation to either wood smoke (WS) or mixed vehicular exhaust (MVE), with filtered air (FA) acting as the control. Intestinal integrity and histology were assessed by observing expression of well- known structural components tight junction proteins (TJPs), matrix metallopeptidase-9 (MMP-9), and gel-forming mucin (MUC2), as well known inflammatory related factors: TNF-α, IL-1β, and toll-like receptor (TLR)-4. Bacterial profiling was done using DNA analysis of microbiota within the ileum, utilizing 16S metagenomics sequencing (Illumina miSeq) technique. Overall results of this experiment suggest that air pollution, both anthropogenic and natural, cause a breach in the intestinal barrier with an increase in inflammatory factors and a decrease in beneficial bacteria. This evidence suggests the possibility of air pollution being a potential causative agent of intestinal disease as well as a possible contributing mechanism for induction of systemic inflammation.