<p dir="ltr">Dietary fibers have been observed to modulate the gut microbiome in ways that prevent and moderate human diseases and confer health benefits onto their human host. How dietary fibers do this is through their structure; gut microbes are equipped with a variety of differ- ent carbohydrate-active enzymes (CAZymes) that allow some to hydrolyze glycosidic bonds, thereby utilizing the dietary fiber. The more complex the dietary fiber, the more diverse the maintained gut microbiota may be, as specialist species may be required for complete hydrol- ysis. Therefore, increasing structural complexity of dietary fibers may increase gut microbial diversity and help prevent diseases. To understand if structural features impact the gut mi- crobiome, a set of resistant glucans varying in structures, including mixed-linkage -glucans, resistant maltodextrins (similar to type IV resistant starch) and polydextroses, which are comprised entirely of glucose, were used as substrates in an in vitro sequential batch fermen- tation using fecal microbiota form three healthy donors as inocula. I measured metabolic outputs, growth curves, and community structures by 16S rRNA amplicon sequencing, which I analyzed for through alpha and beta diversity differences and taxa that overrepresented and increased in each treatment. My results show that, depending on the donor and the resistant glucan, structure does significantly impact the concentrations of short-chain fatty acids (SCFAs) and other metabolites that are produced. Resistant glucan structure also impacts alpha and beta diversity to a degree and linear discriminant analysis (by LEfSe) results also support that specific species have preference towards substrates as well. Next, resistant glucans were supplemented into a high-fat diet, and compared these diets to a low- fat diet (LFD), high-fat diet with cellulose (HFD), and high-fat without cellulose (HWC) in a mouse study using C57BL/6J mice over 4 weeks. Increasing microbial diversity will not only increase diversity in the gut microbiome, but it will also provide protective effects in behavior such as helping to prevent anxiety. I measured weight, metabolic outputs, 16S community structure, changes in alpha and beta diversity, and differential abundances of OTUs and taxa by discriminant analysis Effect Size (LEfSe) and Metastats, and anxiety behaviors using open field and light/dark box tests. Microbial community structure was significantly different in treatment groups from controls. Anxiety for mice in tapioca dextrin 01 (TD01), tapioca dextrin 03 (TD03), and resistant maltodextrin (RMF) treatment groups were gen- erally increased, suggesting that the chemical structure of these resistant dextrins may alter the gut microbiome in ways that may influence behavior.</p><p dir="ltr">My overall results support the hypothesis that the fine structural features of dietary fibers do significantly impact the gut microbiome by selecting for specific microbiota, and may even impact cognition and behavior.</p>

The composition of gut microbes affects host weight, immune function, and disease status, and is sensitive to diet, environment, and pharmaceutical exposure. The gut microbiome modulates the toxicity and bioavailability of chemical stressors, however the effects of chemicals on the gut microbiome of aquatic biota are largely unknown. The Waterloo and Kitchener wastewater treatment plants (WWTPs) release effluents containing antibiotics, pharmaceuticals, and other contaminants into the Grand River (ON) that may negatively affect the gut microbiome of downstream organisms. In this study done in Fall 2018, I collected freshwater mussels (Lasmigona costata), several species of insect larvae, and riparian spiders (Tetragnathidae) from sites upstream and downstream of these WWTPs. The gut microbiome was analyzed following the extraction, PCR amplification, and sequencing of bacterial DNA using the V3-V4 hypervariable regions of the 16S rRNA genetic barcode. Changes in the relative abundance of major gut microbiome phyla were observed in all targeted aquatic organisms downstream of WWTPs except Hydropsychidae. Shannon alpha diversity, a measure of bacterial abundance and evenness, differed significantly among sites for mussels (one-way ANOVA: F=7.894, p=0.001), spiders (F=4.788, p=0.01), Perlidae (F=3.1, p=0.0056), Hydropsychidae (F=3.674, p=0.0014), and Heptageniidae (F=2.715, p=0.0143), but not for Baetidae and Ephemerellidae. In sites downstream of the Waterloo WWTP, alpha diversity decreased in spiders, while in sites downstream of the Kitchener WWTP diversity decreased in mussels and Perlidae, while increasing for spiders. Bray-Curtis beta diversity, a measure of dissimilarity between bacterial communities, was significantly dissimilar among sites in all invertebrate taxa (Permanova: p<0.02). Upstream sites differed from downstream Waterloo sites in spiders, Perlidae, and Hydropsychidae (Adonis pairwise: p<0.05), while upstream mussels, spiders, Perlidae, and Hydropsychidae differed from downstream Kitchener sites (p<0.05). Additionally, effluent-derived bacteria were found in the microbiomes of aquatic invertebrates downstream of the WWTPs and not upstream. Taxa was also a significant driver of bacterial composition and diversity in invertebrates. These results indicate that the gut microbiome of downstream organisms differed from the bacterial composition observed in the same invertebrate taxa upstream of the WWTPs, potentially leading to altered host health. This adds to our understanding of how chemical stressors impact the gut microbiome of aquatic and riparian biota; however, future studies are needed to investigate linkages between the gut microbiome and health of these species. / Thesis / Master of Science (MSc)

