(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.

Background: Polycystic ovary syndrome (PCOS) affects 5–20% of women of reproductive age worldwide and is associated with disorders of glucose metabolism. Hormone and metabolic signaling may be influenced by phytoestrogens, such as isoflavones. Their endocrine effects may modify symptom penetrance in PCOS. Equol is one of the most active isoflavone metabolites, produced by intestinal bacteria, and acts as a selective estrogen receptor modulator. Method: In this interventional study of clinical and biochemical characterization, urine isoflavone levels were measured in PCOS and control women before and three days after a defined isoflavone intervention via soy milk. In this interventional study, bacterial equol production was evaluated using the log(equol: daidzein ratio) and microbiome, metabolic, and predicted metagenome analyses were performed. Results: After isoflavone intervention, predicted stool metagenomic pathways, microbial alpha diversity, and glucose homeostasis in PCOS improved resembling the profile of the control group at baseline. In the whole cohort, larger equol production was associated with lower androgen as well as fertility markers. Conclusion: The dynamics in our metabolic, microbiome, and predicted metagenomic profiles underline the importance of external phytohormones on PCOS characteristics and a potential therapeutic approach or prebiotic in the future.

Despite the wealth of research into strategies for microbiome modulation, studies of microbiome management in pig hosts have found mixed results. A refined understanding of the patterns of microbiome assembly during the host’s early life, when management strategies are most commonly applied, is necessary for the development of successful management practices. Here, we study the development of the pig gut microbial community in a monitoring experiment, sampling the microbiome of pigs in a commercial farm intensively during the first month of life. We found that the community’s taxonomic richness increased linearly with host age. Furthermore, rapid changes across communities occurred in stages, and non-linear patterns in relative abundance were commonly observed among dominant taxa across host age, consistent with primary succession. Our results highlight the importance of understanding the patterns of microbiome assembly during host development, and identify successional stages as windows of opportunity for future research.

<p>Nonribosomal peptides, polyketides, and fatty acids comprise a distinct subset of microbial secondary metabolites produced by similar biosynthetic methods and exhibit broad structural diversity with a high propensity for biological activity. Dedicated studies of these specific microbial small molecules have identified numerous potent actions towards human cells with many clinical translations. Interestingly, most therapeutically used nonribosomal peptides and polyketides were discovered from soil bacteria, meanwhile, bacteria that have co-evolved within a human context, the human microbiota, have barely been explored for secondary metabolites. The central goal of this thesis is to explore the secondary metabolome of human microbiota for nonribosomal peptides and polyketides, which are hypothesized to possess biological activities significant within the human host context. Candidate organisms were chosen for their established connections to human health and evidence suggestive of secondary metabolite production. Specifically, questions about gene to molecule prediction capability, metabolite production, structural diversity, and biological activity were explored from studies of the dental caries linked Streptococcus mutans UA159, from the probiotic Lactobacillus plantarum WCFS1, and the Crohn’s disease associated Faecalibacterium prausnitzii.</p> / Master of Science (MSc)

No description available.

