No description available.

As leveduras Saccharomyces cerevisiae são consideradas importantes matérias primas na nutrição animal pela sua capacidade prebiótica. Os prebióticos são compostos não digeridos pelo organismo animal, mas que são fermentados pelos microrganismos do trato gastrintestinal, cujos produtos são capazes de prover benefícios ao hospedeiro. A fermentação depende de fatores como: substrato utilizado para o seu crescimento, método de fermentação, modo e condição de secagem e idade das células. Assim, os processos produtivos modernos têm como intuito a produção de leveduras com elevado potencial prebiótico. Este estudo objetivou avaliar os efeitos de teores crescentes de leveduras com metabólitos ativos (LSC; baseada na fermentação de substratos específicos) na digestibilidade aparente dos nutrientes da dieta, microbiota e produtos da fermentação fecal e parâmetros imunológicos de gatos adultos. Foram utilizados 27 gatos adultos, idade média de 9,44±5,35 anos, machos e fêmeas, sem raça definida e saudáveis. Os animais foram distribuídos em delineamento em blocos casualizados (idade), constituído de três tratamentos experimentais, denominados: DC (dieta controle), LSC 0,3 (dieta controle com 0,3% de leveduras com metabólitos ativos) e LSC 0,6 (dieta controle com 0,6% de leveduras com metabólitos ativos). Os resultados obtidos foram analisados através do programa computacional Statistical Analysis System (SAS Institute Inc., 2004), sendo considerados significativos valores de p<0,05 e as médias comparadas pelo teste de Tukey. Verificou-se que a inclusão do aditivo alterou apenas a digestibilidade aparente da fibra bruta, da matéria mineral e energia metabolizável (p<0,05). Já em relação aos produtos da fermentação e microbiota das fezes, observou-se redução em ácido lático (p=0,0040) e Clostridium perfringens (p=0,0226) com a inclusão do prebiótico e diminuição do ácido isovalérico (p=0,0144) no tratamento LSC 0,3. Pode-se concluir que o aditivo, nos teores de inclusão avaliados, parece apresentar potencial prebiótico em relação aos produtos da fermentação e microbiota fecal / Saccharomyces cerevisiae yeast are considered important raw materials in animal nutrition due their prebiotic capacity. Prebiotics are compounds not digested by animal organism, but are fermented by microorganisms in the gastrointestinal tract, which products are capable of providing benefits to the host. The fermentation depends on factors such as: substrate used for growth, fermentation method, way and drying condition and age of the cells. Thus, modern production processes have the objective of producing yeast with high prebiotic potential. This study aimed to evaluate the effects of increasing levels of yeast with active metabolites (LSC) based on the fermentation of specific substrates on apparent digestibility of diet nutrients, microbiota and fecal fermentation products and immunological parameters in adult cats. Twenty seven male or female cats with mean weight of 4.19 ± 0.83kg and mean age of 9.44 ± 5.35 years were used and distributed in an unbalanced randomized block design (age), consisting of three experimental treatments, DC (control diet), LSC 0.3 (control diet with 0.3% yeast with active metabolites) and LSC 0.6 (control diet with 0.6% yeast with active metabolites). The results were analyzed using the computer program Statistical Analysis System (SAS Institute Inc., 2004), with significance level of p<0.05 and the averages compared by Tukey test. The inclusion of the additive only changed the apparent digestibility of crude fiber and mineral content (p<0.05). Regarding the fermentation products and microbiota of feces, there was a reduction in lactic acid (p=0,0040) and Clostridium perfringens (p=0,0226) with inclusion of prebiotic and decreased of isovalerate (p=0,0144) in the LSC 0.3 treatment. It can be concluded that the additive, in the levels of inclusion assessed, seems to have prebiotic potential on fecal fermentation products and microbiota

A microbiota intestinal é um ecossistema complexo que geralmente vive em harmonia com seu hospedeiro. É essencial para o desenvolvimento e funcionamento adequado do sistema imunológico da mucosa durante o início da vida, um processo que atualmente é conhecido por ser importante para a imunidade de adultos. A microbiota intestinal compreende aproximadamente 1000 espécies, das quais 80% não são cultiváveis. Tendo em vista a importância de se conhecer a microbiota humana e a utilização da ferramenta da construção da biblioteca genômica RNAr 16S ser relativamente recente, esse estudo tem como objetivo analisar diferentes protocolos para avaliar o uso desta ferramenta para o estudo da microbiota intestinal humana. Para a realização dos ensaios experimentais, o DNA extraído de fezes de seis crianças, em diferentes faixas etárias, foi utilizado para a criação de um pool, o qual foi utilizado nos ensaios de PCR. As bibliotecas RNAr 16S foram construídas utilizando 2 pares de iniciadores bactéria-específicos 27F-1492R e 63F-1387R, variando o tempo de desnaturação inicial de cada reação de amplificação do gene RNAr 16S entre 5 e 10 minutos, e 1 par de iniciadores 341F-518R. Os clones foram selecionados aleatoriamente, parcialmente sequenciados e analisados com base em banco de dados do gene RNAr 16S. A diversidade da microbiota foi menor quando os iniciadores 63F-1387R foram utilizados, em comparação aos resultados dos iniciadores 27F-1492R, no entanto, apenas o par de iniciadores 63F-1387R identificou Bifidobacterium sp., gênero importante para o desenvolvimento da microbiota intestinal humana. Não houve diferenças significativas na diversidade quando o tempo de desnaturação inicial da reação de PCR foi estendido para 10 minutos. Com o uso do par de iniciadores 341F-518R mostrou uma diversidade satisfatória, uma maior riqueza, quando comparada com os outros pares de iniciadores e detectou a presença de Bifidobacterium sp. Os dados obtidos sugerem que mais de um par de iniciadores deve ser empregado para