We investigated the hypothesis that exposure to inhaled diesel exhaust PM can alter the gut microbiome and intestinal integrity, thereby promoting systemic inflammatory response and early CVD risk, which are exacerbated by HF diet. Furthermore, we investigated whether the observed exposure and diet-mediated outcomes could be mitigated through probiotic treatment. We performed an exposure study on C57Bl/6 male mice, placed on either a low fat (LF) diet or a high-fat (HF) diet, and exposed via oropharyngeal aspiration to 35 μg diesel exhaust particles (DEP) suspended in 35 μl of sterile saline or sterile saline controls (CON) twice a week for four weeks. A subset of mice on HF diet were dosed with 0.3 g/day (PRO, ~7.5x108 CFU/day) of probiotic Ecologic® Barrier 849 (Winclove Probiotics) in drinking water during the course of the study. For our first aim, we investigated the alterations in the gut microbiome, measured circulating cytokines and lipopolysaccharide (LPS), and measured CVD biomarkers in the heart. Our results revealed that exposure to inhaled DEP results in gut dysbiosis characterized by expansion of the phyla Verrucomicrobia and Proteobacteria and reduction in Actinobacteria, which was exacerbated by HF diet. Probiotics mitigated the DEP-mediated expansion of Proteobacteria and re-established Actinobacteria in the intestine of HF animals. Furthermore, we determined that exposure to inhaled DEP increases systemic LPS and inflammatory markers IL-1α, IL-3, G-CSF, and TNF-α. Furthermore, we found that inhaled DEP exposure results in increased CVD biomarkers sICAM-1, sP-selectin, and thrombomodulin in the heart. Probiotic treatment was effective in attenuating LPS, inflammatory responses, and CVD biomarkers in HF animals, validating the involvement of the microbiome in mediating inhaled DEP-mediated responses. Considering the effects we observed in the microbiota and systemically of the HF and probiotic treatment animals, we investigated the effects of inhaled DEP on intestinal integrity and inflammation in HF ± PRO animals. Our results showed that inhaled DEP in conjunction with HF diet promotes increased goblet cell and mucin 2 expression, tight junction (TJ) proteins (claudin-3, occludin, and zonula occludens (ZO)-1) expression, matrix metalloproteinase (MMP)-9, toll-like receptor (TLR)-4, and decreased TNF-α and IL-10. Moreover, we found that probiotics promoted intestinal immune response following inhaled DEP exposure characterized by an increase in TNF-α and IL-10 and showed variable expression of TJs in the intestine. In conclusion, we found that inhaled DEP exposure results in changes in gut microbial profiles, altered intestinal integrity, systemic inflammation, and increased CVD biomarkers, which are exacerbated by HF diet. The use of probiotics in this study proved to be pivotal in understanding the microbiome's influences on the regulation of intestinal integrity, intestinal inflammation, systemic inflammation, and cardiovascular system following inhaled DEP exposure with HF diet.

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.

[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

Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biological and Medical Sciences Candidate: Bc. Barbora Zedníková Supervisor: Doc. MUDr. Josef Herink, DrSc. Title of diploma thesis: The role of the gut microbiome in immune-mediated CNS disorders Human body hosts a large number of microorganisms - i.e. Archea, Eukarya, Bacteria and viruses. These microorganisms form microbiome, the total number of the microorganisms is ten times higher than the number of all human cells. Largest part of the microbiome is located in the intestine. The current development of molecular genetics revealed the close relationship between intestinal microbiome and health. Recent studies the most recent studies have pointed to a connection with the pathogenesis of various diseases. This dissertation is focused on the connection between intestinal microbiome and autoimmune diseases of the central nervous system. Research shows that the key factor are the ongoing changes in the composition of microbiome. These changes lead to increased immune stimulation and thereby to inflammatory proliferation.