<p dir="ltr">Crohn’s disease and ulcerative colitis are chronic inflammatory bowel diseases (IBD) with a rising global prevalence, influenced by clinical and demographics factors. The pathogenesis of IBD involves complex interactions between gut microbiome dysbiosis, epithelial cell barrier disruption, and immune hyperactivity, which are poorly understood. This necessitates the development of novel approaches to integrate and model multiple clinical and molecular data modalities from patients, animal models, and <i>in-vitro</i> systems to discover effective biomarkers for disease progression and drug response. As sequencing technologies advance, the amount of molecular and compositional data from paired measurements of host and microbiome systems is exploding. While it is become routine to generate such rich, deep datasets, tools for their interpretation lag behind. Here, I present a computational framework for integrative modeling of microbiome multi-omics data titled: Latent Interacting Variable Effects (LIVE) modeling. LIVE combines various types of microbiome multi-omics data using single-omic latent variables (LV) into a structured meta-model to determine the most predictive combinations of multi-omics features predicting an outcome, patient group, or phenotype. I implemented and tested LIVE using publicly available metagenomic and metabolomics data set from Crohn’s Disease (CD) and ulcerative colitis (UC) status patients in the PRISM and LLDeep cohorts. The findings show that LIVE reduced the number of features interactions from the original datasets for CD to tractable numbers and facilitated prioritization of biological associations between microbes, metabolites, enzymes, clinical variables, and a disease status outcome. LIVE modeling makes a distinct and complementary contribution to the current methods to integrate microbiome data to predict IBD status because of its flexibility to adapt to different types of microbiome multi-omics data, scalability for large and small cohort studies via reliance on latent variables and dimensionality reduction, and the intuitive interpretability of the meta-model integrating -omic data types.</p><p dir="ltr">A novel application of LIVE modeling framework was associated with sex-based differences in UC. Men are 20% more likely to develop this condition and 60% more likely to progress to colitis-associated cancer compared to women. A possible explanation for this observation is differences in estrogen signaling among men and women in which estrogen signaling may be protective against UC. Extracting causal insights into how gut microbes and metabolites regulate host estrogen receptor β (ERβ) signaling can facilitate the study of the gut microbiome’s effects on ERβ’s protective role against UC. Supervised LIVE models<b> </b>ERβ signaling using high-dimensional gut microbiome data by controlling clinical covariates such as: sex and disease status. LIVE models predicted an inhibitory effect on ER-UP and ER-DOWN signaling activities by pairs of gut microbiome features, generating a novel of catalog of metabolites, microbial species and their interactions, capable of modulating ER. Two strongly positively correlated gut microbiome features: <i>Ruminoccocus gnavus</i><i> </i>with acesulfame and <i>Eubacterium rectale</i><i> </i>with 4-Methylcatechol were prioritized as suppressors ER-UP and ER-DOWN signaling activities. An <i>in-vitro</i> experimental validation roadmap is proposed to study the synergistic relationships between metabolites and microbiota suppressors of ERβ signaling in the context of UC. Two i<i>n-vitro</i> systems, HT-29 female colon cancer cell and female epithelial gut organoids are described to evaluate the effect of gut microbiome on ERβ signaling. A detailed experimentation is described per each system including the selection of doses, treatments, metrics, potential interpretations and limitations. This experimental roadmap attempts to compare experimental conditions to study the inhibitory effects of gut microbiome on ERβ signaling and how it could elevate or reduce the risk of developing UC. The intuitive interpretability of the meta-model integrating -omic data types in conjunction with the presented experimental validation roadmap aim to transform an artificial intelligence-generated big data hypothesis into testable experimental predictions.</p>

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.

