Browsing by Subject "Mikroflora"

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Analysis of phosphorus utilization using the host genome and microbiota variability in Japanese quail
(2021) Vollmar, Solveig Deniece; Bennewitz, Jörn
Phosphorus (P) is an essential element for growth and performance of avian species. It is predominantly bound as phytic acids and salts (phytate) in plant seeds. Phytases and other phosphatases can harness P by cleaving P groups. Nonruminants have low endogenous phytase activity in the gastrointestinal tract, and thus, the requirement of this element is not met from exclusive plant-based diets. Therefore, mineral P or phytase enzymes are supplemented in poultry feed. Due to the finite quantities of high quality mineral P worldwide, it is of great economic interest. P supplementation is increasingly causing environmental problems. Past studies investigated the P utilization (PU) of different poultry species. They revealed a high phenotypic variation in PU among individuals. Moderate heritabilities indicates that breeding for this trait is in principle possible. The overall aim of this thesis was to gain a deeper understanding of the variability of P utilization in relation to host genetics, ileal microbiota composition and their interaction in the model species Japanese quail. The objective of chapter two was to verify whether variation in PU in quail is a heritable trait conditioned by a few quantitative trait loci (QTL) with detectable effects. For this purpose, individuals were genome-wide genotyped with a 4k SNP chip, and a linkage map was generated. Based on this map, QTL linkage analysis was performed using multimarker regression analysis in a line-crossing model to map QTL for PU. We identified a few QTL regions with significant effects. Among them was a QTL peak at Coturnix japonica chromosome (CJA) 3 for PU. Several genes were found in the region surrounding this peak, which requires further functional gene analysis. Based on these results, we hypothesized that these traits are polygenically determined due to several small QTL effects, which we could not detect significantly. The overlap of the QTL regions indicated linkage of the traits and confirmed their genetic correlations. With the aim of predicting microbiota-related host traits, chapter three examined the composition of the ileum microbiota and differential abundance analysis (DAA). Based on this study, it was shown that a sex-specific influence on microbiota composition exists. The digesta samples of all animals were dominated by five genera, which contributed to more than 70% of the total ileum microbial community. In examining the microbiota composition of each of the 50 animals with the highest and lowest PU, DAA revealed genera significantly associated with PU. In chapter four, we characterized the influence of performance-related gut microbiota to unravel the microbial architecture of the traits evaluated. The aim of this study was to determine whether the variation in PU is partly driven by the microbial community in the ileum. We used microbial mixed linear models to estimate microbiabilities (m^2). This determines the fraction of phenotypic variance that can be explained by the gut microbiota. The estimation of m^2 was 0.15 for PU and was highly significant. It was also highly significant for feed intake, body weight gain and feed per gain. This model was bivariately extended and showed a high microbial correlation of the traits. Based on both results, the ileum microbiota composition plays a substantial role in PU as well as in performance traits, and there is a considerable animal microbiota correlation, showing that the microbiota affects multiple traits. The microbial drivers of this microbial fraction were identified by applying microbiome-wide association studies (MWAS). By back-solving the microbial linear mixed model, we approximated the effect of single OTUs on the phenotypic traits from the microbial model solutions. An MWAS at the genus level uncovered several traits associated with bacterial genera. Subsequently, we assessed whether the microbial community in the ileum is a heritable host trait that can be used for breeding individuals with improved PU. In chapter five we applied QTL analysis using specific genera to examine whether they are linked with genomic SNP markers. These QTL analyses revealed a link between some microbiota species and host genomic regions of chromosomes and SNP markers. By estimating significant heritabilities for some genera, we were able to provide evidence for the hypothesis that the microbial community and microbial features are at least partially related to host genetics. We predicted the animal microbial effects on PU and correlated performance traits by applying microbial best linear unbiased predictions (M-BLUP). In addition, genomic best linear unbiased predictions (G-BLUP) were used to predict the SNP effect for the predicted animal microbial effect. A combination of those two may help to predict genomic breeding values of the microbiota effects for future hologenomic breeding programs.