o estudo da microbiota fecal quando se utilizar a biblioteca de RNAr 16S como ferramenta. / The intestinal microbiota is a complex ecosystem that usually lives in harmony with its host. It is essential for the development and proper functioning of the mucosal immune system during beginning of the life, a process that is currently known to be important for overall immunity of adults. The intestinal microbiota comprises about 1000 species, 80% of which are not cultivable. Given the importance of understanding the human microbiota and that the use of the tool library construction genomic 16S rRNA is relatively recent, this study aims to analyze different protocols to evaluate the use of this tool to study of the human intestinal microbiota. To perform the experimental tests, the DNA extracted from feces of six children, in different age groups, was used for the creation of a pool, which was used in the PCR assays. The 16S rRNA libraries were constructed using 2 pairs of primers specific-bacterium 27F-1492R and 63F-1387R, varying the denaturation time of each initial amplification reaction between 5 and 10 minutes, and the pair of primers 341F - 518R.The clones were randomly selected, partially sequenced and analyzed based on database 16S rRNA gene. The diversity of the microbiota was lower when the primers 63F - 1387R were used when compared with the results of the primers 27F - 1492R. However, only the pair 63F - 1387R was able to identified Bifidobacterium spp., important genus for the development of the microbiota from human gut. No significant differences in diversity were observed when the time of initial denaturation of the PCR reaction was extended to 10 minutes. By using the pair of primers 341F - 518R a satisfactory diversity, with detection of Bifidobacterium spp., and greater richness were observed, compared with the other pairs of primer. The data suggests that more than one pair of primers should be used for the study of fecal microbiota when using the library of 16S rRNA as a tool.

As leveduras Saccharomyces cerevisiae são consideradas importantes matérias primas na nutrição animal pela sua capacidade prebiótica. Os prebióticos são compostos não digeridos pelo organismo animal, mas que são fermentados pelos microrganismos do trato gastrintestinal, cujos produtos são capazes de prover benefícios ao hospedeiro. A fermentação depende de fatores como: substrato utilizado para o seu crescimento, método de fermentação, modo e condição de secagem e idade das células. Assim, os processos produtivos modernos têm como intuito a produção de leveduras com elevado potencial prebiótico. Este estudo objetivou avaliar os efeitos de teores crescentes de leveduras com metabólitos ativos (LSC; baseada na fermentação de substratos específicos) na digestibilidade aparente dos nutrientes da dieta, microbiota e produtos da fermentação fecal e parâmetros imunológicos de gatos adultos. Foram utilizados 27 gatos adultos, idade média de 9,44±5,35 anos, machos e fêmeas, sem raça definida e saudáveis. Os animais foram distribuídos em delineamento em blocos casualizados (idade), constituído de três tratamentos experimentais, denominados: DC (dieta controle), LSC 0,3 (dieta controle com 0,3% de leveduras com metabólitos ativos) e LSC 0,6 (dieta controle com 0,6% de leveduras com metabólitos ativos). Os resultados obtidos foram analisados através do programa computacional Statistical Analysis System (SAS Institute Inc., 2004), sendo considerados significativos valores de p<0,05 e as médias comparadas pelo teste de Tukey. Verificou-se que a inclusão do aditivo alterou apenas a digestibilidade aparente da fibra bruta, da matéria mineral e energia metabolizável (p<0,05). Já em relação aos produtos da fermentação e microbiota das fezes, observou-se redução em ácido lático (p=0,0040) e Clostridium perfringens (p=0,0226) com a inclusão do prebiótico e diminuição do ácido isovalérico (p=0,0144) no tratamento LSC 0,3. Pode-se concluir que o aditivo, nos teores de inclusão avaliados, parece apresentar potencial prebiótico em relação aos produtos da fermentação e microbiota fecal / Saccharomyces cerevisiae yeast are considered important raw materials in animal nutrition due their prebiotic capacity. Prebiotics are compounds not digested by animal organism, but are fermented by microorganisms in the gastrointestinal tract, which products are capable of providing benefits to the host. The fermentation depends on factors such as: substrate used for growth, fermentation method, way and drying condition and age of the cells. Thus, modern production processes have the objective of producing yeast with high prebiotic potential. This study aimed to evaluate the effects of increasing levels of yeast with active metabolites (LSC) based on the fermentation of specific substrates on apparent digestibility of diet nutrients, microbiota and fecal fermentation products and immunological parameters in adult cats. Twenty seven male or female cats with mean weight of 4.19 ± 0.83kg and mean age of 9.44 ± 5.35 years were used and distributed in an unbalanced randomized block design (age), consisting of three experimental treatments, DC (control diet), LSC 0.3 (control diet with 0.3% yeast with active metabolites) and LSC 0.6 (control diet with 0.6% yeast with active metabolites). The results were analyzed using the computer program Statistical Analysis System (SAS Institute Inc., 2004), with significance level of p<0.05 and the averages compared by Tukey test. The inclusion of the additive only changed the apparent digestibility of crude fiber and mineral content (p<0.05). Regarding the fermentation products and microbiota of feces, there was a reduction in lactic acid (p=0,0040) and Clostridium perfringens (p=0,0226) with inclusion of prebiotic and decreased of isovalerate (p=0,0144) in the LSC 0.3 treatment. It can be concluded that the additive, in the levels of inclusion assessed, seems to have prebiotic potential on fecal fermentation products and microbiota

