Browsing by Subject "microbiome"

Tiivistelmä – Referat – Abstract Mental disorders are among the leading causes of global disease burden and years lived with disability. Their pathogenesis is poorly understood and there are enormous challenges in the development of biomarkers to aid in diagnosis and more effective therapeutic options. It has been documented that the microbiota-gut-brain axis shows alterations in mental disorders such as anxiety, depression, autism spectrum disorders, bipolar disorder and schizophrenia. Here we study the gut microbiota of individuals with axis I mental disorders and their unaffected siblings by 16S RNA gene amplicon sequencing. In the Central Valley of Costa Rica, a total of 37 participants were recruited and diagnosed using a Best Estimate Diagnosis protocol. For each of the individuals diagnosed with a mental disorder a healthy sibling was selected after matching by age and gender. A total of 13 pairs of 26 siblings, affected and unaffected, was used for the analysis. In a subsequent analysis, individuals were also divided into the three categories of “unaffected” (UA), “affected without psychosis” (AA) and “affected with psychosis” (AP). They underwent clinical assessments about their habits and diet and about resilience (Connor-Davidson Resilience Scale), current status (SADS-C) and disability (WHODAS 2.0). Their fecal samples were collected freshly and stored at -80°C. DNA was extracted, libraries constructed by PCR and subjected for Illumina MiSeq 300 paired-end 16S RNA amplicon sequencing for analysis of the gut microbiota. The sequencing data were analyzed using the R packages mare and vegan for gut microbiota composition, diversity and richness, taking into account the identified confounders. All participants were of Hispanic ethnicity, residents of the San José Greater Metropolitan Area, adults and 69% of them were women. Affected individuals had major depression, bipolar affective disorder, psychosis non-otherwise specified or schizoaffective disorder. Based on beta-diversity analysis as a measure of the community-level microbiota variation, it was found that the use of levothyroxine (R2=0.08, p=0.005) and of irbesartan (R2=0.068 ,p=0.001) had a significant impact on the microbiota composition and hence the use of these drugs was included as confounder in further analyses. Several statistically significant differences in the relative abundance of intestinal bacteria were identified: Differences were found in the relative abundance of bacterial families Peptostreptococcaceae, Ruminococcaceae, Porphyromonadaceae, and in bacterial genera Pseudomonas, Barnesiella, Odoribacter, Paludibacter, Lactococcus, Clostridium, Acidaminococcus and Haemophilus. Our results indicate that affected individuals have more pro-inflammatory Proteobacteria (Pseudomonas) and less bacteria associated to healthy phenotype, such as Barnesiella and Ruminococcaceae, the former being dose-dependently depleted in AP and AA compared to UA. Furthermore, we documented decreased bacterial richness among affected participants while no significant differences were detected in alpha diversity. Our study identified significant differences in the microbiota of individuals affected by mental illness when comparing to their healthy siblings. The results may have important implications for the holistic understanding of mental health and its diagnosis and therapeutics. Larger studies to confirm these findings would be justified.

The early life gut microbiota plays a major role in establishing neonatal immunity and child’s long-term health. However, relatively little is still known about the role of individual bacteria as most studies so far have focused on characterizing the diversity and the individual and temporal variations of the infant gut microbiome. The genus Bacteroides is of particular interest since its abundance is remarkably decreased in infants born via C-section, and relatively little is known about the genomic and phenotypic characteristics of early Bacteroides colonizers despite their anticipated role in the increased morbidity following C-section birth. This thesis aims to contribute to the isolation and characterization of Bacteroides strains from infant and mother stool samples from the Health and Early Life Microbiota (HELMi) cohort study using culture-based and metagenomic approaches. Gram-negative bacteria were isolated from stool samples of 9-week-old infants and identified by Sanger sequencing. In total, seven isolates identified as unique species of Bacteroides, isolated from infant samples or previously from mother samples in late pregnancy, were then characterized for their potential to activate innate immunity in vitro by using HEK-Blue™ hTLR2-hTLR6 reporter cells either as live cells or filtered culture media. Whole genome shotgun sequenced stool metagenomes obtained from 88 infants during the first year of life were leveraged as well. A computational pipeline able to scale to the large size of the dataset was developed to obtain metagenome assembled genomes (MAGs) from the metagenomes. MAGs obtained from Bacteroides species were further taxonomically and functionally annotated. Among the seven Bacteroides spp. isolated from HELMi mother and infant samples, the majority were able to activate the TLR2/6 receptor in vitro. The isolates varied in their potential to activate the receptor via their cell surface molecules and substances they excreted to the culture media. In addition, over 2500 MAGs could be retrieved from the infant metagenomes, of which 18 belonged to Bacteroides spp. Based on predicted open reading frames, majority of the identified proteins of these MAGs were involved in housekeeping functions. Most of predicted proteins involved in cellular metabolism were, however, related to carbohydrate metabolism, amino acid metabolism, and glycan metabolism, stressing the role of Bacteroides spp. in the gut as important and versatile carbohydrate consumers. The results indicate that the Bacteroides spp. colonizing infant gut have an immunologically and metabolically active role. Further work is needed to characterize the molecules responsible for the TLR2/6 activation as well as the nature of the downstream immune responses elicited by the isolated Bacteroides spp.