[ES] En esta tesis, se realizaron análisis genómicos, metagenómicos y metabolómicos en líneas de conejo seleccionadas de forma divergente para alta y baja VE del tamaño de la camada (TC). Estos animales mostraron diferencias en su potencial de resiliencia. Por ello, estas poblaciones divergentes son un excelente material biológico para estudiar la resiliencia animal a través de la VE. Se realizaron estudios de asociación del genoma (GWAS) utilizando la regresión de un solo marcador y la regresión bayesiana de múltiples marcadores. Cuatro regiones genómicas se asociaron con la VE en el cromosoma 3 de Oryctolagus cuniculus (OCU), OCU7, OCU10 y OCU14, explicando el 8,6% de la varianza genética total para la VE. Además, el estudio de huellas de selección (SS) identificó 134 regiones genómicas que podrían estar bajo selección para la VE. El solapamiento entre ambos estudios se identificó en el OCU3, donde también se encontraron mutaciones funcionales para los genes DOCK2, INSYN2B y FOXI1. Los genes candidatos de GWAS y SS fueron aquellos con mutaciones funcionales identificadas mediante el análisis de secuenciación del genoma completo (WGS) con pools de ADN. Los genes candidatos destacados mostraron funciones biológicas relacionadas con el desarrollo de estructuras sensoriales, la respuesta inmunitaria, la respuesta al estrés y el sistema nervioso. Todas ellas son funciones relevantes para modular la resiliencia de los animales. Por otra parte, los estudios metagenómicos y metabolómicos mostraron que la selección para la VE modificó el microbioma intestinal y la composición de su metaboloma. Las especies microbianas beneficiosas como Alistipes prutedinis, Alistipes shahii, Odoribacter splanchnicus y Limosilactobacillus fermentum eran más abundantes en la población resiliente. En cambio, las especies microbianas nocivas, como Acetatifactor muris y Eggerthella sp, fueron más abundantes en los animales no resistentes. Los genes relacionados con la formación de biofilms, el metabolismo de aminoácidos aromáticos (fenilalanina, triptófano y tirosina) y el metabolismo del glutamato también se expresaron de forma diferencial entre las poblaciones de conejos. Además, se identificaron 15 metabolitos intestinales como potenciales biomarcadores para discriminar y predecir adecuadamente entre las poblaciones de conejos resistentes y no resistentes. Cinco de ellos, el equol, el 3-(4-hidroxifenil)lactato, el 5-aminovalerato, la N6-acetilisina y la serina son metabolitos de origen microbiano. Este es el primer estudio que desvela importantes mecanismos biológicos de la resiliencia animal generada por la selección de la VE de TC. El genoma y el microbioma intestinal y la composición del metaboloma se modificaron a lo largo del proceso de selección, afectando a la respuesta inmunitaria y al estrés. Se encontraron resultados coincidentes entre los estudios metagenómicos y del metaboloma. Por otro lado, en esta tesis desarrollamos por primera vez una herramienta flexible para simular la coevolución del genoma y el microbioma a través de un proceso de selección. La clave de esta herramienta fue la implementación de la herencia del microbioma. Está construida en R y basada en AlphaSimR para que el usuario pueda modificar el código e implementar diferentes escenarios. Esta tesis es el primer paso para desarrollar futuras estrategias y nuevas investigaciones para mejorar la resiliencia de los animales. Una selección que combine información genómica y metagenómica puede mejorar la respuesta de selección. Además, los metabolitos derivados del intestino con evidencia de crosstalk pueden utilizarse como biomarcadores para identificar animales resilientes por plasma, evitando la extracción de muestras fecales para determinar la composición del microbioma. Si estos estudios tienen éxito, estas estrategias podrían mejorar la resiliencia de los animales con el objetivo de buscar un sistema ganadero más sostenible. / [CA] En aquesta tesi, es van realitzar anàlisis genòmiques, metagenòmiques i metabolòmiques en línies de conill seleccionades de manera divergent per a alta i baixa VE de la grandària de la ventrada (GV). Aquests animals van mostrar diferències en el seu potencial de resiliència. Per això, aquestes poblacions divergents són un excel·lent material biològic per a estudiar la resiliència animal a través de la VE. Es van realitzar estudis d'associació del genoma (GWAS) utilitzant la regressió d'un solo marcador i la regressió bayesiana de múltiples marcadors. Quatre regions genòmiques es van associar amb la VE en el cromosoma 3 de Oryctolagus cuniculus (OCU), OCU7, OCU10 i OCU14, explicant el 8,6% de la variància genètica total per a la VE. A més, l'estudi de petjades de selecció (SS) va identificar 134 regions genòmiques que podrien estar sota selecció per a la VE. El solapament entre tots dos estudis es va identificar en l'OCU3, on també es van trobar mutacions funcionals per als gens DOCK2, INSYN2B i FOXI1. Els gens candidats de GWAS i SS van ser aquells amb mutacions funcionals identificades mitjançant l'anàlisi de seqüenciació del genoma complet (WGS) amb pools d'ADN. Els gens candidats destacats van mostrar funcions biològiques relacionades amb el desenvolupament d'estructures sensorials, la resposta immunitària, la resposta a l'estrés i el sistema nerviós. Totes elles són funcions rellevants per a modular la resiliència dels animals. D'altra banda, els estudis metagenòmiques i *metabolòmiques van mostrar que la selecció per a la VE va modificar el microbioma intestinal i la composició de la seua metaboloma. Les espècies microbianes beneficioses com Alistipes prutedinis, Alistipes shahii, Odoribacter splanchnicus i Limosilactobacillus fermentum eren més abundants en la població resilient. En canvi, les espècies microbianes nocives, com Acetatifactor muris i Eggerthella sp, van ser més abundants en els animals no resistents. Els gens relacionats amb la formació de biofilms, el metabolisme d'aminoàcids aromàtics (fenilalanina, triptòfan i tirosina) i el metabolisme del glutamat també es van expressar de manera diferencial entre les poblacions de conills. A més, es van identificar 15 