Submitted by Renata Lopes (renatasil82@gmail.com) on 2017-03-29T19:15:57Z No. of bitstreams: 1 patriciaguedesgarcia.pdf: 1420428 bytes, checksum: 551d89ea9ff226d1f121abd878c41208 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-03-30T11:25:09Z (GMT) No. of bitstreams: 1 patriciaguedesgarcia.pdf: 1420428 bytes, checksum: 551d89ea9ff226d1f121abd878c41208 (MD5) / Made available in DSpace on 2017-03-30T11:25:09Z (GMT). No. of bitstreams: 1 patriciaguedesgarcia.pdf: 1420428 bytes, checksum: 551d89ea9ff226d1f121abd878c41208 (MD5) Previous issue date: 2009-11-20 / Diarréia aguda é um problema de saúde pública, causa importante de morbidade e mortalidade, sobretudo, em países em desenvolvimento. A etiologia das doenças diarréicas é variada e entre os patógenos bacterianos destacam-se as linhagens diarreiogênicas ou patotipos de Escherichia coli. Foi investigada a ocorrência de E. coli comensal ou diarreiogênica em 141 amostras fecais de crianças de zero até cinco anos de idade de origem comunitária em Juiz de Fora, MG. Dos espécimes, 84 foram provenientes de doadores com manifestação clínica de diarréia aguda (CD) e 57 de doadores clinicamente saudáveis, sem diarréia (SD). Após identificação e caracterização microbiana, foi determinado o seu perfil de susceptibilidade a antimicrobianos. As linhagens bacterianas foram identificadas fenotípica e genotipicamente por métodos bioquímicos clássicos e moleculares, respectivamente, e a caracterização dos patotipos de interesse no trato gastrintestinal foi feita por metodologia molecular. O perfil de susceptibilidade a antimicrobianos foi determinado pelo método de Kirby-Bauer, segundo recomendações do CLSI (2007). Considerando-se todo o grupo amostral, 136 linhagens de E. coli foram recuperadas de fezes CD (36,8% diarreiogências) e 84 de fezes SD (30,9% diarreiogênicas). A confirmação da identificação bioquímica por metodologia molecular mostrou uma correlação de 97,3% entre as duas metodologias. E. coli enteroagregativa (EAEC) foi o patotipo mais freqüentemente recuperado no grupo CD (16,2%) e o único isolado no grupo SD (30,9%). E. coli enteropatogênica (EPEC) atípica foi o segundo patotipo mais isolado no grupo CD (10,3%), seguido de E. coli produtora de toxina Shiga (STEC) (7,4%) e E. coli enterotoxigênica (ETEC) (2,9%). Nenhuma linhagem de EPEC típica e E. coli enteroinvasiva (EIEC) foi recuperada. A investigação do perfil de susceptibilidade aos antimicrobianos demonstra elevada resistência a fármacos, como ampicilina, tetraciclina e sulfametoxazol-trimetoprim, enquanto que resistência intermediária foi observada contra a ampicilina/sulbactam, cefalotina, tetraciclina e amicacina e gentamicina. Os antimicrobianos mais eficazes foram ceftazidima, ceftriaxona, imipenem e piperacilina/tazobactan, para os quais não foi verificada resistência bacteriana. A diferenciação entre os patotipos de E. coli é de grande importância, uma vez que estes microrganismos estão implicados em diarréias agudas em crianças e, quase sempre, necessitarão de tratamento medicamentoso. De maneira geral, nossos resultados indicam a relevância da participação de agentes bacterianos diarreiogênicos na gênese da diarréia aguda na infância, em crianças de 0 a 5 anos. A alta resistência a antimicrobianos observada em nosso trabalho suscita uma ampla discussão sobre uso indiscriminado/indevido de antibióticos e os riscos inerentes da automedicação. / Acute diarrhea is a public health problem, important cause of morbidity and mortality, especially in developing countries. The etiology is varied and considering the bacterial pathogens, the diarrheagenic Escherichia coli pathotypes are among the most important. In this study we have investigated the occurrence of commensal or diarrheagenic E. coli in 141 fecal samples from children of zero to five years of age in Juiz de Fora, MG. Of the specimens, 84 were obtained from clinically injured donors with acute diarrhea (CD) and 57 from clinically healthy donors without diarrhea (SD). After microbial identification and characterization the bacterial antimicrobial susceptibility patterns were determined. The bacterial strains were phenotipically and genotipically indentified by conventional biochemical methods and molecular approach, respectively. The E. coli pathotypes were also genetically characterized by molecular methodology. The antimicrobial susceptibility patterns were determined by the Kirby-Bauer method, according to the CLSI guidelines (2007). Considering all fecal specimens, 136 E. coli strains were isolated and identified (36.8%, CD) and 84 (30.9%, SD). Confirmation of identification by molecular approach showed 97,3% of correlation between the two methodologies. Enteroaggregative E. coli (EAEC) was the most frequently recovered pathotype identified in the acute diarrhea group (16.2%) whereas the only recovered pathotype identified in the feces from healthy donors (30.9%). Enteropathogenic E. coli (EPEC) was the second most isolated pathotype group (10.3%), followed by STEC (7.4%) and ETEC (2.9%). No typical EPEC or EIEC strains were recovered. The antimicrobial susceptibility patterns shown high antimicrobial resistance rates against ampicillin, tetracycline, and sulfamethoxazole-trimethoprim. Intermediary resistance was observed against ampicillin/sulbactam, cefalotin, tetracycline, amikacin and gentamicin. The most effective antimicrobials were ceftazidime ceftriaxone, imipenem and piperacillin/tazobactam, for which no bacterial resistance was observed. The differentiation between the diarrheagenic E. coli pathotypes is of great importance, since they are involved in acute diarrheal diseases in children and, almost often will require antimicrobial chemotherapy. Overall, our results may indicate the importance of the bacterial agents in the etiology of diarrheic diseases in childhood, mainly considering children of 0 to 5 years. The high antimicrobial resistance observed in our study raises a broad discussion on the indiscriminate or improper use of antimicrobials, besides the risks of self-medication.