Comprehensive characterization of microbiota in the gastrointestinal tract of quails and two high yielding laying hen breeds
(2023) Roth, Christoph Florian; Camarinha-Silva, Amélia
The microbiomes composition in the gastrointestinal tract (GIT) is subject to several changes and influences. In addition to breed, sex, or diet, age affects the GIT microbiome dynamics of laying hens and quails. From the first day, the microbiome develops and increases its bacterial load to thousands of species. Then, depending on the diet fed, the animals microbiome and associated active bacteria vary and directly influence the animals nutrient uptake and efficiency. Omics technologies give insights into changes in microbes in the GIT (crop, gizzard, duodenum, ileum, caeca). In addition, they can reveal how feed supplements such as calcium (Ca) or phosphorus (P) can affect host health and performance through alterations in the microbiome. The Japanese quail has been an established animal model for nutritional and biological studies in poultry for the last 60 years. In particular, its short development time makes it a convenient model for microbiome research. However, compared to broiler microbiome research, the quail microbiome is still poorly understood. Animals of the breed Coturnix japonica were housed under the same conditions, fed a diet with P below recommendation, and the ileum microbiota characterized. Microbiota relations with gender and higher or lower predisposition of the birds for PU, CaU, FI, BWG, and FC were described (Chapter II). In addition, these performance parameters influenced the relative average abundance of bacteria like Candidatus Arthromitus, Bacillus, and Leuconostoc. Gender affects specific bacterial groups of the GIT, such as Lactobacillus, Streptococcus, Escherichia, and Clostridium, which differ in average abundance between male and female quails. Despite the comprehensive microbiota analysis, the interplay between animal genetics, diet, sex, and microbiome functionality is not yet understood. The laying hen breeds Lohmann LSL-Classic and Lohmann Brown-Classic are used worldwide. Little is known about the interaction with microbiome composition, performance, dietary effects, and changes during the productive life that might help develop feeding strategies and microbiome responses on a large scale. Because of the importance of P and Ca in poultry diet, the research in Chapter III was conducted to challenge laying hens with reduced dietary P and Ca and describe the effect on GIT active microbiota. The breed was the primary driver of microbial differences. A core microbiome of active bacteria, present along the complete GIT, was revealed for the first time and consisted of five bacteria detected in 97% of all samples, including digesta and mucosa samples (uncl. Lactobacillus, Megamonas funiformis, Ligilactobacillus salivarius, Lactobacillus helveticus, uncl. Fuscatenibacter). Furthermore, significant microbial differences between the GIT sections and between the breeds were described. Minor dietary effects of the P and Ca reduction on the microbiota showed that a further decrease in Ca and P supplementation might be possible without affecting the gut microbial composition and bird performance. Furthermore, the microbiome of laying hens was characterized at five productive stages (weeks 10, 16, 24, 30, and 60) to analyze the age effect on the GIT microbiome (Chapter IV). Although the two breeds of laying hens were offered the same diet and housed under similar conditions, the active microbiota composition changed between the analyzed productive stages, the breed and the GIT sections. The major shift occurred between weeks 16 and 24 and supported the hypothesis of bacterial fluctuations due to the onset of the laying period. Those changes occurred mainly in the abundance of the genera Lactobacillus and Ligilactobacillus. However, it remains unclear whether the dietary changes, due to the development of the birds, influenced the microbiota shifts or if the anatomical and physiological modifications influenced the GIT microbiota. Furthermore, the shotgun metagenomic analysis revealed differences in regulatory functions and pathways between breeds, sections, and the two production stages. Different relative abundance levels of the microbial composition were observed between the RNA-based targeted sequencing and the DNA-based shotgun metagenomics. In conclusion, the comprehensive characterization of the microbiota in the GIT of quails and two high-yielding breeds of laying hens contributes to a broader knowledge of the microbiome dynamics within the fowl GIT. Age and breed play a more important role than diet in influencing the dynamics of microbial composition in laying hens, and individual performance and sex in quails. Research characterizing the microbiome in poultry and its effect on diet and host genetics will help improve feeding and breeding strategies in the future and reduce excretion of nutrients into the environment while ensuring overall animal health.
Genomic and microbial analyses of quantitative traits in poultry
(2023) Haas, Valentin Peter; Bennewitz, Jörn
Feed and nutrient efficiency will become increasingly important in poultry production in the coming years. In addition to feed efficiency, particular attention is paid to phosphorus (P) in nonruminants. Especially growing animals have a high demand of P but through the low usability of plant-based P sources for nonruminants, mineral P is added to their feeds. Due to worldwide limited mineral P sources, the high environmental impact of P in excretions and high supplementation costs, a better utilization of P from feed components is required. Animals’ P utilization (PU) is known to be influenced by the host genetics and by gastrointestinal microbiota. The overall aim of this thesis was to investigate the relationships between host genetics, gastrointestinal microbiota composition and quantitative traits with the focus on PU and related traits in F2 cross Japanese quail (Coturnix japonica). Japanese quail represent a model species for agriculturally important poultry species. In Chapter one, a genetic linkage map for 4k genome-wide distributed SNPs in the study design was constructed and quantitative trait loci (QTL) linkage mapping for performance as well as bone ash traits using a multi-marker regression approach was conducted. Several genome-wide significant QTL were mapped, and subsequent single marker association analyses were performed to find trait associated marker within the significant QTL regions. The analyses revealed a polygenic nature of the traits with few significant QTL and many undetectable QTL. Some overlapping QTL regions for different traits were found, which agreed with the genetic correlations between the traits. Potential candidate genes within the discovered QTL regions were identified and discussed. Chapter two provided a new perspective on utilization and efficiency traits by incorporating gastrointestinal microbiota and investigated the links between host