metabòlits intestinals com a potencials biomarcadores per a discriminar i predir adequadament entre les poblacions de conills resistents i no resistents. Cinc d'ells, el equol, el 3-(4-hidroxifenil)lactat, el 5-aminovalerato, la N6-acetilisina i la serina són metabòlits d'origen microbià. Aquest és el primer estudi que revela importants mecanismes biològics de la resiliència animal generada per la selecció de la VE de GC. El genoma i el microbioma intestinal i la composició del metaboloma es van modificar al llarg del procés de selecció, afectant la resposta immunitària i a l'estrés. Es van trobar resultats coincidents entre els estudis metagenòmiques i del metaboloma. D'altra banda, en aquesta tesi desenvolupem per primera vegada una eina flexible per a simular la coevolució del genoma i el microbioma a través d'un procés de selecció. La clau d'aquesta eina va ser la implementació de l'herència del microbioma. Està construïda en R i basada en AlphaSimR perquè l'usuari puga modificar el codi i implementar diferents escenaris. Aquesta tesi és el primer pas per a desenvolupar futures estratègies i noves investigacions per a millorar la resiliència dels animals. Una selecció que combine informació genòmica i metagenòmique pot millorar la resposta de selecció. A més, els metabòlits derivats de l'intestí amb evidència de crosstalk poden utilitzar-se com biomarcadores per a identificar animals resilients per plasma, evitant l'extracció de mostres fecals per a determinar la composició del microbioma. Si aquests estudis tenen èxit, aquestes estratègies podrien millorar la resiliència dels animals amb l'objectiu de buscar un sistema ramader més sostenible. / [EN] Disclosing the biological mechanisms of the VE can help to gain some insight into the biological basics of animal resilience. In this thesis, genomic, metagenomic, and metabolomic analyses were performed on rabbit lines divergently selected for high and low VE of litter size (LS). These animals showed differences in their resilience potential. Thus, these divergent populations are an excellent biological material for studying animal resilience through the VE. Genome-wide association studies (GWAS) were performed using single marker regression, and Bayesian multiple marker regression approaches. Four genomic regions were associated with the VE in the Oryctolagus cuniculus chromosome (OCU) 3, OCU7, OCU10, and OCU14, explaining 8.6% of the total genetic variance for the VE. In addition, the signature of selection (SS) study identified 134 genomic regions which could be under selection for VE. Overlapping between both studies was placed in the OCU3, where functional mutations for the DOCK2, INSYN2B and FOXI1 genes were also found. Candidate genes from GWAS and SS were those with functional mutations identified using whole genome sequencing (WGS) analysis with pools of DNA. Highlighted candidate genes showed biological functions related to the development of sensory structures, the immune response, the stress response, and the nervous system. All of them are relevant functions to modulate animal resilience. On the other hand, metagenomic and metabolomic studies showed that the selection for VE modified the gut microbiome and metabolome composition. Beneficial microbial species such as Alistipes prutedinis, Alistipes shahii, Odoribacter splanchnicus and Limosilactobacillus fermentum were more abundant in the resilient population. In contrast, harmful microbial species such as Acetatifactor muris and Eggerthella sp were more abundant in the non-resilient animals. Genes related to biofilm formation, aromatic amino acid metabolism (Phenylalanine, tryptophan, and tyrosine), and glutamate metabolism were also differentially expressed between the rabbit populations. Furthermore, 15 gut metabolites were identified as potential biomarkers to properly discriminate and predict between the resilient and non-resilient rabbit populations. Five of them, the equol, 3-(4-hydroxyphenyl)lactate, 5-aminovalerate, N6-acetyllisine, and serine were microbial-derived metabolites. This is the first study unravelling important biological mechanisms under the animal resilience generated by VE of LS selection. Genome and gut microbiome and metabolome composition were modified throughout the selection process, affecting the immune and stress response. Overlapping results were found between the metagenomic and metabolome studies. On the other hand, in this thesis, we developed a flexible tool for simulating the coevolution of the genome and microbiome across a selection process for the first time. The key of this tool was the implementation of the microbiome inheritance. It is constructed in R and based on AlphaSimR so the user can modify the code and implement different scenarios. This thesis is the first step to develop future strategies and further research to improve animal resilience. A selection combining genomic and metagenomic information may improve the selection response. Moreover, gut-derived metabolites with evidence of crosstalk can be used as biomarkers to identify resilient animals by plasma, avoiding the extraction of faecal samples to determine the microbiome composition. If these studies suceed, these strategies could improve animal resilience with the aim of search a more sustainable livestock system. Lastly, the simulation tool developed could help unravel the microbiome's implications in animal breeding programs. / This study was supported by projects AGL2014-5592, C2-1-P and C2-2-P, and AGL2017-86083, C2-1-P and C2-2-P, funded by the Spanish Ministerio de Ciencia e Innovación (MIC)-Agencia Estatal de Investigación (AEI) and the European Regional Development Fund (FEDER). Projects PID2020-115558GB-C21, funded by the Spanish Ministerio de Ciencia e Innovación (MIC)-Agencia Estatal de Investigación (AEI) and the European Regional Development Funds (FEDER) FPU17/01196 scholarship from the Spanish Ministry of Science, Innovation and Universities. / Casto Rebollo, C. (2023). Unravelling the Environmental Variance of Litter Size Through the Genome and Gut Microbiome [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/192460

