Browsing by Author "Pakola, Ida"

Dysbiosis is a condition where gut microbiota’s diversity and stability are decreased and the composition of bacterial community is altered as compared to healthy microbiota. Dysbiosis can cause serious harm to host’s immune system and it is linked to inflammatory bowel diseases (IBD). In IBD, the immune system is disturbed and there is a constant inflammation of the gut. Currently IBD is treated by antibiotics and immunomodulators, but it could be preferable to use treatments which aim to restore a healthy microbiota, because dysbiosis has been found to play role in maintaining the continuous inflammation in gut. One potential treatment to restore the healthy microbiota is bacterial therapy. Anti-inflammatory bacterial species of healthy gut could be potential components of bacterial therapy product for IBD-targeted drug. The aim of this study was to isolate spore-forming anti-inflammatory bacteria from fecal material that had been used previously in fecal microbiota transplantation. The aim was to isolate intestinal bacteria, which could be used as a treatment for IBD in the future. Anti-inflammatory properties of spore-formers were an object of interest because the aim was to study could bacterial spores be used in IBD-targeted bacterial therapy. For isolating bacterial spores from the sample, two selection methods were used, ethanol-treatment and heat shock. Treated samples were cultivated on six different media. Bacterial isolates were picked from the plates followed by anti-inflammatory screening to select potentially anti-inflammatory isolates. In anti-inflammatory screening the effect of an isolate on lipopolysaccharide induced IL-8-production of HT-29-cell line was measured. Potentially anti-inflammatory isolates were purified to pure cultures followed by sequencing of their 16S rRNA gene (rDNA). Anti-inflammatory effect of pure cultures on HT-29-cells were tested again with three parallel reactions. Selection methods didn’t work out as well as expected. Based on the 16S rDNA sequences of bacterial populations collected from different media, two treatments managed to select a good portion of Firmicutes, but none of the potentially anti-inflammatory pure culture isolates were spore-formers. According to the 16S rDNA sequences most of the purified strains belonged to the genera Staphylococcus or Enterococcus. Only a small proportion of the potentially anti-inflammatory isolates were confirmed as anti-inflammatory. In this study, six anti-inflammatory Enterococcus faecalis -strains were isolated and purified. In the future, these strains need to be studied further to assess their potential in bacteriotherapeutic applications and clinical use. The methods and results of this study can be used to further optimize the procedures to isolate various intestinal bacteria.