The aim of this study was to examine the leaf endophytic bacteria in Plantago lanceolata. The first aim was to get a comprehensive picture of the bacterial diversity, by screening for the different bacterial genera inside the leaves. Furthermore, I aimed to examine the effect of soil and maternal genotype on the endophytic community within P. lanceolata leaves and search for clues of vertical inheritance of endophytes from parent to offspring via seeds. I studied the endophytic bacteria by extracting DNA from the plant leaves and by trying to amplify any bacterial DNA present to get a view of the bacterial diversity in the leaves. My aim was to compare the bacterial community of the mother plants to that of their offspring and also to compare the bacterial communities of plants grown in different soil conditions. Furthermore, I tried to study how the soil conditions affect the growth of P. lanceolata seedlings. I collected seeds and leaf samples of P. lanceolata from Åland, Southwestern Finland, from a population that is part of the ongoing long-term metapopulation research started in Åland in the early 90’s. I marked 21 plant individuals (hereafter referred to as the “mother plants”) in the field in June, when collecting the first leaf samples. In August I collected all seeds from the same plant individuals and a second set of leaf samples. I also collected soil samples from the same location. With the seeds collected from the wild population I executed a growth experiment in Viikki, Helsinki. I grew one set of seeds in twice autoclaved sand (hereafter referred to as the “sterile soil”) and another set in twice autoclaved sand mixed with soil collected from the Åland population (hereafter referred to as the “environmental soil”). I surface sterilized all seeds and then sowed each in their own growth pot and placed them in a growth chamber. During the experiment I took measurements of the leaves. At end of the growth experiment, I took samples of the leaves and surface sterilized them to exclude any epiphytic microorganisms from the analysis. I also surface sterilized the leaf samples taken from the mother plants. I then extracted DNA from the leaf samples and run PCR to amplify certain regions of the bacterial 16S rDNA gene, that is widely used in bacterial taxonomy. The obtained DNA reads where then clustered into Operational Taxonomic Units (OTUs) and assigned taxonomy using SILVA reference database. Mitochondria and chloroplasts of eukaryotic organisms also harbour 16S rDNA regions, so the challenge of studies looking at endophytic bacteria is to exclude the 16S regions of mitochondria and chloroplasts. This proved to be a problem in my study also. More than 86% of all DNA reads obtained turned out to be from P. lanceolata mitochondria and more than 12% from P. lanceolata chloroplasts. Only a bit more than 1% of the reads were eubacterial. This effectively hindered reliable analysis of the endophyte community. I nevertheless analysed the observed eubacterial diversity although the results must be taken as only preliminary and with utmost caution. The eubacterial reads clustered into 218 OTUs, representing 71 different bacterial genera. Six most common genera constituted over 83% of eubacterial reads. Most of these bacteria, most notably Shewanella, Ralstonia and Halomonas, could be identified as being clearly contaminants and not real endophytes. For the 65 less common bacterial genera I performed community analysis using Bray-Curtis Dissimilarity index and Analysis of Similarities (ANOSIM). The results showed that there was a significant difference between the different soil treatments (P = 0.014, R = 0.3787) and also between the two growth chambers (P = 0.011, R = 0.5493). I found no effect of maternal genotype on the bacterial community. Therefore, I observed no sign of vertical inheritance of endophytes. The growth experiment results showed that germination percentage was significantly lower in the environmental soil than in the sterile soil for all genotypes (F = 10.78, P = 0.0012). However, seedling in the environmental soil grew bigger than the seedlings in the sterile soil (F = 10.91, P < 0.0001). For future studies on similar topics, validating molecular methods before large scale sequencing could yield more reliable results. Size fractionating the DNA products of the first PCR round could exclude most mitochondrial sequences and hence allow better analysis of endophytes. This would enable studying interesting questions on coevolution and ecology of host-endophyte interactions. Although I did find some differences in the bacterial communities of different treatments, these results must be considered with caution and as only preliminary.

