Browsing by master's degree program "Mikrobiologian ja mikrobibiotekniikan maisteriohjelma"

Antibiotic resistance is a growing threat to global health due to overuse and misuse of antibiotics leading to untreatable or difficult to treat infections. Natural environments are an important reservoir of antibiotic resistance. The release of antibiotics into the environment promotes the development of antibiotic resistant bacteria and environmental occurrence of antibiotic resistance genes (ARGs). ARGs are common in nature and prevalent in aquatic environments such as surface waters and effluent. Cyanobacteria are widely found in marine, freshwater, and terrestrial environments. Since their ubiquitous presence in water environments cyanobacteria are exposed to antibiotic pollution and are in contact with resistant bacteria. The role of cyanobacteria in the antimicrobial resistome and dissemination of ARGs has only been studied recently. This work aimed to evaluate the antibiotic susceptibilities of 51 cyanobacterial strains against different classes of antibiotics, using liquid batch cultures, antibiotic discs, and bioinformatics approaches. Cyanobacterial strains used in this work were sensitive to most of the tested antibiotics. However, majority of the strains also showed resistance against trimethoprim and novobiocin. Overall, there was little variation in the antibiotic resistances observed between strains but differences in sensitivity to different antibiotics was observed between species and strains with most differences seen with Nostoc spp. According to bioinformatic tools used (CARD database and BLASTp) FosA protein was found only in strains showing resistance against fosfomycin but not in any sensitive phenotypes and therefore fosA gene was selected as the most promising putative resistance gene for subsequent assays. To determine whether the fosA from cyanobacteria could confer resistance to fosfomycin, the fosA gene from Nostoc sp. XPORK 5A was cloned into pET28a(+) expression vector under the control of T7 promoter and subsequently native cyanobacterial promoter. The ability of Escherichia coli BL21 (DE3) carrying each plasmid constructs to grow in the presence of fosfomycin was determined with agar plates and growth curve assay. E. coli transformants containing the fosA gene and T7 promoter conferred high-level resistance to fosfomycin showing ability to grow at the highest concentrations tested (1mg/ml) on agar plates and (500 µg/ml) in growth curve assay. FosA protein expression from the native cyanobacterial promoter appeared to be weaker and conferred lower-level resistance to fosfomycin (≥ 10 µg/ml). The results of this study provide more information about the antibiotic susceptibility of cyanobacteria. In addition, replicating a horizontal transfer of the fosA gene from cyanobacteria to proteobacteria conferred resistance to fosfomycin and these results may indicate that also nonpathogenic cyanobacteria could act as a source of fosA antibiotic resistance genes.

Saprotrophic wood-decaying fungi access nutritional carbon by degrading lignocellulosic biomass with ligninolytic and carbohydrate active enzymes. Research of species-species level interactions of the fungi is crucial to understand ecosystem functions and carbon cycling. Furthermore, research results of wood-decaying fungi and interspecific fungal interactions can be beneficial for development of biotechnological applications. In this study, two brown rot species Fomitopsis pinicola and Fomitopsis betulina, two white rot species Phlebia radiata and Fomes fomentarius and one soft rot species Schizophyllum commune were studied in combination cultures on birch wood substrate for 12 weeks. Interactions of the fungi were studied by analysing laccase, manganese peroxidase, xylanase, and iron reduction activities, and by analysing the expression of genes coding for lignocellulose-degrading enzymes. pH of the cultures was measured to estimate the metabolic activity of the fungal species. Suppression of xylanase and iron reduction activities demonstrated dominance of the white rot species over the other decay type species in the co-cultures. In addition to the depressed enzyme activities, increase in white rot production of laccase and manganese peroxidase activities indicated combative interactions in the co-cultures. This study evidenced species and growth-time dependent changes in signature enzyme activities and in gene expression of wood-decaying fungi during interspecific fungal interactions.

Semliki Forest virus (SFV) is a well-studied model virus of medically important mosquito-borne alphaviruses, like chikungunya virus and Sindbis virus. SFV replicates within membrane invaginations called spherules at the host plasma membrane, facilitated by the virus-encoded nsP1 protein. The objectives of this MSc thesis were to identify candidate host proteins interacting with nsP1 based on previous proteomics work and develop a screening workflow to identify stable nsP1 interactors. The overarching aim is to improve our understanding of the roles of host proteins in spherule formation and replication complex functions. The screening workflow involved knockdown assays to assess the antiviral and proviral effects of potential host interactors, followed by cell viability assays for toxicity assessment. Selected promising hits were further investigated for protein interaction with nsP1, which required cloning for mammalian expression and tagging with Myc epitope. Knockdowns resulted in several statistically significantly proviral and antiviral host factors, and all the knockdowns were non-toxic to the cells. Among the significant proviral hits, four promising candidate host proteins were cloned and expressed. Pull-down assays did not reveal stable interactions, suggesting transient or indirect interactions between these host proteins and nsP1. The lack of co-localisation with SFV replication complex supported this finding. This work sheds light on possible transient or indirect interactors of nsP1. The screening workflow effectively identified protein-protein interactions and can be applied to screen additional proteins. Future studies should employ methods suitable for studying transient interactors to gain further insights. This would enhance our understanding of key host proteins in SFV spherule formation and replication, potentially leading to novel antiviral therapies targeting alphavirus replication.