genetics, gastrointestinal microbiota and quantitative traits. We demonstrated the host genetic influences on parts of the microbial colonization localized in the ileum by estimating heritabilities and mapping QTL regions. From 59 bacterial genera, 24 showed a significant heritability and six genome-wide significant QTL were found. Structural equation models (SEM) were applied to determine causal relationships between the heritable part of the microbiota and efficiency traits. Furthermore, accuracies of different microbial and genomic trait predictions were compared and a hologenomic selection approach was investigated based on the host genome and the heritable part of the ileum microbiota composition. This chapter confirmed the indirect influence of host genetics via the microbiota composition on the quantitative traits. Chapter three further extended the approaches to identify causalities from chapter two. Bayesian learning algorithms were used to discover causal networks. In this approach, microbial diversity was considered as an additional quantitative trait and analyzed jointly with the efficiency traits in order to model and identify their directional relationships. The detected directional relationships were confirmed using SEM and extended to SEM association analyses to separate total SNP effects on a trait into direct or indirect SNP effects mediated by upstream traits. This chapter showed that up to one half of the total SNP effects on a trait are composed of indirect SNP effects via mediating traits. A method for detecting causal relationships between microbial and efficiency traits was established, allowing separation of direct and indirect SNP effects. Chapter four includes an invited review on the major genetic-statistical studies involving the gut microbiota information of nonruminants. The review discussed the analyses conducted in chapter one to three and places the analyses published in these chapters in the context of other statistical approaches. Chapter four completed the microbial genetic approaches published to date and discussed the potential use of microbial information in poultry and pig breeding. The general discussion includes further results not presented in any of the chapters and discusses the general findings across the chapters.
Genomische und mikrobielle Analysen von Effizienzmerkmalen beim Schwein
(2022) Weishaar, Ramona Ribanna; Bennewitz, Jörn
Most traits in animal breeding, including efficiency traits in pigs, are influenced by many genes with small effect and have moderate heritabilities between 0.1 and 0.5, which enables efficient selection. These so-called quantitative traits are influenced by genetic factors and environmental factors. The use of next-generation sequencing methods, such as 16S rRNA sequencing to analyse the gut microbiome of livestock, allows identification and analysis of the gut microbiota. It has been shown that the composition of the microbiota in the gastrointestinal tract is heritable and has an influence on efficiency traits. Thus, the animal genome influences the phenotype not only directly by altering metabolic pathways, but also indirectly by changing the composition of the microbiota. This increases the interest in implementing gut microbiota into existing breeding strategies as an explanatory variable. The potential of an efficient utilization and absorption of nutrients varies between individuals. Differences in nutrient absorption depend on feed intake, digestion of dietary components in the stomach and intestine, and intake of digested nutrients from the gastrointestinal tract into blood and lymphatic vessels. Undigested nitrogen is excreted as urea and can be detected by blood urea nitrogen (BUN). The BUN is correlated with efficiency traits and there exist differences between pig breeds. Thus, therefore the BUN would be conceivable as an easier recordable trait for nitrogen utilisation efficiency in pig breeding. In the first chapter of this study, an existing data set of the Department for Animal Genetics and Breeding of the University of Hohenheim was used. This is a data set with 207 phenotyped and genotyped Piétrain sows. The relationship between gut microbial composition, efficiency traits and the porcine genome is investigated using quantitative genetic methods. The heritabilities of the traits FVW, RFI, TZ, and FI ranged from 0.11 to 0.47. The microbiabilities of the traits were significant and ranged from 0.16 to 0.45. In a further step, the previously generated microbial animal effects were used as observation vector for a genomic mixed model. Subsequently, heritabilities for the microbial animal effect were estimated, ranging from 0.20 to 0.61. The similarity of the heritabilities and microbiabilities suggests that the traits are influenced to a similar extent by both genetics and gut microbiota and that the microbial animal effect is determined by the host. These results are underlined by the identification of genera and phyla with significant effects on efficiency traits. The microbial architecture of the traits demonstrated a poly-microbial nature, there are many OTUs with small effects involved in the variation of the observed traits. Genomic Best Linear Unbiased Predictions (G-BLUP) and Microbial Best Linear Unbiased Predictions (M-BLUP) were performed to predict complex traits. The accuracies of M-BLUP and G-BLUP were all in a similar range between 0.14-0.41. This shows that gut microbiota could be used to predict performance traits or be included as a variable in the existing models of breeding value estimation to realize an increase in accuracies. The second part of the paper analysed a dataset from a research project called "ProtiPig". The data set included 475 sows and castrates of crossbreds of German Landrace x Piétrain and was analysed for protein utilization efficiency and nitrogen(N)-utilization efficiency. N-utilization efficiency is a trait that is difficult to record. Because conventional metabolic cage methods are a very complex procedure and difficult to integrate in the standard recording, it was tested whether the BUN is suitable as a proxy trait. Moderate to medium heritabilities could be estimated for all traits and ranged from 0.13 to 0.49. The genome-wide association studies showed that the traits were polygenic. For the BUN, SNPs could be detected that were above the genome-wide significance level. Significant genetic and phenotypic correlations were found between some traits. In particular, the heritabilities of BUNs and the significant genetic correlation between BUN and N-utilization efficiency indicate an opportunity to use the BUN to select for improved N-utilization efficiency. Before the research results generated here can be implemented in breeding practice, further questions must be clarified. In addition, a larger number of animals is needed to validate the results. The results presented here demonstrate the potential of microbial-assisted breeding value estimation and the use of BUN to identify selection candidates for breeding for increased efficiency.
