Imbalance of intestinal microbiota is called dysbiosis. Signs of dysbiosis are altered abundance of different bacterial species and reduced diversity together with altered interactions between bacterial species and microbiota and the host. Dysbiosis of intestinal microbiota is connected to many intestinal diseases and today many studies are focused to find so called “next generation” probiotics to be used for the alleviation of dysbiosis instead of traditional antibiotic treatments. The study was made in the Human Microbiome Research Program, Faculty of Medicine, University of Helsinki. Aim of the study was to isolate spore-forming bacterial species for the treatment of intestinal inflammation and infections with bacterial therapy. For this purpose, feces from a healthy adult who had acted as a donor for fecal microbiota transplantation was used to isolate spore-forming commensal bacteria. The isolated bacteria were identified and their ability to adhere into intestinal epithelium and strengthen it was investigated. Also anti-inflammatory potential of these isolated bacterial strains was investigated. For isolating bacteria three different heat treatments and ethanol and methanol treatments were used as a pre-treatment step. Pre-treated samples were cultivated on YCFA-media and isolates were picked from plates at different growth points for further cultivation. Selected isolates were purified, their DNA was isolated and they were identified by partial 16S rRNA -gene sequencing. From these identified isolates four isolates were chosen for further investigation and their full length 16S rRNA -gene was sequenced. These isolates were studied also by using API and aerotolerance tests. Potential anti-inflammatory and adhesion properties of the isolates were investigated by attenuation, adhesion and TER-experiments. In the isolation, the effect of different pre-treatments on the recovery of isolates was clear and based on sequencing isolates that were spore-forming anaerobic bacteria were selected for further investigation. Three of the isolates were Clostridium butyricum and one Blautia wexlerae species. Anti- and pro-inflammatory properties of these isolates were very different depending on isolate and one of them was potentially anti-inflammatory. Isolates also adhered differentially and two of them possibly strengthened gut epithelial barrier so they are promising for further research and in the future investigation with these isolates continues. Experience and results with different cultivation methods can be used to for further development of cultivation for anaerobic intestinal bacteria.

Microbiota inhabiting the colon fermentate carbohydrates, proteins and endogenous substrates to volatile fatty acids (VFA) and produce energy for the microbial growth. Because all species of bacteria ferment some component of the digesta and produce various VFAs, alterations in microflora may modify these fermentative end products. Thus, measuring the amount and type of VFA produced gives an instrument which reflects changes in the bacterial microbiota of the intestine. This study set out to explain the connections between diet composition and the formation of VFAs. The general hypothesis was that different food compositions cause differences in the VFA profile, and this may have systemic effect on animal health. Graeco Latin Square design study with 5 healthy Beagles was performed, feeding high protein (diet A, starch 54 g/kg, crude protein 609 g/kg), high carbohydrate (diet B, starch 438 g/kg, crude protein 194 g/kg), and a balanced commercial (diet C, starch 277 g/kg, crude protein 264 g/kg) diets for three weeks each. The diet C was used also for the baseline. VFA, fecal dry matter and fecal consistency score were assessed. All dogs had formed feces during diets B and C but diarrhea during diet A, leading to significant differences in fecal consistency score between the diets (p < 0.0001). The results indicate that alterations in diet had a large influence on the amount and quality of VFAs produced. Mixed-effect model analysis shows that the diets had a statistically significant (p<0.05) influence on all of the VFAs produced excluding butyric acid. The most significant changes from the baseline diet were seen with the high protein diet. Compared to the baseline diet, valeric acid production increased 24-fold, isobutyric acid by 79.5% and isovaleric acid by 42.4 %. Production of propionic acid decreased by 43.3%, acetic acid by 25.0%, and butyric acid by 10.2 %. In previous studies similar changes in VFA profile have been coupled with various intestinal diseases as well as inhibition in biotin absorption. Furthermore, this might have an influence on inflammatory response at the cellular level. Thus, changes in VFA profile may have an influence at least on the local intestinal health. The total amount of fatty acids decreased on both experimental diets. It seems that having moderate protein and carbohydate levels in the diet is a virtue and more is not necessarily better. This study provides additions to existing understanding of the relationship between diet composition and the formation of VFAs in the intestine. The findings suggest that observing the alterations in VFA levels formed in the intestine and therefore present in feces, may provide an instrument to indirectly observe changes in the bacterial microbiota of the intestine. Thus, there is a need to find the link between the changes in VFA profiles and colonic microbiota, and bacterial diversity in feces by using molecular methods. Having this greater level of understanding would lead to more robust insights into the role of intestinal microbiota in animal health, and to potential advances in the prevention and curing of related diseases.

Non-alcoholic fatty liver disease (NAFLD) is currently the most common liver disease in the western world. The human intestinal microbiota possesses enormous metabolic and immunomodulatory capabilities, and together with increased intestinal permeability, changes in the microbiota have been linked to the development of NAFLD. However, human studies so far have yielded contradictory findings regarding the compositional microbiota changes and provided little mechanistic understanding due to the predominance of cross-sectional studies. The aim of this study was to study human intestinal microbiota and gut permeability in NAFLD. Real-time PCR was employed to quantify the key intestinal bacterial groups in overweight or obese subjects with (n = 12) and without (n = 19) NAFLD, and in response to hypercaloric overfeeding, where participants were provided with three compositionally distinct diets to temporarily increase liver fat. In addition to the comparative analysis, the microbiota results were correlated to serum markers of intestinal permeability and metabolic endotoxemia, as well as clinical parameters related to NAFLD. The results show that host lipid metabolism and the gut microbiota, specifically Bacteroidetes and Clostridium cluster XIVa, are firmly intercorrelated. Bacteroidetes were found to be less abundant in subjects with NAFLD and correlate negatively with liver fat and serum triglycerides at baseline. Clostridium cluster XIVa, a dominant Firmicute group, was positively associated with serum triglycerides and pro-inflammatory markers but negatively with intestinal permeability. The relative abundance of Bacteroidetes as well as the markers of metabolic endotoxemia changed significantly in response to overfeeding, while no diet-induced systematic effects were found in Clostridium cluster XIVa, total bacteria, Escherichia coli group, Bifidobacterium or gut permeability. Our results based on a targeted microbiota analysis suggest that the role of the intestinal microbiota and gut permeability on triggering metabolic disarrangement and NAFLD in humans is inferior to other stimuli, such as diet.