A microbiota intestinal é um ecossistema complexo que geralmente vive em harmonia com seu hospedeiro. É essencial para o desenvolvimento e funcionamento adequado do sistema imunológico da mucosa durante o início da vida, um processo que atualmente é conhecido por ser importante para a imunidade de adultos. A microbiota intestinal compreende aproximadamente 1000 espécies, das quais 80% não são cultiváveis. Tendo em vista a importância de se conhecer a microbiota humana e a utilização da ferramenta da construção da biblioteca genômica RNAr 16S ser relativamente recente, esse estudo tem como objetivo analisar diferentes protocolos para avaliar o uso desta ferramenta para o estudo da microbiota intestinal humana. Para a realização dos ensaios experimentais, o DNA extraído de fezes de seis crianças, em diferentes faixas etárias, foi utilizado para a criação de um pool, o qual foi utilizado nos ensaios de PCR. As bibliotecas RNAr 16S foram construídas utilizando 2 pares de iniciadores bactéria-específicos 27F-1492R e 63F-1387R, variando o tempo de desnaturação inicial de cada reação de amplificação do gene RNAr 16S entre 5 e 10 minutos, e 1 par de iniciadores 341F-518R. Os clones foram selecionados aleatoriamente, parcialmente sequenciados e analisados com base em banco de dados do gene RNAr 16S. A diversidade da microbiota foi menor quando os iniciadores 63F-1387R foram utilizados, em comparação aos resultados dos iniciadores 27F-1492R, no entanto, apenas o par de iniciadores 63F-1387R identificou Bifidobacterium sp., gênero importante para o desenvolvimento da microbiota intestinal humana. Não houve diferenças significativas na diversidade quando o tempo de desnaturação inicial da reação de PCR foi estendido para 10 minutos. Com o uso do par de iniciadores 341F-518R mostrou uma diversidade satisfatória, uma maior riqueza, quando comparada com os outros pares de iniciadores e detectou a presença de Bifidobacterium sp. Os dados obtidos sugerem que mais de um par de iniciadores deve ser empregado para o estudo da microbiota fecal quando se utilizar a biblioteca de RNAr 16S como ferramenta. / The intestinal microbiota is a complex ecosystem that usually lives in harmony with its host. It is essential for the development and proper functioning of the mucosal immune system during beginning of the life, a process that is currently known to be important for overall immunity of adults. The intestinal microbiota comprises about 1000 species, 80% of which are not cultivable. Given the importance of understanding the human microbiota and that the use of the tool library construction genomic 16S rRNA is relatively recent, this study aims to analyze different protocols to evaluate the use of this tool to study of the human intestinal microbiota. To perform the experimental tests, the DNA extracted from feces of six children, in different age groups, was used for the creation of a pool, which was used in the PCR assays. The 16S rRNA libraries were constructed using 2 pairs of primers specific-bacterium 27F-1492R and 63F-1387R, varying the denaturation time of each initial amplification reaction between 5 and 10 minutes, and the pair of primers 341F - 518R.The clones were randomly selected, partially sequenced and analyzed based on database 16S rRNA gene. The diversity of the microbiota was lower when the primers 63F - 1387R were used when compared with the results of the primers 27F - 1492R. However, only the pair 63F - 1387R was able to identified Bifidobacterium spp., important genus for the development of the microbiota from human gut. No significant differences in diversity were observed when the time of initial denaturation of the PCR reaction was extended to 10 minutes. By using the pair of primers 341F - 518R a satisfactory diversity, with detection of Bifidobacterium spp., and greater richness were observed, compared with the other pairs of primer. The data suggests that more than one pair of primers should be used for the study of fecal microbiota when using the library of 16S rRNA as a tool.

Changes in the human gastrointestinal microbiome are associated with several diseases. To infer causality, experiments in representative models are essential, but widely used animal models exhibit limitations. Here we present a modular, microfluidics-based model (HuMiX, human-microbial crosstalk), which allows co-culture of human and microbial cells under conditions representative of the gastrointestinal human-microbe interface. We demonstrate the ability of HuMiX to recapitulate in vivo transcriptional, metabolic and immunological responses in human intestinal epithelial cells following their co-culture with the commensal Lactobacillus rhamnosus GG (LGG) grown under anaerobic conditions. In addition, we show that the co-culture of human epithelial cells with the obligate anaerobe Bacteroides caccae and LGG results in a transcriptional response, which is distinct from that of a co-culture solely comprising LGG. HuMiX facilitates investigations of host-microbe molecular interactions and provides insights into a range of fundamental research questions linking the gastrointestinal microbiome to human health and disease.

No description available.

