Browsing by Subject "dysbiosis"
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(2023)Abstract Faculty: Faculty of Agriculture and Forestry* and Faculty of Medicine *coordination Degree programme: Master′s program in Microbiology and Microbial Biotechnology Author: Johanna Potila Title: Characterization of potentially therapeutic bacteria from a healthy fecal donor. Level: Master′s thesis Month and year: August 2023 Number of pages: 40 Keywords: Clostridioides difficile, dysbiosis, FMT, next-generation probiotics, adhesion, anti-inflammatory Supervisors: PhD Kaisa Hiippala, PhD, Docent Reetta Satokari and PhD Pauliina Lankinen Where deposited: E-thesis University of Helsinki Abstract: Recurrent Clostridioides difficile infection (rCDI) is a healthcare-associated infection related to antibiotic use, that causes significant morbidity and mortality. Fecal microbiota transplantation (FMT) is the most effective treatment for rCDI and it is successful in nearly 90% of patients. However, there are some risks related to FMT use such as the potential risk of transferring pathogens or other phenotypes despite donor screening. Defined bacterial mixtures consisting of endogenous commensal gut microbes with beneficial properties could be used instead of FMT to mitigate the risks and improve the availability of the treatment. 12 bacterial strains previously isolated from a healthy fecal donor were characterized in this study. At first, oxygen tolerance and culturability of the isolates in several different media were examined. The second aim was to investigate if these isolates are safe for bacteriotherapeutic use by testing hemolytic properties, antibiotic susceptibilities and proinflammatory properties. The third objective was to investigate potential beneficial properties such as adherence of the isolates to mucus and epithelial cell lines and anti-inflammatory effects on epithelial cells. Caco-2 and HT-29 cell lines were used as a model of intestinal epithelial cells. Growth was abundant on standard brain heart infusion (BHI) medium supplemented with 0,5% yeast extract and more than half of the isolates tolerated the 4-hour oxygen exposure. These results suggest that many of the strains have good production characteristics. All 12 isolates were non-hemolytic and most of them were susceptible to many commonly used anti-microbials such as amoxycillin/clavulanic acid and piperacillin/tazobactam. Low induction of interleukin-8 (IL-8) release from HT-29 cells was observed for all the isolates which indicates no pro-inflammatory effect. These safety tests suggest that the isolates are safe for therapeutical use. Adhesion to mucus and intestinal epithelial cells (HT-29, Caco-2) was low to moderate (2-7%), which can potentially promote their colonization in the gut. No attenuation of Escherichia coli lipopolysaccharide (LPS)-induced IL-8 release from HT-29 cells was observed, which indicates that characterized strains do not have anti-inflammatory effects on epithelial cells. However, it is likely that they have some other important roles in the gut e.g., in cross-feeding networks and can thus help with restoration of a healthy, diverse gut ecosystem. In conclusion, the characterized isolates could be suitable for bacteriotherapeutic use in the treatment of rCDI.
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(2017)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.
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