Bile acids are crucial for the uptake of dietary lipids and can shape the gut-microbiome composition. This latter function is associated with the toxicity of bile acids and can be modulated by bile acid modifying bacteria such as Eggerthella lenta, but the molecular details of the interaction of bacteria depending on bile acid modifications are not well understood. In order to unravel the molecular response to bile acids and their metabolites, we cultivated eight strains from a human intestinal microbiome model alone and in co-culture with Eggerthella lenta in the presence of cholic acid (CA) and deoxycholic acid (DCA). We observed growth inhibition of particularly gram-positive strains such as Clostridium ramosum and the gram-variable Anaerostipes cacae by CA and DCA stress. C. ramosum was alleviated through co-culturing with Eggerthella lenta. We approached effects on the membrane by zeta potential and genotoxic and metabolic effects by (meta)proteomic and metabolomic analyses. Co-culturing with Eggerthella lenta decreased both CA and DCA by the formation of oxidized and epimerized bile acids. Eggerthella lenta also produces microbial bile salt conjugates in a co-cultured species-specific manner. This study highlights how the interaction with other bacteria can influence the functionality of bacteria.

Surveys of the four major soilborne pathogens of strawberry (Fusarium oxysporum f. sp. fragariae, Macrophomina phaseolina, Phytophthora spp., and Verticillium dahliae) to determine their relative prevalence were conducted in Watsonville-Salinas, CA in 2021 and in Santa Maria, CA in 2022. All four major pathogens were detected at relatively similar prevalence in Watsonville-Salinas, between 22% and 31% of sampled fields. In Santa Maria, M. phaseolina was far more prevalent at 52% of sampled fields, the other three falling between 14% and 17%. Additionally replicated greenhouse and field trials were conducted to evaluate the effects of wheat as a single season cover crop on Macrophomina root rot of strawberry and the soil microbiome. Greenhouse trials and the first year of the field trial are described here and demonstrate a lack of substantial disease mitigation or pathogen reduction in the soil following wheat growth compared to no-treatment control. Significant changes were seen in the soil microbiome following wheat growth, including the significant amplification of several bacterial species known to be antagonistic to plant-pathogenic fungi.