The objective was to evaluate how different silage additives can manipulate the ensiling process and the profile of bacterial communities of grass silages under varying management conditions. Silages were made from mixed timothy (Phleum pratense) and meadow fescue (Festuca pratensis) grass to laboratory scale silos using two compaction levels. The tightly compacted grass was also contaminated with soil and dairy cow faeces. Four additive treatments were used including control without additive (CONT), formic acid based additive (FA), homofermentative strains of lactic acid bacteria (LAB) and salt based additive (SALT). Tight compaction resulted on average in lower pH and ethanol concentration in silages than loose compaction mostly caused by changes in CONT silages. Soil contamination clearly affected CONT and SALT silages by stimulating extensive fermentation and thus decreasing pH and amount of residual water-soluble carbohydrates (WSC) compared to non-contaminated silages. In all conditions, FA restricted fermentation resulting in silages with high WSC and reduced total fermentation products concentration. Soil contamination improved aerobic stability of silages compared to non-contaminated ones because of higher acetic acid concentration in contaminated silages. Abundance of selected 16 bacteria in raw material was low, with Sphingomonas and Stenotrophomonas genera being the most abundant. After fermentation both Lactobacillaceae family and as part of it Lactobacillus genus were dominant with Sphingomonas genus in most of the silages. FA decreased the abundance of Lactobacillaceae family whereas LAB increased it. Soil contamination reduced the amount of other Lactobacillaceae family but boosted the growth of Lactobacillus genus. Lactobacillus presented negative correlations with Mycoplana, Devosia and Sphingomonas. Five bacteria were connected to desirable fermentation pattern and they all were part of same phylum Firmicute. All other selected bacteria had negative correlation with low pH and amount of lactic and total fermentation acids in silage. Use of additives improved fermentation quality of silages ensiled under different management conditions. Different types of additives resulted in varied bacterial profiles. Results confirmed the importance of tight compaction and good hygiene for stable fermentation. Strong correlations between bacterial communities and fermentation quality parameters provided clear insight of the role of the most abundant populations on the fermentation process of grass silage.

Background Short-chain fatty acids (SCFAs), such as butyrate, acetate and propionate, mainly originate from dietary fibre fermentation in the colon. In recent years, additional gastrointestinal (local) and systemic effects of SCFAs have been observed in vitro and in both animals and humans in vivo. Faecal SCFAs have been associated with several conditions and diseases, including irritable bowel syndrome, type 2 diabetes, colorectal cancer, and cardiovascular diseases. Aim(s) The BEAN MAN study was carried out to assess the effects of the partial replacement of red and processed meat with legumes on nutrient intake, nutritional status, and the risk factors of type 2 diabetes, colorectal cancer and cardiovascular diseases. This thesis was part of the BEAN MAN study and investigated the effects of partially replacing red and processed meat with legumes on the concentration and composition of faecal SCFAs in healthy, working-age Finnish men. As a secondary outcome, the intake of dietary fibre and soluble polysaccharides were assessed and correlations between the total and individual SCFAs were analysed. Methods The BEAN MAN study was a 6-week randomized dietary intervention, with two diet groups in a parallel design. The Conventional Finnish diet group (Conventional FIN) was given 760 g/week of red and processed meat, corresponding the average meat consumption of Finnish men. The Flexitarian/Legume group (Flex/Leg) received 200 g/week of red and processed meat, and legume products to provide an equal amount of protein as 560 g of red meat. The faecal samples obtained from the participants before and at the end of the intervention period were freeze-dried, and SCFA were extracted and analysed using gas chromatography. Dietary fibre intake was analysed based on 4-day food records. Results At the endpoint, no significant differences in total (p = 0.560) nor individual SCFAs concentration and composition between the two diet groups were observed (ANCOVA, baseline as a covariate). The dietary fibre intake of the Flex/Leg group was higher (p < 0,001) when compared to the Conventional FIN. This was not the case for soluble polysaccharides. No correlation between the dietary fibre intake and the concentration of total or individual SCFA was found. Conclusion The concentration and composition of faecal SCFAs were not significantly affected by the partial replacement of red and processed meat with legumes during the 6-week intervention. This may highlight a more complex relationship between diet, microbiota and the production of SCFA.