High-throughput sequencing techniques to analyze microbial communities in the gastrointestinal tract of broiler chickens
(2018) Borda Molina, Daniel Enrique; Camarinha-Silva, Amélia
Broiler chicken represents an excellent case-study to elucidate the inter-communication between the host and its microbial communities. The general aim of this thesis was to describe the changes in bacterial community structure that occurred in chickens, in response to different experimental diets. An update of the state of the art of the chicken gastrointestinal microbiota was done in chapter 2. The composition and functionality are described through the most recent technologies that provide taxonomic information at DNA level using 16S rRNA genes. Gene catalogs and their abundance are deciphered through shotgun metagenome sequencing, which is still at its infancy and only eight publications have been published so far. At the protein level, only two studies were found that contribute metaproteomic information. Thanks to these technologies many studies were able to focus on answering how feed supplementations altered the microbes in GIT sections. The second part presented in chapter 3 comprises an extensive investigation of the broiler chicken microbiota composition in digesta and mucosa of individual samples under varying supplementation of calcium, phosphorus, and phytase. The dietary impact on the distribution of the microbial communities was studied in the crop, ileum, and caecum through illumina sequencing of the 16S rRNA gene. One important outcome was the high variability in the microbial composition between individual samples. Significant differences were observed between the digesta and mucosa samples, supporting the hypothesis that being close to the host, mucosa-associated communities show a different composition. A calcium effect on the performance was observed, where values for body weight gain and feed conversion were lower in comparison to the other treatments. Microbial communities in the crop mucosa revealed a dietary effect, while in the digesta samples no significant changes were seen. Regarding the ileum mucosa, there was an effect of P addition on the microbial distribution. As expected, caeca-derived samples showed an increase in the diversity indexes when compared to the ileum and crop and butyrate producers were detected in higher abundance. A lower microbial diversity in the crop was linked to lower growth performance regarding the supplementation of Ca. Hence, each dietary treatment affected the microbial communities; nevertheless, none of the dietary treatments displayed a consistent effect across the studied gut sections. Additionally, the effects of supplementing different proteases and one phytase on the microbial community of the ileum of broiler chickens was assessed. Thus, the specific aim of chapter 4 was to determine how enzyme supplementation affects the microbiota composition in the ileum of broilers and whether these effects were related to differences in pre-caecal AA digestibility. Three different protease sources at a low and high level were included. The microbial taxonomy was assessed through 16S rRNA gene Illumina amplicon sequencing. Performance results revealed a significant increase in growth and feed efficiency in broilers fed with phytase only and the high dosage of protease C, in comparison to the control. Most of the AA showed a significant difference between the control diet and protease C at high dosage and phytase diets. Effects on microbiota composition were observed at the genus level for some protease and phytase supplementations. The genera Streptococcus, Lactobacillus, and uncultured Clostridiaceae were responsible for these differences. This study demonstrates that effects of enzyme supplementation were evident in the terminal small intestine microbiota composition, and, to a lesser extent, in pc AA digestibility. However, the changes in microbiota composition and pc AA digestibility could not be correlated which may indicate the absence of a causal relationship. Finally, an outlook with metagenome sequencing is presented in chapter 5, to further characterize the result of feeding strategies. Metabolism information, essential to microbial activities registered 50% of abundant genes in the supplemented diets while being reduced to 40% in the control samples Phosphatases pathways and butyrate production increased in the supplemented diets while calcium signaling pathway was higher in the control. In conclusion, within this project a method of standardization to study the microbiota along the gastrointestinal tract of broiler chickens was successfully established. The obtained results revealed a significant impact of both, enzyme and mineral supplementation in the individual sections of the GIT. Also, it was proved that even if the GIT works as an interconnected system, its compartmentalization creates different environmental conditions which influence the microbiota. This study provides insights into the responses of the bacteria and their functionality which were stimulated by the feed supplementations.
Impact of process parameters on the sourdough microbiota, selection of suitable starter strains, and description of the novel yeast Cryptococcus thermophilus sp. nov.