Submitted by Ana Damasceno (ana.damasceno@unipampa.edu.br) on 2017-06-07T18:12:48Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Baixa diversidade e sucessão microbiana anormal estão associadas à enterocolite necrosante em recém-nascidos prematuros.pdf: 1587508 bytes, checksum: c407e4cf94f25b2272a7d25213f72873 (MD5) / Made available in DSpace on 2017-06-07T18:12:48Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Baixa diversidade e sucessão microbiana anormal estão associadas à enterocolite necrosante em recém-nascidos prematuros.pdf: 1587508 bytes, checksum: c407e4cf94f25b2272a7d25213f72873 (MD5) Previous issue date: 2017-04-07 / As múltiplas causas de Enterocolite Necrosante (NEC) e seus indicativos clínicos utilizados para o diagnóstico ainda se mantêm elusivos. Biomarcadores alternativos para o diagnóstico precoce de NEC em recém-nascidos prematuros e um melhor entendimento dos fatores de risco para o desenvolvimento de NEC são desafios emergentes. Em uma tentativa de contribuir para a solução deste problema, neste trabalho nós rastreamos as mudanças no microbioma dos recém-nascidos (diversidade microbiana, abundância e estrutura) com NEC, iniciando com a primeira evacuação (mecônio) e continuando até a liberação, e comparamos essas mudanças com os prematuros sem o diagnóstico de NEC. Um estudo metataxonomico foi conduzido usando 88 amostras fecais, a partir da primeira evacuação até a 5ª semana de vida, obtidas de 25 recém-nascidos prematuros (14 controles e 11 casos de NEC) selecionados de um grupo de 52 prematuros. Nossos dados revelaram que casos de NEC apresentaram baixa diversidade e uma transição anormal da comunidade microbiana até o diagnóstico de NEC. Um microrganismo pertencendo a família Enterobacteriaceae foi consistentemente mais abundante em prematuros com NEC do que nos controles, mesmo nas amostras de mecônio, e foi considerado um constituinte chave da comunidade microbiana correlacionada com a doença. Finalmente, nos também detectamos uma distorção na associação micróbio-micróbio nas amostras de mecônio dos casos de NEC. Portanto, nossos dados sugerem que a detecção precoce de elevada dominância de Enterobacteriaceae, baixa diversidade e associações micróbio-micróbio nos primeiros dias de vida poderiam ser utilizados como indicativo de risco de desenvolvimento de Enterocolite Necrosante nas UTIs neonatais brasileiras. / The multiple causes of Necrotizing Enterocolitis (NEC) as well as the clinical predictors used for diagnosis have remained elusive to date. Alternative biomarkers for early diagnosis of NEC in premature infants and a better understanding of risk factors for NEC development are emergent challenges. In attempt to contribute to solve this problem, in this work we tracked the changes in the newborn’s microbiome (microbial diversity, abundance and structure) with Necrotizing Enterocolitis beginning with the first stool (meconium) continuing until discharge and compare those changes with preterns without NEC diagnosis. A metataxonomy study was conducted using 88 fecal samples from the first stool (meconium) until the 5th week of life obtained from 25 preterm babies (14 controls and 11 NEC cases) selected from a cohort of 52 premature infants. Our data revealed low microbial diversity in NEC cases and an abnormal transition of the microbial community until NEC diagnosis. A microbial phylotype belonging to the Enterobacteriaceae family were consistently more abundant in NEC than in the controls even in meconium samples and was considered a key constituent of the microbial community that correlated with the disease. Finally, we also detected a disruption of microbial-microbial associations in the meconium samples of NEC cases. Thus, our data suggests that early detection of high dominance of Enterobacteriaceae, low diversity and altered microbial-microbial associations at the first days of life could be used as an indicative of risk of preterm development of Necrotizing Enterocolitis in Brazilian NICU’s.