OBJECTIVES and RESEARCH QUESTION. Human parechovirus 3 (HPeV3) is a (+)ssRNA icosahaedrally symmetric virus which causes meningoencephalitis and sepsis in children and neonates. As it causes the most severe symptoms among parechoviruses it is attracting more attention (4). Currently there are no approved broad treatment strategies against parechoviruses, however recent research by Rhoden et al., 2017, reported the antiviral activity of posaconazole (PSZ) against HPeV3 in cell culture. Posaconazole is an antifungal drug approved for use against Candida and Aspergillus infections. It targets lanosterol-14alpha-demethylase and prevents the production of ergosterol, a lipid vital for fungal membranes not present in mammalian cells (24). In mammalian cells PSZ accumulates at the endoplasmic reticulum (ER) and binds to the oxysterol-binding protein (OSBP) and Niemann-pick type C1 (NPC1) (59, 28, 30). The drug may affect cellular components and thusly block parechoviral infection or could bind to the viral capsid. METHODS. To test viral capsid-binding hypothesis PSZ activity was tested in a range of concentrations against two HPeV3 isolates and HPeV1 Harris in Vero and HT29 cell lines. HPeV3 isolate 152037 was purified on a CsCl step gradient and imaged by cryo electron microscopy (cryo-EM). Single particle analysis was done in Scipion (40) and acquired density maps visualized in UCSF Chimera (49). Atomic model of a different isolate of HPeV3 (PDB ID: 6GV4, 16) was changed at 6 sites and fitted to density maps from this work in Coot (52). Maps were subtracted in search of density that would represent PSZ. RESULTS. PSZ was effective against both HPeV3 isolates at 1 μM in Vero cells when added to the virus prior to infection, however not in HT29 cells. At higher concentrations (>10 μM) PSZ formed crystals which limited the concentration that can be used for cryo-EM. In order to test the hypothesis of PSZ being a capsid binder 3 datasets were collected, HPeV3 control, HPeV3+DMSO and HPeV3+PSZ (4 μM) with final resolutions after single particle analysis of 3.3 Å, 3.9 Å and 3.4 Å respectively. Subtraction of maps yielded no difference that would represent PSZ. DISCUSSION and CONCLUSION. PSZ does not appear to be a capsid binder although it appears to work early in the infection. Absence of PSZ density in HPeV3+PSZ density map could be due to low saturation and images containing PSZ were filtered out in image processing. Another possibility is low affinity of PSZ for the capsid. As PSZ binds various membranes it is possible that it blocks HPeV3 infection by targeting cell components. Additional experiments could be performed in the future in order to provide insight into which stages of infection PSZ affects.

Global warming affects permafrost in the Arctic regions, where melting organic carbon storages will increasingly contribute to the emission of greenhouse gases. Little is known about tundra soil microbial communities, but Acidobacteria and viruses seem to have important roles there. Here, for the first time, we isolated five Acidobacteria infecting viruses from Kilpisjärvi tundra soils using host strains previously isolated from the same area. Three viruses were isolated on Edaphobacter sp. X5P2, one on Edaphobacter sp. M8UP27, and one on Granulicella sp. X4BP1. The viruses had circular double-stranded DNA genomes 63,196–308,711 bp in length and 51–58% GC content. From 108 to 348 putative ORFs were predicted, 54–72% of which were sequences unique to each virus. Annotations indicated that all five phages most likely have tailed virions. The diversity of viruses present in the studied soils was estimated with the metagenome analysis. Only 0.1% (627) of all assembled metagenomic contigs were phage-positive. The gene-sharing network analysis showed approximately genus-level clustering between the virus isolates and a few metagenomic viral contigs, but overall, all (except one) viral contigs clustered only with each other, not with any known viruses from the NCBI database. No taxonomical assignments could be done for the metagenomic viral contigs, highlighting overall undersampling of soil viruses. Further detailed studies on virus-host interactions are needed to understand the impact of viruses on host abundance and metabolism in Arctic soils, as well as the microbial input into biogeochemical cycles.