Einleitung Angesichts des besorgniserregenden Anstiegs der weltweiten Inzidenz von Übergewicht und Adipositas sowie der damit assoziierten Komorbiditäten, bedarf es neben weitreichenden präventiven Maßnahmen vor allem der Entwicklung neuer, nachhaltig effizienter Strategien zum langfristigen Gewichts- und Stoffwechselmanagement. Als wichtiger Ausgangspunkt für solch non-invasive Behandlungsalternativen, könnte das Verständnis der zugrundeliegenden Wirkmechanismen der „bariatrisch-metabolischen Chirurgie“, mit dem Roux-en-Y Magenbypass (Roux-en-Y gastric bypass, RYGB) als eines der am häufigsten angewandten Verfahren, genutzt werden. Zumal der langfristige Erfolg der RYGB-Operation nicht nur auf die restriktiv-malabsorptiven Modifikationen zurückzuführen ist, sondern auch mit zahlreichen vom Gewichtsverlust unabhängigen Faktoren, wie einer erheblichen Umgestaltung der mikrobiellen Darmbesiedlung, in Verbindung gebracht wird. Inwieweit es sich hierbei um eine kausale Verbindung zwischen der RYGB-modulierten intestinalen Mikrobiota und den durch den chirurgischen Eingriff induzierten Gesundheitsvorteilen handelt, blieb bislang jedoch ungeklärt. Ziel der Untersuchung Die vorliegende Arbeit dient der Darstellung der funktionellen Beziehung zwischen der RYGB-spezifischen Darm-Mikrobiota und den positiven therapeutischen Effekten der Roux-en-Y Magenbypass-Operation bei diätetisch-induzierter Adipositas. Hierbei ist vor allem die Rolle der postoperativen „RYGB-Mikrobiota“ beim Erreichen der nachhaltigen Gewichtsreduktion und verbesserten Stoffwechselfunktion von besonderem Interesse. Im Weiteren wird die Mikrobiota-basierte Übertragbarkeit jener vorteilhaften Auswirkungen des bariatrischen Eingriffs auf ein konventionelles Adipositasmodell untersucht. Ergebnisse Mithilfe der gezielten Dezimierung der mikrobiellen Darmflora von RYGB-operierten Tieren, ließ sich die grundlegende Bedeutung derselben für die postoperativen Verbesserungen der Gewichts-, Stoffwechsel- und Energiekontrolle des Wirtes verdeutlichen. So führte die kontinuierliche Antibiotikagabe zu einer signifikanten Minderung der positiven klinischen Effekte des Eingriffes, was als Beweis für die essenzielle Bedeutung der intestinalen „RYGB-Mikrobiota“ zum Erreichen des vollen Ausmaßes der therapeutischen Wirksamkeit der Operation bewertet werden kann. Basierend auf Versuchen, die die Übertragbarkeit bariatrischer Therapieeffekte auf keimfreie, metabolisch unbelastete Versuchstiere belegen, gelang es uns zudem nachzuweisen, dass der fäkale Mikrobiota-Transfer der post-RYGB veränderten mikrobiellen Darmbesiedlung auch bei konventionell aufgezogenen, nicht-operierten Kleinnagern mit diätetisch-induzierter Adipositas ausreicht, um die positiven Auswirkungen der Operation auf die Stoffwechselgesundheit des Empfängers nachzuahmen. Schlussfolgerungen So unterstreichen die Ergebnisse unseres konventionellen Adipositas-Krankheitsmodells zum einen die entscheidende Rolle der intestinalen Mikrobiota als Schlüsselfaktor für den Operationserfolg des Roux-en-Y Magenbypasses und veranschaulichen zum anderen eine innovative Möglichkeit zur Behandlung von Adipositas und den damit verbundenen Stoffwechselerkrankungen auf der Grundlage von Mikrobiota-Modulation.:INHALTSVERZEICHNIS 1 EINLEITUNG 2 LITERATURÜBERSICHT 2.1 Adipositas in unserer Gesellschaft 2.2 Therapiemöglichkeiten von Adipositas 2.2.1 konservative Behandlungsansätze 2.2.2 bariatrische Chirurgie 2.3 Auswirkungen der Roux-en-Y Magenbypass-Operation auf das Körpergewicht, den Stoffwechsel und die intestinale Mikrobiota bei Adipositas 2.4 Rolle der intestinalen Mikrobiota bei physiologischen und pathologischen Stoffwechselprozessen im Wirtsorganismus 2.5 Behandlung von Darmerkrankungen und extraintestinalen Krankheiten durch fäkalen Mikrobiota-Transfer 2.6 Evidenz für die Übertragung des metabolischen Phänotyps durch fäkalen Mikrobiota-Transfer 2.7 Bisherige Evidenz zur Wirkung der nach RYGB-Operation modulierten intestinalen Mikrobiota auf den Wirtsstoffwechsel 2.8 Fragestellungen und Hypothesen 3 TIERE, MATERIALIEN UND METHODEN 3.1 Versuchstiere 3.2 Materialien 3.2.1 Materialien des allgemeinen Versuchsablaufs und der In-vivo-Tests 3.2.2 Materialien der Roux-en-Y Magenbypass-Operation 3.2.3 Materialien der Ex-vivo-Analysen 3.3 Methoden 3.3.1 Allgemeiner Versuchsablauf 3.3.2 Roux-en-Y Magenbypass-Operation in der Ratte 3.3.2.1 Präoperative Versorgung 3.3.2.2 OP-Ablauf 3.3.2.3 Postoperative Versorgung 3.3.3 In-vivo-Tests 3.3.3.1 Glukosetoleranztest 3.3.3.2 Insulintoleranztest 3.3.3.3 Kälteexposition und Wärmebildaufnahme 3.3.3.4 Echo MRI 3.3.4 Ex-vivo-Analysen 3.3.4.1 Gewebeentnahme und -konservierung 3.3.4.2 Histologische Gewebeaufarbeitung 3.3.4.2.1 Generierung histologischer Gewebeschnitte 3.3.4.2.2 Immunhistochemische/Histologische Färbeverfahren 3.3.4.2.3 Mikroskopische Gewebsanalyse 3.3.4.3 Absorptionsphotometrie 3.3.4.4 Statistische Auswertung 4 ERGEBNISSE 4.1 Dezimierung der intestinalen Mikrobiota post-RYGB beeinflusst die therapeutischen Effekte der operativen Intervention