(2013) Vogelmann, Stephanie Anke; Hertel, Christian
The microbiota of a ripe sourdough consists of lactic acid bacteria (LAB), especially of the genus Lactobacillus, and yeasts. Their composition is influenced by the interplay of species or strains, the kind of substrate as well as the process parameters temperature, dough yield, redox potential, refreshment time, and number of propagation steps (Hammes and Gänzle, 1997). As taste and quality of sourdough breads are mainly influenced by the fermentation microbiota, intense research has been focused on determination of sourdough associated species and search for new starter cultures. In recent years, economic competition pressure and new consumer demands have led to steady research for new cereal products, especially with health benefit or for people suffering from celiac disease. For these reasons, alternative cereals like oat and barley (both toxic for celiac disease patients) as well as the celiac disease compatible cereals rice and maize, sorghum and millets, the pseudocereals amaranth, quinoa and buckwheat as well as cassava got into the focus of interest. However, information about the microbiota of sourdoughs fermented with buckwheat, amaranth, quinoa, oat or barley is not available except for the following recent studies: a study about the microbiota of amaranth sourdoughs by Sterr et al. (2009), a study about barley sourdough by Zannini et al. (2009), a study about oat sourdoughs by Huettner et al. (2010) and a study about buckwheat and teff sourdoughs by Moroni et al. (2011). The microbiota of sourdoughs from the other mentioned cereals as well as cassava was multiply characterised but not systematically. Fermentation conditions were partly not clearly defined, and identification of species was often based on physiological criteria only, known to be insufficient for the exact classification of LAB. Thus, in this thesis, the influence of the process parameters substrate, temperature, refreshment time, amount of backslopping dough as well as the interplay between the different species or strains were examined and potential starter strains were selected. In Chapter III, the effect of the substrate on the sourdough microbiota was examined and suitable starter cultures for fermentation of non-bread cereals and pseudocereals were selected. Eleven different flours from wheat, rye, oat, barley, millet, rice, maize, amaranth, quinoa, buckwheat and cassava were inoculated with a starter mixture containing numerous LAB and yeasts. Sourdoughs were fermented at 30 °C and refreshed every 24 hours until the microbiota was stable. Species were identified by PCR-DGGE as well as bacteriological culture and RAPD-PCR, followed by 16S/26S rRNA sequence analysis. In these fermentations, the dominant yeast was Saccharomyces cerevisiae; Issatchenkia (I.) orientalis was only competitive in the quinoa and the maize sourdough. No yeasts were found in the buckwheat and the oat sourdough. The dominant LAB species were Lactobacillus (L.) paralimentarius in the pseudocereal sourdoughs, L. fermentum, L. helveticus and L. pontis in the cereal sourdoughs, and L. fermentum, L. plantarum and L. spicheri in the cassava sourdough. Competitive LAB and yeasts were inserted as starters for a further fermentation using new flours from rice, maize, millet and the pseudocereals. After ten days of fermentation, most of the starter strains were still dominant, but L. pontis and L. helveticus could not compete with the other species. It is remarkable that from the numerous starter strains which all were adapted to or isolated from sourdoughs, only a few were competitive in these fermentations; but if, then in most cases in a lot of different flours. In Chapter IV, the effects of the exogenous process parameters substrate, refreshment time, temperature, amount of backslopping dough as well as competing species on the two microbial associations L. sanfranciscensis ? Candida (C.) humilis and L. reuteri ? L. johnsonii ? I. orientalis were examined. Both associations had previously been found to be competitive in sourdough (Kline and Sugihara, 1971a; Nout and Creemers-Molenaar, 1987; Gobbetti et al., 1994a; Garofalo et al., 2008; Böcker et al., 1990; Meroth et al., 2003a). 28 sourdough batches were fermented under defined conditions until the microbiota was stable. Dominant LAB and yeasts were characterized by bacteriological culture, RAPD-PCR and 16S/26S rRNA gene sequence analysis. The process parameters for the association L. sanfranciscensis ? C. humilis could be defined as follows: rye bran, rye flour or wheat flour as substrate, temperatures between 20 and 30 °C, refreshment times of 12 to 24 hours and amounts of backslopping dough from 5 to 20 %. In addition, the association was predominating against all competing lactic acid bacteria and yeasts. The association L. reuteri ? L. johnsonii ? I. orientalis was competitive at temperatures of 35 to 40 °C, refreshment times of 12 to 24 hours and the substrates rye bran, wheat flour and rye flour, but only with sufficient oxygen supply. Cell counts of I. orientalis fell rapidly under the detection limit when using high amounts of doughs (small ratio of surface to volume) and refreshment times of 12 hours. The fermentations depicted in Chapter III and IV give new information about the influence of process parameters on the sourdough microbiota. The studies show that the sourdough microbiota is markedly influenced by the process parameters and kind and quality of substrate. The competitiveness of a single LAB or yeast is strain specific. Interactions between microorganisms also play an important role. However, for the search for suitable starter strains, it would be beneficial to know the reasons, why a single LAB or yeast strain is better adapted to specific process parameters or substrates than others. One of the starter sourdoughs used for fermentation I described in Chapter III was a sourdough made from cassava flour, inoculated with several LAB. No yeast had been inserted, but several yeasts were isolated from the ripe sourdough, which are supposed to originate from the cassava flour. An unknown yeast species constituted 10 % of the isolated yeasts which is described as novel species Cryptococcus thermophilus sp. nov. in Chapter V. This yeast is characterized by budding on small neck-like structures, no fermentative ability, growth at 42 °C and without vitamins, a major ubiquinone of Q-10, as well as the production of green or blue fluorescent substances in the growth medium. It is distinct from related species by the ability to assimilate raffinose and cadaverine, the inability to assimilate soluble starch, xylitol, galactitol, butane-2,3-diol, sodium nitrite and lysine, and the inability to produce starch-like substances. The closest relatives are the yeasts belonging to the Cryptococcus humicola complex.
