Browsing by master's degree program "Magisterprogrammet i mikrobiologi och mikrobiell bioteknik"

The emergence of zoonotic viruses has been a great concern and threat for global public health. It is important to be aware of the persistence and circulation of zoonoses and their natural hosts in their habitants where wildlife-human interaction can more likely occur. Not many studies have been published regarding the situation in Ireland. In this study, we concentrated in three viral zoonoses, poxviruses, hantaviruses, and arenaviruses, that were screened and detected from Irish rodents specifically bank voles (Myodes glareolus) and wood mice (Apodemus sylvaticus) collected in spring and fall 2021 and spring 2022. By using serological approach such as indirect immunofluorescence assay (IFA), 683 rodent heart samples were screened. Based on serological screening, molecular assays were performed to detect each virus from tissue samples. As a result of the serological screening, 34 antibody-positives either for pox- (CPXV), hanta- (PUUV, DOBV), and arenaviruses (LCMV) were found from which 15 were CXPV, five PUUV, 11 DOBV and three LCMV. The seroprevalence was ranging from 0.4% to 2.2%. When it comes to molecular detection, no viral genome was identified in any of the tissue samples. Although the seroprevalences were low, this study presents the first evidence of CXPV, PUUV, DOBV and LCMV exposures found in Ireland. These findings suggest that there is a possibility of circulation of these viruses among wild rodents and therefore novel risks for viral transmission should be considered. Further research requires increased effort to monitor these viruses and their wildlife hosts in Ireland.

Antibiotic resistance is a global threat which could lead us into a “post-antibiotic” era. Although antibiotic resistance is a natural and ancient phenomenon, the widespread use of antibiotics has accelerated the evolutive selective pressure for antibiotic-resistant bacteria and antibiotic-resistant genes (ARGs). The EU is promoting using of bio-based fertilizers (BBFs) due to sustainability goals, yet these are materials that may contain bacteria carrying ARGs as well as antibiotic-producers such as Streptomyces. The role of antibiotic-producing bacteria in the dissemination of antibiotic resistance has not been extensively studied and therefore it is not fully understood. This work aimed to investigate phenotypic and genotypic characteristics of eight bacterial isolates from two different BBFs, which were composted biosolids, to gain a preliminary understanding of the role of antibiotic producers as potential mediators of the resistance. The phenotypic characteristics of isolates were studied using various media and temperatures. Whole-genome sequencing and bioinformatic approaches were used to investigate the genotypic features. One isolate from an ancient latrine, sampled during archeological excavation, was included in the bioinformatic analyzes to be compared with the genotypic similarity of the modern isolates. The phenotypic characteristics of the isolates were typical of Streptomyces, such as the formation, color, and composition of aerial and substrate mycelium. According to bioinformatic tools used (GTDB, ResFinder, MGE, and antiSMASH databases and BLASTn), the isolates were identified as Streptomyces spp. Several ARGs were found in the isolates: a total of 11 types of beta-lactam, 4 types of aminoglycoside, 5 types of glycopeptide, 7 types of macrolide, 4 types of phenicol, 2 types of quinolones, and 9 types of tetracycline resistance genes passed the qualitative screening. Ten different mobile genetic element (MGE) components were also found in the isolates. In addition, from 15 to 29 potential clusters of secondary metabolites were identified in the genomes of the isolates, some of which are known to have antibiotic activity. The results of this study suggest that antibiotic producers might play a role in maintaining and possibly even transmitting resistance genes in composted biosolids. Although this was only a preliminary and limited study on this topic, it suggests that antibiotic producers may have contributed to the dissemination and evolution of antibiotic resistance. Thus, investigating the co-evolution of antibiotic producers and antimicrobial resistant bacteria should be in the interest of scientists from here on. Perhaps understanding the interactions of the producers and resistant bacteria could open new pathways for controlling the spread of antibiotic resistance.