The alpaca is a small South American camelid (SAC) that is an important production animal in Peru, especially among the highly impoverished communities of the high Andes, and raised for its fiber and meat. Alpacas are highly reliant on the microbes within their digestive tracts to digest the plant material they consume; volatile fatty acids (VFAs) are released as a byproduct of this microbial fermentation and used as a major source of energy by the alpaca. To explore optimal parameters for alpaca microbiome analysis, performed 16S rRNA gene surveys on alpaca C1 and fecal samples that had been extracted using one of three different DNA extraction methods (PowerFecal® DNA Isolation Kit (MO BIO); ZR Fecal DNA MiniPrep™ (Zymo); and a non-commercial extraction method called salting out) and amplified using one of two different polymerase enzyme mixes (AccuPrime™ Pfx SuperMix and 5 PRIME HotMasterMix). We found that choice of polymerase enzyme had a profound effect on the recovered microbiome, with the majority of 5 PRIME-amplified fecal samples failing to amplify. Extraction method had an effect on the recovered microbiome of fecal samples (but not C1 samples), with samples extracted using the MO BIO kit and the salting out method recovering different communities. The Zymo extraction kit returned microbial communities comparable to each of the other extraction methods. These results suggested that the AccuPrime enzyme and either the MO BIO or Zymo kits were optimal for alpaca gut microbiome analysis. We also performed two 16S rRNA gene surveys, the first from alpacas fed either a grass hay (GH) or alfalfa hay (AH) diet, and the second a C1 survey of alpacas fed two-week periods of mixed grass hay plus one of four supplements. We discovered body site and diet effects on the microbiota of alpacas fed either the GH or AH diet, with samples grouping by general body site (C1, small intestine, and distal intestine) and diet. However, we found no significant effect on the C1 microbiome of alpacas administered grain supplements. To study how energy extraction related to the microbiome, we correlated OTUs from GH/AH-fed alpaca with C1 VFA abundances. We discovered no significant correlations, and a 16S survey of low body condition (LBC) and good body condition (GBC) alpacas showed no difference in C1 microbial communities. We concluded that the microbiota of the alpaca digestive tract follow trends seen in microbiome studies of ruminants, but found no evidence of a relationship between body condition, energy extraction, and the C1 microbiome in alpacas.

Human biotherapy (HBT) or fecal transplants have been shown to be an effective treatment for patients with recurrent Clostridium difficile infection (CDI). This study examines the microbial populations present in CDI patients pre- and post-HBT by extracting bacterial DNA from stool samples and performing pyrosequencing of the 16S rRNA gene. We then compared these microbial populations to those of the donors. We examined 19 pairs of patient samples, of which 14 were clinically cured of CDI, and 5 patients were failures. The successful treatment of CDI was associated with an increase in diversity and richness of the patient's fecal microbiome. The majority of those cured showed an increase in the proportion of Firmicutes and decrease in the proportion of Proteobacteria, although varying antibiotic exposure and innate variability between patients was observed. / MSc thesis / NSERC, CIHR, St. Joseph's Healthcare Hamilton

In human microbiome analysis, sequencing of bacterial 16S rRNA genes has revealed a role for the gut microbiota in maintaining health and contributing to various pathologies. Novel community analysis techniques must be evaluated in terms of bias, sensitivity, and reproducibility and compared to existing techniques to be effectively implemented. Next- generation sequencing technologies offer many advantages over traditional fingerprinting methods, but this extensive evaluation required for the most efficacious use of data has not been performed previously. Illumina libraries were generated from the V3 region of the 16S rRNA gene of samples taken from 12 unique sites within the gastrointestinal tract for each of 4 individuals. Fingerprint data were generated from these samples and prominent bands were sequenced. Sequenced bands were matched with OTUs within their respective libraries. The results demonstrate that denaturing gradient gel electrophoresis (DGGE) represents relatively abundant bacterial taxa (>0.1%) beta-diversity of all samples was compared using Principal Coordinates Analysis (PCoA) of UniFrac distances and Multi-Response Permutation Procedure (MRPP) was applied to measure sample cluster strength and significance; indicator species analysis of fingerprint bands and Illumina OTUs were also compared. The results demonstrate overall similarities between community profiling methods but also indicate that sequence data were not subject to the same limitations observed with the DGGE method (i.e., only abundant taxa bands are resolved, unable to distinguish disparate samples). In addition, the effect of stochastic fluctuations in ???????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????? differ for DGGE and next-generation sequencing. I compared pooled and individual reactions for samples of high and low template concentration for both Illumina and DGGE using the combined V3-V4 region of the 16S rRNA gene, and demonstrated that template concentration has a greater impact on reproducibility than pooling. This research shows congruity between two disparate molecular methods, identifies sources of bias, and establishes new guidelines for minimizing bias in microbial community analyses.