Browsing by Person "Burbach, Katharina"

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The porcine intestinal microbiota : studies on diversity and dietary impact
(2018) Burbach, Katharina; Seifert, Jana
The entirety of microbial communities within the gastrointestinal tract is referred to as intestinal microbiota and is predominantly composed of bacteria. Interactions between the microbiota, the host and the diet are essential for maintaining a healthy and functional intestinal ecosystem. The overarching aim of this thesis was the characterization of the porcine intestinal microbiota and further to enhance knowledge about the effects of varying diets. High-throughput sequencing of the 16S rRNA gene facilitates exploration of the taxonomic composition of the microbiota. However, the respective findings may be impaired by methodological variations. Thus, within this thesis, commercial DNA extraction kits are evaluated for their suitability in porcine microbiota analysis. The tested extractions yield into variations of quantity and quality of DNA. The DNA extracts are further used to elucidate the structure of the microbiota by a rapid fingerprinting (Terminal Restriction Fragment Length Polymorphism) and high-resolution sequencing (Illumina amplicon sequencing). While different variable regions of the 16S rRNA gene vary in the taxonomical resolution, sequencing analyses exhibit a good comparability of the two regions V1-V2 and the V5-V6. Furthermore, the microbiota profiles reveal a high consistency by the fingerprinting and sequencing approach but are distinguished by the different DNA extraction kits. Based on criteria of DNA extraction and the depicted microbiota composition, it is recommended to use the FastDNA SPIN Kit for Soil for further analysis of porcine intestinal microbiota. Subsequently, these methodological findings are applied to investigate the impact of varying diets. Illumina amplicon sequencing of the V1-V2 region of the 16S rRNA gene reveals different microbiota structures when diets are solely composed of rye or triticale. Besides the taxonomic analyses of ileal digesta and fecal samples, the concentrations of bacterial metabolites in feces are determined. In summary, rye promotes an increased abundance of saccharolytic bacteria like Lactobacillus, Bifidobacterium, and Prevotella and results in higher concentrations of bacterial metabolites in fecal samples. In contrast, a diet based on triticale is associated with an increased abundance of Clostridium sensu stricto, which may indicate an enhanced cellulolytic potential of the microbiota. When the crude protein content is increased (18%), compared to a lower content (14%), an increased abundance of Lactobacillus is demonstrated in microbiota of ileal digesta samples. However, the content of crude protein did not affect the overall microbiota significantly. In addition, dietary supplementation with probiotic Bacillus spp. shows no effect. In conclusion, these dietary effects on microbiota are considered together with results of a protein digestibility analysis. Moreover, an impact of dietary calcium and phosphorus in combination with different sources of dietary protein is analyzed by fingerprinting approach of digesta samples. Here, the content of calcium-phosphorus shows significant effects on the microbiota of caecal digesta and the putative identities of discriminative variables are determined by a cloning-sequencing approach. Similar, 16S rRNA gene sequencing reveals a significant impact of dietary calcium-phosphorus on the overall fecal microbiota without indicating specific discriminating variables. In combination with the results of a meta-proteomic approach, a gradual adaptation on dietary changes is indicated and consequently, a prolonged adaptation time of three to four weeks is recommended for diet-microbiota studies. This thesis includes a comprehensive analysis of the microbiota across and along the gastrointestinal tract of piglets and explores the dietary inclusion of four levels of insect larvae meal. Feeding insects represent an alternative source of dietary protein, whereby the increased content of chitin indicates a potential shift in microbiota composition compared to a control diet. However, in this case, the structural analysis demonstrates no effects on the overall microbiota’s structure. However, a pairwise comparison between diets reveals significant effects on the microbiota of digesta samples of the small intestine. Dietary inclusion of 5% insect meal increases the abundance of Lactobacillus, whereas the control treatment promotes Bifidobacterium. In conclusion, the results of the present thesis emphasize the importance of standardization within 16S rRNA gene based studies of the porcine intestinal microbiota. Furthermore, the necessity of studying various sampling sites combined with multidisciplinary approaches is demonstrated.