The gut microbiota is important for human health, participating in many important functions, such as digestion, and is strongly modulated by the diet. The consumption of red and processed meat should be reduced due to both health and environmental reasons. Red meat could be partially replaced with legumes, as they are rich in protein. In addition, legumes are a good source of fibre and increasing their consumption would increase fibre intake. Here we aimed to study the effects of a partial replacement of red and processed meat with legumes on the gut microbiota composition in Finnish healthy men. The study was a 6-week randomized clinical trial in parallel design and included two groups with diet supplemented either with red and processed meat (760 g/wk) or a lower amount of red and processed meat (200 g/wk) and legume products containing the corresponding amount protein as in 560 grams of red and processed meat. Both diets provided 25% of the participants’ daily protein intake. The microbiota composition was analysed before and at the end of the intervention period from faecal samples. In total 102 participants finished the study. The group with the diet containing legume products showed a significant reduction in alpha diversity (p=0.029) and in the relative abundance of the genus Prevotella (false discovery rate (FDR)-corrected p-value (p-FDR) =0.130) and Ruminococcaceae NK4A214 group (p-FDR=0.130) when comparing before and after the intervention period. No significant changes were seen in the meat-based diet group. When comparing the two diet groups at the end of the intervention period we observed a significantly higher relative abundance of the genus Agathobacter (p-FDR=0.023), Coprococcus ( p-FDR=0.154) and Ruminiclostridium (p-FDR=0.154) in the meat-based diet group, while the genus Bacteroides (p-FDR=0.112) and Ruminococcaceae UCG.013 group (pFDR=0.066) showed higher relative abundance in the legume-based diet group. In conclusion, our results show that even a partial replacement of red and processed meat affects the composition of the gut microbiota.

The resilient nature of enterococci has made once a harmless commensal bacterium into a significant nosocomial pathogen. The increasing antibiotic resistance among Enterococcus faecalis and Enterococcus faecium has created pressure to find alternative therapies to traditional antibiotics. Phage therapy that utilizes virulent bacteriophages has been seen as a promising alternative to treat enterococcal infections due to their intrinsic specificity and ability to eradicate bacterial biofilm. In this thesis, therapeutic potential and limitations of previously isolated Enterococcus phages were further assessed. Genome analysis of the Enterococcus hosts revealed the presence of prophage regions, antibiotic and toxin resistance genes, and pathogenicity-related genes associated with invasion and intracellular resistance in the host genomes. Despite the presence of prophages and antibiotic-resistance genes, no concerning findings were made suggesting the potential suitability of Enterococcus hosts for phage propagation. Evaluation of phages against preformed (1-day-old) Enterococcus biofilms yielded diverse results, as some phages demonstrated high efficacy in biofilm elimination and prevention, while others were less effective. Additionally, experiments on phage-antibiotic interactions unveiled synergistic effects and promising potential in preventing the emergence of phage and/or antibiotic resistance. Furthermore, the impact of phage resistance on phage adsorption and susceptibility to other phages used in this study was examined, revealing varying mechanisms bacteria employ to evade phage infection. Understanding the intricate interplay between phages and bacterial hosts can enhance targeted therapeutic strategies by potentially refining the design of phage cocktails or phage-antibiotic combinations, thereby providing more effective treatment options against challenging enterococcal infections. Furthermore, as the demand for alternative treatment options in clinical settings grows, ongoing investigation is crucial to ensure the successful integration of therapeutic phages into medical practice.

Tiivistelmä — Referat — Abstract Intestine renews itself from intestinal stem cells (ISCs) in response to cell damage and disease. Damaged and dead cells are replaced by ISCs through cell division followed by daughter cell differentiation. Disturbances in this process can lead to diseases such as cancer. Hexosamine biosynthetic pathway (HBP) is a mediator of systemic insulin signaling induced ISC proliferation. However, the full molecular mechanism of HBP mediated ISC proliferation is yet to be discovered. Deciphering the mechanisms of the regulation responsible for ISC renewal could pave way for disease etiology dependent on the rate of ISCs proliferation. In this project I use Drosophila genetic tools to elucidate transcriptional and translational level control of HBP regulated ISC proliferation. Glutamine Fructose-6-phosphate Amidotransferase (GFAT) activity limits the rate of N-Acetylglucosamine GlcNAc and consequently ISC proliferation. Thus, gfat2Δ1 mutant flies are used to study molecular regulation of HBP. Full midguts of Drosophila will be imaged using Aurox Clarity Spinning-disc Confocal system. Confocal 3D images will be analyzed using an image analysis software called linear analysis of midgut (LAM) to retain region specific data. Exploring ISC proliferation in relation to nutrient sensing pathways in the full midgut level is still novel and LAM provides region specific data in previously unprecedented detail. The end product of HBP pathway Uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) was supplemented within the diet of the flies to observe its effect to the gfat2Δ1 ISC phenotype. UDP-GlcNac did not rescue the gfat2Δ1 ISCs. Usage of Drosophila genetic tools elucidated previously unknown transcriptional level regulation of HBP induced ISC proliferation: ATF4 knockdown in gfat2Δ1 mutant ISCs rescued gfat2Δ1 attenuated ISC division. In addition, ATF4 was indicated to possibly regulate the gfat2Δ1 phenotype via the regulation of growth through ribosome biogenesis and 4EBP translation inhibition. This study revealed the mediator of HBP, the transcription factor ATF4 to be the modulator of ISC proliferation.