Strain-resolved analysis of the human intestinal microbiota
(2022) Podlesny, Daniel; Fricke, Florian W.
The gut microbiota is ascribed a crucial role in human health, particularly in regulating immune and inflammatory responses, which is why it is being associated with a wide range of diseases, including obesity, diabetes, and cancer. Nonetheless, fundamental ecological questions of microbiome establishment, stability and resilience, as well as its transmission across hosts and generations remain incompletely understood, partly due to the lack of methods for high-resolution microbiome profiling. New insights in this field can therefore directly contribute to the development of bacterial and microbiota-based therapies. This work introduces SameStr, a novel bioinformatic program for strain-resolved metagenomics that allows for the specific tracking of microbes across samples, enabling the detection and quantification of microbial transmission and persistence, as well as the observation of direct strain competition. Deployed across cohorts to process over 4200 metagenomes, SameStr enabled analysis of the microbiome with unprecedented phylogenetic resolution. The data included both publicly available metagenomes and sequence data generated in collaboration with our research partners, and was examined using multivariate statistics and machine learning frameworks. First, the establishment and development of the neonatal microbiota was studied, revealing a birth mode-dependent vertical transmission of the maternal microbiota. The microbiota of neonates born by cesarean section was characterized by increased relative abundance of oxygen-tolerant and atypical organisms and showed signs of a delayed establishment of a strictly anaerobic gut environment in these children. Such birth mode-dependent differences diminished over time, yet were measurable within the first two years of life. Furthermore, strain analysis verified the transmission and colonization of parental microbes, which indicated a possible lifelong colonization by microbes from selected species. The temporal persistence of microbes was also characterized in healthy adults, revealing similar taxonomy-dependent patterns of stability. For some species, persistence has been demonstrated both in children and in adults over a period of at least two years. These species are known for their capability to metabolize host-derived glycans found both in breastmilk and intestinal mucus, pointing to a potential strategy for effective cross-generational microbiota transmission, and warranting additional research to assess the implications of their disturbed transfer for long-term health. Since their specificity allows assignment to individual hosts, fingerprints of individual microbial strains offer the potential to be used in forensics and data quality control applications. Finally, to gain new insights into the microbiota dynamics during Fecal Microbiota Transplantation (FMT), microbial strain transmission was analyzed in the context of a diverse set of patient, microbiome, and clinical conditions. In the analyzed studies, FMT was used for the experimental treatment of a variety of diseases, including colonization with drug-resistant and pathogenic microbes, metabolic and inflammatory bowel diseases, and as an adjunct to the immunotherapeutic treatment of cancer. Analyses uncovered what appear to be the universal drivers of post-FMT microbiota assembly, including clinical and ecological factors that are important for successful transplantation of donor strains. In particular, the relevance of the microbiota dysbiosis of the recipient was emphasized, which was inducible by pre-treating the patient with antibiotics or laxatives. Presumably, this can open up ecological niches in the patients intestines, which favors colonization with donor strains. Colonization rates did not play a role for the treatment success of recurrent C. difficile infections and inflammatory bowel disease, but indicated a trend associated with an improved immune response in cancer patients. Concerningly, the transfer of an atypical and potentially pro-inflammatory microbial community from one donor was also observed, calling for further investigations into the immediate and long-term clinical consequences of FMT. These analyses demonstrate the advantages of a strain-based microbiome analysis. Due to the achieved methodological accuracy, strain-resolved microbial dynamics could be precisely disentangled when comparing longitudinal samples from healthy adults as well as parent-child and patient-donor pairs. This revealed taxonomic, clinical, and ecological factors that are critical to microbiome assembly, including microbial transmission, persistence, and competition. Together, these findings lay the groundwork for future developments of precision personalized microbiota modulation therapies.
