Faculty of Agriculture and Forestry

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.

This thesis examines the effects of Internationally Transferred Mitigation Outcomes (ITMOs) trade –embedded in the market mechanisms of Article 6 of the Paris Agreement– in assisting countries to achieve their Nationally Determined Contributions (NDCs), incentivize increased mitigation ambition, and contribute to the long-term goals of the Paris Agreement. The study takes place within a context characterized by highly heterogeneous and non-Paris aligned NDCs, reflecting current global circumstances. The study employs a conceptual framework that integrates the Social Cost of Carbon (SCC), Marginal Abatement Cost (MAC) curves, and Marginal Benefit (MB) curves. This framework is used to analyze the impact of ITMO trading on the ambition of NDCs and the potential for global mitigation. The research reveals that the roles countries play in the ITMO market—whether as buyers or sellers—is significantly influenced by the varied nature of NDCs, each country’s unique domestic marginal abatement costs, and how their mitigation ambition aligns with the SCC. In addition, it was found that ITMO trading can potentially trigger additional mitigation and escalate target ambitions. However, the success of this mechanism heavily depends on certain conditions, restrictions, and alignment with Article 6 principles. For instance, allowing negative reoptimization could lead to countries manipulating their NDCs for short-term gain, thus undermining long-term climate goals. In contrast, scenarios permitting positive reoptimization and restricting trade to beyond-NDC targets can encourage higher ambition levels in line with the Paris Agreement goals. However, the study also highlighted significant challenges and limitations. The risk of overselling could undermine future mitigation efforts, and managing these complexities requires a thorough understanding of each country’s domestic mitigation context. Moreover, the varied nature of NDCs adds a layer of complexity to ITMO trading, necessitating a meticulous examination of each country’s future actions. The study recommends future research to devise methods for better aligning NDCs to facilitate more effective ITMO trading. This could involve an extensive exploration of potential safeguards for ITMO trade such as transfer limits, ambition coefficients, surplus targets, and taxes. Evaluating their effectiveness could offer valuable policy implications for ITMO trade regulation. Future studies should also delve into the uncertainties and dynamic intertemporal effects that impact ITMO trading, aspects not accounted for in the current conceptual framework. By broadening the focus to include imperfect markets and real-world uncertainties, we can strive towards more robust and effective climate policies.

Various disturbances of varying durations can occur in production lines. Objective of this master´s thesis was to research how disturbances in production lines possibly affect the microbiological and sensory quality of the product. The study examined products that had been standing at room temperature for 0, 30, 60 and 210 minutes. These samples were examined at time points 0, 20, 40 and 60 days. Three different batches were used for the sampling purposes. In the microbiology section contaminating microbes, yeast, molds, enterobacteriaceae and lactic acid bacteria were studied. The identification of colonies was performed using the Maldi-TOF method. For the sensory study, evaluations were conducted at the company factory using difference-from-control method. The aim of sensory study was to determine whether temperature fluctuations alter the sensory quality. A total of 29 participants took part in the sensory study. The participants discovered variation between samples and the fluctuations in temperature did cause growth in contaminating microbes and lactic acid bacteria on the culture media. However, there was no significant growth observed in molds and yeasts. No enterobacteriaceae were detected during the research. In the sensory study, the evaluators did not find significant differences between reference sample and sample which was exposed to room temperature for 30 minutes. The results did not support the hypothesis of the sensory aspect of this thesis. It should be noted that possible greater number of participants or different sensory research method could have revealed more information. Overall, the research revealed information about the effects of temperature on cheese, particularly the crucial time frame within which cheese can be kept at room temperature before quality deteriorates. The results indicate that temperature variations during processing phase have impact on the overall quality. Furthermore, the study confirmed the absence of harmful bacteria and highlighted that temperature fluctuations influence the sensory quality especially in terms of the product´s appearance.