auf den Wirtsorganismus 4.1.1 Dezimierung der „RYGB-Mikrobiota“ verändert den Einfluss der Operation auf die Futterpräferenz, Energiezufuhr und das Körpergewicht 4.1.2 Dezimierung der „RYGB-Mikrobiota“ modifiziert die Auswirkungen der chirurgischen Intervention auf die Glukose-Homöostase 4.1.3 Dezimierung der „RYGB-Mikrobiota“ beeinflusst die Folgen des bariatrischen Eingriffs auf den Lipidmetabolismus 4.1.4 Auswirkung der Dezimierung der „RYGB-Mikrobiota“ auf die postoperativ auftretenden Veränderungen des Energiehaushaltes (Thermogenese) 4.1.5 Dezimierung der „RYGB-Mikrobiota“ verändert die Effekte der Operation auf die Morphologie des Darms 4.2 Übertragung der post-RYGB veränderten intestinalen Mikrobiota auf ein konventionelles Adipositasmodell imitiert die therapeutischen Effekte der operativen Intervention 4.2.1 Einfluss des Transfers der fäkalen „RYGB-Mikrobiota“ auf die Futterpräferenz, Energiezufuhr und das Körpergewicht im konventionellen Adipositasmodell 4.2.2 Auswirkung des Transfers der fäkalen „RYGB-Mikrobiota“ auf die Glukose- Homöostase im konventionellen Adipositasmodell 4.2.3 Beeinflussung des Lipidmetabolismus durch den Transfer der fäkalen „RYGB-Mikrobiota“ im konventionellen Adipositasmodell 4.2.4 Veränderung des Energiehaushaltes (Thermogenese) durch den Transfer der fäkalen „RYGB-Mikrobiota“ im konventionellen Adipositasmodell 4.2.5 Transfer der fäkalen „RYGB-Mikrobiota“ modifiziert die Morphologie des Darms im konventionellen Adipositasmodell 5 DISKUSSION 5.1 Kausale Rolle der postoperativ veränderten intestinalen „RYGB-Mikrobiota“ für den Therapieerfolg der Operation 5.2 Therapeutische Bedeutung der RYGB-spezifischen intestinalen Mikrobiota auf Gewicht und Stoffwechsel bei konventioneller Adipositas 5.3 Fazit und Ausblick 6 ZUSAMMENFASSUNG 7 SUMMARY 8 LITERATURVERZEICHNIS / Introduction In view of the alarming increase in the global incidence of overweight and obesity and the associated comorbidities, there is a particular need for the development of new, sustainably efficient strategies for long-term weight and metabolic management, in addition to far-reaching preventive measures. As an important starting point for such noninvasive treatment alternatives, the understanding of the underlying mechanisms of action of 'bariatric metabolic surgery', with Roux-en-Y gastric bypass (RYGB) as one of the most commonly used procedures, could be used. Especially since the long-term success of RYGB surgery is not only due to the restrictive malabsorptive modifications, but also associated with numerous factors independent of weight loss, such as significant transformation of the intestinal microbial colonization. However, the extent to which this constitutes a causal link between the RYGB-modulated intestinal microbiota and the health benefits induced by the surgical intervention has so far remained unclear. Aims of the Study The aim of this investigation is to present the functional relationship between the RYGB-specific gut microbiota and the beneficial therapeutic effects of Roux-en-Y gastric bypass surgery for diet-induced obesity. Here, the role of the postoperative 'RYGB-microbiota' in achieving sustained weight loss and improved metabolic function is of particular interest. Furthermore, the microbiota-based transferability of those beneficial effects of bariatric surgery to a conventional obesity model is investigated. Results Using deliberate depletion of the intestinal microbial flora of RYGB-operated animals, our experiments in the RYGB small animal model allowed us to elucidate the fundamental importance of surgery-induced modulation of the intestinal microbiota for the postoperative improvements in the host's weight as well as metabolic, and energy control. In fact, continuous administration of antibiotics significantly attenuated the beneficial clinical effects of the Roux-en-Y gastric bypass, which can be interpreted as evidence of the essential importance of the intestinal 'RYGB-microbiota' for the full extent of the therapeutic efficacy of surgery. Based on experiments demonstrating the transferability of the effects of bariatric therapy to germ-free, metabolically unaffected experimental animals, we were also able to demonstrate that fecal microbiota transfer of the altered post-RYGB intestinal microbial colonization was sufficient to mimic the beneficial effects of surgery on the metabolic health of the recipient, even in non-operated, conventionally reared small rodents with diet-induced obesity. Conclusions Thus, the results of our conventional obesity disease model both highlight the critical role of intestinal microbiota as a key factor in the surgical success of Roux-en-Y gastric bypass and illustrate an innovative way to treat obesity and associated metabolic diseases through microbiota modulation.:INHALTSVERZEICHNIS 1 EINLEITUNG 2 LITERATURÜBERSICHT 2.1 Adipositas in unserer Gesellschaft 2.2 Therapiemöglichkeiten von Adipositas 2.2.1 konservative Behandlungsansätze 2.2.2 bariatrische Chirurgie 2.3 Auswirkungen der Roux-en-Y Magenbypass-Operation