The effect of aging in the murine gut microbiome
(2020) Hernández Arriaga, Angélica; Camarinha-Silva, Amélia
Aging is characterized by several physiological changes. During the lifespan, the biological systems from the body of humans and other animals remain dynamic. Throughout the early stages of life, the microbiome develops into a complex ecosystem with thousands of species. Variations related to diet, environmental changes, medications affect the diversity and composition of the microbiota through the lifespan. Some old individuals with higher incidence of chronic diseases have a loss of the stability of the microbiome and an imbalance occurs between the different colonizers of the gut, also named dysbiosis. One of the most distinctive changes occurring with age is the prevalent low grade inflammation, which is named inflamm-aging. This not only changes the microbial composition of the GIT but also affects the permeability. Murine models are well established and help us to understand the complex dynamics between the host and the microbial communities inhabiting the gastrointestinal tract. These models allow us to analyze microbial communities from tissue and mucosa, from all sections of the gut, which is limited in humans. Methods standardization is an important topic in microbiome research. In chapter 2 it was compared the efficiency of two sample methods, cotton swab and tissue biopsy, in characterizing the mouth microbiota. In recent years, the mouth microbiome is being seen as a diagnostic tool for not only oral diseases but also systemic diseases. As physiological changes occur with aging, the microbiome from the mouth is affected and there is an increase of pathogens present in the oral surfaces. In murine models, cotton swab is a common tool used for sampling the microbiome of the oral cavity. In our study, we observed similar microbial community structure using both methodologies. However, the species Streptococcus danieliae, Moraxella osloensis, and some unclassified members of Streptococcus were affected by the different sampling procedures. In this trial, we included mice at two different ages, 2 months old being considered young and 15 months old considered middle aged mice. We observed changes in the genera Actinobacillus, Neisseria, Staphylococcus, and Streptococcus related to the age of the animal and the sampling type. These results showed the importance of sampling standardization in microbiome research and that age has a strong effect on the microbial ecology of the oral cavity. In chapter 3, it was studied the bacterial communities from duodenum and colon of mice at 2, 15, 24 and 30 months of age in combination with the results of the expression levels of antimicrobial peptides in small intestine and markers of intestinal barrier function. Besides, in this chapter were also assessed the indices of liver damage, inflammation and expression levels of lipopolysaccharide binding protein (Lbp) as well as of toll-like receptors (Tlr) 1-9 in liver tissues. At 24 and 30 months of age there was an increase in inflammation, they developed fibrosis and the levels of endotoxin in plasma were higher. Regarding changes in the microbiome, the duodenum had more changes than the colon related to age. Allobacullum, Bifidobacterium, Olsenella, Corynebacterium were the genera that differed statistically in the duodenum through the murine lifespan. Fewer changes were observed in the colon, as Allobaculum was the only genus that showed differences between young and old mice. Additionaly, it was analyzed the impact of aging in the active microbial communities of mouth, duodenum and colon at 2, 9, 15, 24 and 30 months of age (chapter 4). Changes were observed at every age and different taxonomical levels, with a greater shift at 15 months of age. This is related to the age of the mice, as at middle age systemic changes related to the aging process start to occur. At old ages, there was an increment of the pathobiontic species Helicobacter hepaticus and Helicobacter ganmani in the duodenum and colon. The oral, duodenal and colonic microbial communities are important pieces of information that can be related to the health status of the host. Research that focuses on assessing the changes in the different niches and not only in the feces, gives a broader overview of the microbial community of the host.
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.
The production of melezitose in honeydew and its impact on honey bees (Apis mellifera L.)