Trichoderma reesei is an industrially widely utilized filamentous fungus currently used mainly for production of cellulases and xylanases. As T. reesei is a well-known, powerful and robust protein production organism, it is also being developed for heterologous expression of food protein and various enzymes by substituting the main cellulase coding sequence with the sequence of the produced protein. Transcription factor -based regulation of cellulase production in the fungus is well known, whereas thus far another member of Sordariomycetes, Neurospora crassa, has been the main model organism for noncoding RNA (ncRNA) studies in filamentous fungi. To find new ncRNA-derived regulatory mechanisms involved in cellulase expression in T. reesei, previously characterized potential micro-RNA-like RNAs (milRNAs) as well as other potential ncRNAs near cellulolytic pathway genes were deleted using CRISPR-Cas9. These candidates were based on existing total RNA expression data aligned with the Trichoderma reesei genome (Trire v2.0). The effects of these deletions on cellulase production, protease production, total protein production, and growth were analyzed by well-established methods. One purified deletion strain with unchanged growth in cellulase-inducing conditions (2% lactose) was established. Interestingly, this strain displayed low protein production (16%) together with abolished activity of cellulases (3%) and proteases (5%) compared to the parent strain. These findings warrant further studies on the nature of the deleted area, which could perhaps be an unannotated exon based on the RNA expression data. Another strain with a different deletion had abolished cellulase activity with otherwise largely unchanged properties, and all except for one of the analyzed deletion strains had significantly (p<0.05) diminished cellulase activity despite largely unchanged growth. Further investigation of the deleted areas is needed to elucidate the functions of these regions in more detail than was possible in the scope of this work. Seven potential noncoding RNAs were deleted in this work with interesting results that are likely to be useful in improvement of T. reesei production strains in the future.

Tick-borne diseases pose a major One Health threat in Finland. Tick-borne pathogens are transmitted through a tick-bite, impacting human, companion animal, livestock, and wildlife health. The geographical distributions of many tick species are shifting as a result of climate change. In northern Europe, tick distributions are expanding northward due to previously inhospitable climates becoming warmer. This has led to enhanced contact between ticks, humans and companion animals – increasing the incidence of tick-borne disease transmission. Established tick-borne pathogens such as TBEV and Borrelia spp. are considered endemic across Finland. However, the diversity of viruses found within ticks in Finland remains largely unexplored. Thus, here we aimed to characterize the viromes of the dominant tick species found in Finland. We used Next-generation sequencing methods to characterize the viral components of Ixodes Ricinus and Ixodes persulcatus ticks associated with companion dogs across Finland. In total, 4 different pools consisting of 36 ticks were sequenced. Our results identified 9 unique viruses belonging to the families Nairoviridae, Phenuiviridae, Iflaviridae and Partitiviridae. We identified numerous recently characterized nairo-like viruses, including Pustyn virus and Gakugsa tick virus. We were able to bridge the gap on the geographical spread of many of these viruses, including reporting Sulina virus and Norway nairovirus 1 for the first time after their discovery. Phylogenetic analyses revealed close relationships between the recently characterized RNA viruses, and a high degree of conservation across wide geographical ranges. As previously highlighted, the pathogenicity of these newly identified viruses is not yet established. However, our results indicate many of the viruses identified are closely related to viruses associated with acute febrile illness in humans. Due to our small sample size, we are unable to imply the virome composition for all ticks in Finland. However, we highlight a high degree of viral diversity even within a small sample size of ticks. Further research is urgently required to establish whether these recently characterised viruses pose a threat to human or animal health in Finland.