auf das Körpergewicht, den Stoffwechsel und die intestinale Mikrobiota bei Adipositas 2.4 Rolle der intestinalen Mikrobiota bei physiologischen und pathologischen Stoffwechselprozessen im Wirtsorganismus 2.5 Behandlung von Darmerkrankungen und extraintestinalen Krankheiten durch fäkalen Mikrobiota-Transfer 2.6 Evidenz für die Übertragung des metabolischen Phänotyps durch fäkalen Mikrobiota-Transfer 2.7 Bisherige Evidenz zur Wirkung der nach RYGB-Operation modulierten intestinalen Mikrobiota auf den Wirtsstoffwechsel 2.8 Fragestellungen und Hypothesen 3 TIERE, MATERIALIEN UND METHODEN 3.1 Versuchstiere 3.2 Materialien 3.2.1 Materialien des allgemeinen Versuchsablaufs und der In-vivo-Tests 3.2.2 Materialien der Roux-en-Y Magenbypass-Operation 3.2.3 Materialien der Ex-vivo-Analysen 3.3 Methoden 3.3.1 Allgemeiner Versuchsablauf 3.3.2 Roux-en-Y Magenbypass-Operation in der Ratte 3.3.2.1 Präoperative Versorgung 3.3.2.2 OP-Ablauf 3.3.2.3 Postoperative Versorgung 3.3.3 In-vivo-Tests 3.3.3.1 Glukosetoleranztest 3.3.3.2 Insulintoleranztest 3.3.3.3 Kälteexposition und Wärmebildaufnahme 3.3.3.4 Echo MRI 3.3.4 Ex-vivo-Analysen 3.3.4.1 Gewebeentnahme und -konservierung 3.3.4.2 Histologische Gewebeaufarbeitung 3.3.4.2.1 Generierung histologischer Gewebeschnitte 3.3.4.2.2 Immunhistochemische/Histologische Färbeverfahren 3.3.4.2.3 Mikroskopische Gewebsanalyse 3.3.4.3 Absorptionsphotometrie 3.3.4.4 Statistische Auswertung 4 ERGEBNISSE 4.1 Dezimierung der intestinalen Mikrobiota post-RYGB beeinflusst die therapeutischen Effekte der operativen Intervention auf den Wirtsorganismus 4.1.1 Dezimierung der „RYGB-Mikrobiota“ verändert den Einfluss der Operation auf die Futterpräferenz, Energiezufuhr und das Körpergewicht 4.1.2 Dezimierung der „RYGB-Mikrobiota“ modifiziert die Auswirkungen der chirurgischen Intervention auf die Glukose-Homöostase 4.1.3 Dezimierung der „RYGB-Mikrobiota“ beeinflusst die Folgen des bariatrischen Eingriffs auf den Lipidmetabolismus 4.1.4 Auswirkung der Dezimierung der „RYGB-Mikrobiota“ auf die postoperativ auftretenden Veränderungen des Energiehaushaltes (Thermogenese) 4.1.5 Dezimierung der „RYGB-Mikrobiota“ verändert die Effekte der Operation auf die Morphologie des Darms 4.2 Übertragung der post-RYGB veränderten intestinalen Mikrobiota auf ein konventionelles Adipositasmodell imitiert die therapeutischen Effekte der operativen Intervention 4.2.1 Einfluss des Transfers der fäkalen „RYGB-Mikrobiota“ auf die Futterpräferenz, Energiezufuhr und das Körpergewicht im konventionellen Adipositasmodell 4.2.2 Auswirkung des Transfers der fäkalen „RYGB-Mikrobiota“ auf die Glukose- Homöostase im konventionellen Adipositasmodell 4.2.3 Beeinflussung des Lipidmetabolismus durch den Transfer der fäkalen „RYGB-Mikrobiota“ im konventionellen Adipositasmodell 4.2.4 Veränderung des Energiehaushaltes (Thermogenese) durch den Transfer der fäkalen „RYGB-Mikrobiota“ im konventionellen Adipositasmodell 4.2.5 Transfer der fäkalen „RYGB-Mikrobiota“ modifiziert die Morphologie des Darms im konventionellen Adipositasmodell 5 DISKUSSION 5.1 Kausale Rolle der postoperativ veränderten intestinalen „RYGB-Mikrobiota“ für den Therapieerfolg der Operation 5.2 Therapeutische Bedeutung der RYGB-spezifischen intestinalen Mikrobiota auf Gewicht und Stoffwechsel bei konventioneller Adipositas 5.3 Fazit und Ausblick 6 ZUSAMMENFASSUNG 7 SUMMARY 8 LITERATURVERZEICHNIS

Le microbiote intestinal équin joue un rôle important dans le maintien de la santé de l'hôte. Le microbiote intestinal est composé de nombreux micro-organismes tels que les bactéries, les virus, les champignons et les archées. Cependant, la majorité de ces cellules microbiennes sont bactériennes, et par conséquent, de nombreuses études, y compris la présente, se concentrent sur l'exploration des communautés bactériennes dans l'intestin. Un déséquilibre du microbiote intestinal, appelé dysbiose, a été observé dans plusieurs conditions, telles que la colite, après l’administration d'antibiotiques ou la modification du régime alimentaire. La restauration du microbiote peut être effectuée par la transplantation de microbiote fécal (FMT). Des études utilisant les recommandations actuelles pour la FMT ont montré une récupération clinique chez les chevaux souffrant de diarrhée, mais le microbiote reste largement inchangé après la FMT et aucune étude randomisée avec contrôle placébo n'a été réalisée. Les hypothèses de ce projet étaient que le traitement avec une FMT concentrée corrigera la dysbiose plus rapidement qu’une FMT conventionnelle et le véhicule, et que le microbiote intestinal des chevaux traités avec une FMT concentrée ressemblera au microbiote intestinal du cheval donneur. L'objectif de ce projet était de développer un protocole pour améliorer la FMT chez les chevaux, en augmentant la concentration de bactéries présentes dans les selles du donneur par centrifugation, et de le tester chez les chevaux atteints de dysbiose intestinale induite par les antibiotiques. L'antibiotique triméthoprime sulfadiazine (TMS) a été administré à neuf