(2021) Seeburger, Victoria; Hasselmann, Martin
Honeydew honey is a honey type which is of high economic importance in Europe. Phloem sap feeding insects of the order Hemiptera (true bugs) excrete honeydew, the key component of honeydew honey. Beekeepers move their hives between forest regions so that their bees can process the honeydew into honey. In case of high osmolality in the phloem sap of the hemipterans’ host trees, they counteract osmotic pressure by osmoregulation and produce oligosaccharides such as melezitose. Melezitose-rich honeydew honey is a major issue for beekeepers; it crystallises and obstructs the combs, leading to an economical loss. Nevertheless, precise analyses of the conditions of the occurrence of melezitose have not been realised. Furthermore, it is not known which impacts the trisaccharide has on honey bee health and the honeydew flow disease documented in beekeepers’ journals can have one explanation in the nutrition on melezitose. In order to determine influence factors for the emergence of melezitose, more than 600 honeydew droplets from defined honeydew producer species were collected under different environmental conditions (hemipteran species (host tree specific), natural area, air temperature, relative humidity, altitude, time of the year and of the day) between 2016 and 2019. The sugar spectra were analysed by high performance anion exchange chromatography with pulsed amperometric detection. To obtain the impact of melezitose on honey bee health, additional feeding experiments with daily evaluation of food uptake, gut-body weight ratio and mortality have been realised between 2017 and 2019. Additionally, comprehensive 16S rRNA Illumina sequencing of the gut microbial community has been performed. Remarkable differences could be found in the amount of melezitose between honeydew samples collected from different honeydew producer species and according to different environmental conditions. Air temperature increases and decreases in relative humidity increased the melezitose production in honeydew by the observed seven hemipteran species. Both, scale insect species on Picea abies and aphid species on Abies alba produced significantly less honeydew containing melezitose than aphid species on Picea abies. Additionally, honeydew with increased melezitose content was significantly more frequent collected in natural areas with limited water reservoir capacities, at higher altitudes and years with low precipitation. All results lead to the conclusion that hemipteran species produce more melezitose when the host trees have less access to water, increasing the osmolality of the phloem sap and indirectly enhancing the osmoregulation with producing melezitose by hemipteran species. Bees fed with melezitose showed increased food uptake and higher gut-body weight ratio than the control groups. Furthermore, melezitose feeding caused disease symptoms such as swollen abdomen, abdomen tipping and impaired movement and a significantly higher mortality than in control groups. Gut microbiota analyses indicated a shift of the bacterial species Lactobacillus Firm-4 and Lactobacillus kunkeei in favour of Lactobacillus Firm-5 in melezitose fed bees. This PhD project provides the important knowledge about the indicators that point out an enhanced melezitose production. This is a valuable contribution to design a warning system for beekeepers that will help to prevent harmful nutrition for honey bees or crystallised honey in the future by timely removal of bee colonies from local regions at risk. Additionally, feeding experiments point out the high effort that is required for the degradation process of the large-molecule melezitose. This effort might lead to a higher uptake of food, heavier guts, shorter lifespan and a higher susceptibility to intestinal diseases. Finally, an evidence was presented that the lactic acid bacteria of the gut microbiota are significantly involved in the digestion of melezitose.
Toll-like Receptor 9 (TLR9) activation and the innate immune response to microbial and human DNA
(2023) Hsu, Emily; Fricke, Florian W.
The human Toll-like Receptor 9 (TLR9) is an endosomal Pattern Recognition Receptor (PRR) that recognizes DNA sequences containing the unmethylated Cytosine-Guanine (CpG) dimers, which are present in greater abundance in most bacterial genomes compared to those of vertebrates. Specific CpG-containing sequences are strongly stimulatory of human TLR9, as shown in published studies using synthetic oligonucleotides (ODN) and DNA from bacterial species of varying genomic CpG concentration. Human TLR9 activation was experimentally examined in this thesis using DNA extracted from different bacterial sources, human DNA from Caco-2 cells, known immunostimulatory ODN, and short ODN. In vitro assays using fragment length-standardized microbial genomic DNA on HEK-Dual TLR9 Cells and human peripheral blood mononuclear cells (PBMCs) revealed that TLR9 activation strongly correlated to CpG concentration of the input DNA, with an additional influence of CpG-containing 5-mer TCGTT concentration. When DNA of varying origins and fragment lengths were used together, however, complex dynamics of TLR9 activation, co-activation, and repression were observed, which were less predictable than expected from genomic CpG concentration alone. DNase I-treated microbial DNA fragments of less than 15 bp of length were non-activating on their own, but co-activated human TLR9 together with ODN-2006 in Ramos Blue (B) cells. Similarly, human DNA fragments at the length of 50-200 bp co-activated human TLR9 with both ODN-2006 and Escherichia coli DNA in HEK-dual TLR9 cells. In contrast, large human DNA fragments at over 10000 bp of length repressed TLR9 activation by ODN-2006 in Ramos Blue cells. Finally, a preliminary study was conducted in HT-29 cells on the effect of TLR9 activation on the invasion of Fusobacterium nucleatum, an opportunistic gut pathogen with a very low genomic CpG concentration at 0.296%, using ODN-2006 and human DNA as TLR9 activators. While increased presence of intracellular Fusobacterium nucleatum upon treatment with both ODN-2006 and human DNA was noted, more studies are needed to confirm TLR9 activation as a cause of greater bacterial invasion. The human colon is the location of the largest microbial population of the human body, which provides a rich source of non-human DNA in contact with human TLR9 present in intestinal epithelial cells, plasmacytoid dendritic cells (pDCs), and B lymphocytes. Additionally, the daily mass shedding and death of human intestinal epithelial cells provide large amounts of human DNA, which when combined with microbial DNA could result in co-activation and possible autoimmunity. The thesis thus provided an in vitro model of TLR9 activation by complex DNA of varying origins and fragment lengths likely to present in the human gut environment, and prepared a working basis for future studies of TLR9 activation by human fecal metagenomic DNA.