chevaux pour induire une dysbiose intestinale. Les chevaux ont été séparés en trois groupes: les chevaux recevant une FMT concentrée (cFMT, n = 3); les chevaux recevant la FMT fraîche (fFMT), selon les recommandations actuelles (n = 3); et les chevaux recevant un véhicule (VEH) avec 10% de glycérol dans une solution saline à 0,9% (n=3). Des échantillons fécaux ont été prélevés avant et après l'administration du TMS, ainsi qu'avant, pendant et après la transplantation. Le séquençage a été réalisé à l'aide de la plateforme Illumina MiSeq et les données analysées à l'aide du logiciel Mothur. Tel qu’attendu, l'antibiotique TMS a significativement diminué la richesse microbienne chez tous les chevaux. De manière inattendue, la composition des suspensions fécales des donneurs cFMT et fFMT était significativement différente de la composition de base des receveurs cFMT et fFMT, respectivement. La composition du microbiote des chevaux ayant reçu une transplantation fécale (concentrée ou non) était significativement différente après la transplantation, alors que ce n’était pas le cas chez les chevaux ayant reçu le véhicule. En outre, l’abondance relative de Escherichia était significativement plus élevée dans les suspensions fécales du donneur cFMT par rapport aux suspensions fécales du donneur fFMT. Les principales limites de ce projet sont la petite taille des groupes et l'exposition des selles des donneurs à l'oxygène et à la congélation-décongélation. En outre, le modèle de dysbiose peut ne pas être optimal pour tester l'efficacité de la FMT, et des études réalisant la FMT chez les chevaux souffrant de diarrhée sont nécessaire. Cette étude a contribué à la recherche de nouvelles approches pour améliorer la FMT chez les chevaux. Le faible effet mesuré avec les deux protocoles de FMT et l’augmentation de Escherichia démontre que les protocoles actuels doivent être optimisés avant de pouvoir recommander la FMT pour traiter et prévenir la dysbiose chez les chevaux. / The equine gut microbiota plays an important role in maintaining the health of the host. The gut microbiota is composed of many microorganisms such as bacteria, viruses, fungi, and archaea. However, the majority of these microbial cells are bacterial cells, and consequently, many studies, including the present one, focus on exploring bacterial communities in the gut. An imbalance of the gut microbiota, termed dysbiosis, has been observed in several conditions such as colitis, colic, after antibiotic administration, or diet modification. Restoration of the gut to a healthy state can be performed through fecal microbiota transplantation (FMT). Studies using current recommendations for FMT have shown clinical recovery in horses with diarrhea, but the microbiota remains largely unchanged after FMT and no controlled studies have been performed. The hypotheses of this project were that treatment with concentrated FMT will correct dysbiosis faster than conventional FMT and the vehicle, and that the gut microbiota of horses treated with concentrated FMT will resemble the gut microbiota of the donor. The objective of this project was to develop an improved protocol for FMT in horses, by increasing the concentration of bacteria found in the donor stool using centrifugation, and to test it in horses with antibiotic-induced intestinal dysbiosis. The antibiotic trimethoprim sulfadiazine (TMS) was administered to nine horses to induce intestinal dysbiosis. Horses were separated into three groups: horses receiving concentrated FMT (cFMT) (n=3); horses receiving fresh FMT (fFMT), as per current recommendations (n=3); horses receiving a vehicle (VEH) with 10% glycerol in 0.9% saline (n=3). Fecal samples were collected before and after antibiotic administration, as well as before, during, and after transplantation. Sequencing was performed using the Illumina MiSeq platform and data analysed using the software Mothur. As expected, the antibiotic TMS significantly decreased the richness in all horses (P < 0.05). Unexpectedly, the membership of the cFMT and fFMT donor fecal suspensions was significantly different from cFMT and fFMT recipients’ baseline membership, respectively. The membership of the cFMT and fFMT recipient horses was significantly different after transplantation, while the vehicle recipients were not. In addition, the Escherichia genus was found in significantly higher relative abundances in the cFMT donor fecal suspensions when compared to the fFMT donor fecal suspensions. The main limitations of this study are the small sample size and exposure of cFMT donor stool to oxygen and freeze-thawing. In addition, the dysbiosis model may not be optimal to test the efficacy of FMT, and studies performing FMT in horses with diarrhea are warranted. This study contributed to the search for novel approaches to improve FMT in horses. The weak effect of both FMT protocols on the gut microbiota and the increase in Escherichia suggest that further clinical studies are needed before FMT can be recommended to treat and prevent dysbiosis in horses.