Bacteriophages are viruses that infect bacteria. With the ever-increasing threat of antibiotic resistance, they have emerged as a promising alternative treatment. Many phage genomes contain modified bases. They prevent digestion by restriction enzymes allowing the resistance of these viruses to bacterial defence mechanisms. YerA41, a phage that infects Yersinia ruckeri, contains a genome that could not be amplified using any of the DNA polymerases available in the market. Neither restrictions enzymes were able to digest it. These properties led to the assumption that YerA41 genome is not conventional and is likely to contain modified nucleotides. In order to replicate its genome, YerA41 should possess its own DNA polymerase that would be able to use, YerA41 genome as template. If so, it would be able to use other modified genomes as well. Hence, this DNA polymerase could become a very valuable biotechnological tool. In this study we isolated and optimised the purification of DNAP01, one of the putative DNA polymerases encoded by YerA41 genome. In addition, this work shows, with the help of different experiments, how DNAP01 is a novel DNA polymerase able to use YerA41 DNA as template. This is the first time an enzyme of this nature has been described and isolated.

Coevolution, the reciprocal evolution of species, is a significant evolutionary phenomenon, and it has been known since the days of Darwin. These days it can be studied using experimental evolution in laboratory-regulated environments where the “fossil populations” which are preserved during the experiments can be compared with contemporary populations and with each other. Bacteria and unicellular eukaryotic predators are suitable for the research of predator-prey interactions including of antagonistic coevolution. This is due to their short generation time and thus the fast evolution. In my Master’s thesis, I examined the changes caused by antagonistic coevolution in a log-term predator selection experiment in the bacterium Pseudomonas fluorescens and the ciliate Tetrahymena thermophila. I examined the ecological stability between the populations using time-shift experiments. I also examined growth curve parameters for the bacterial population as well as its metabolic activity, diversity through colony morphology, and the ability of the non-evolved and coevolved ciliate to consume bacteria. Part of the experiments were performed using as control a bacterial population which had evolved without predator. Based on previous research, I hypothesized that coevolution would increase the stability of the community and the diversity of the bacterial populations. I expected the carrying capacity, maximum growth rate, and metabolic activity, in turn, to decrease over time. I observed that coevolution stabilized the dynamics, as was expected, and this was associated with increased diversity in the prey population. As the latter has been observed to be the reason for increased stability, the results here support earlier observations. The carrying capacity and area under the growth curve decreased as expected, but the maximum growth rate did not change over time. There was also no difference in the growth of the ciliates, regardless of evolutionary history, on the bacteria from different time points. Because of uncertainties arising from the experimental design, some of the results cannot be confirmed to have been caused by coevolution. The results increase the knowledge regarding the effects (co)evolutionary history can have on ecology and the phenotypic traits of populations. The differences and similarities in the results compared to earlier studies indicate that the effects of coevolution change in time and differ between short-term and long-term settings. Further studies are required to provide more unequivocal support for the presence of coevolution and elucidate its precise phenotypic and molecular drivers.

Brewers’ spent grains (BSG) are by-products of the brewing industry. Utilization of BSG in food applications is challenging, due to its poor technological characteristics. Because of their water retaining properties, interactions with matrix components and impact on texture formation, bacterial exopolysaccharides (EPS) represent a promising tool for improvement of BSG properties. Among bacterial exopolysaccharides, dextran produced in situ by lactic acid bacteria (LAB) during fermentation has shown major improvements in technological and sensorial features of products prepared from various types of plant materials. The nutritious composition of BSG may support the growth of LAB and enable in situ dextran production. The aim of this study was to establish and examine the synthesis of dextran by LAB in BSG. Sixteen dextran producing LAB strains were screened for viscosity formation in BSG fermentation. The strains showing the highest viscosity formation were further assessed for fermentation performance. The more suitable fermentation temperature was traced by comparing the viscosifying performance of selected starters at 20 and 25 °C. Dextran amount was determined semi-quantitatively from selected fermented samples showing optimal results, and the presence of oligosaccharides was assessed. Sucrose, glucose, maltose and fructose amounts were analyzed to observe the relation between sugar consumption and dextran and oligosaccharides formation. Weissella confusa strains A16 and 2LABPTO5 and Leuconostoc pseudomesenteroides strain DSM20193 appeared the most promising starters for viscosity formation and thus dextran synthesis in this matrix. From the examined fermentation temperatures, strains showed the highest potential for dextran synthesis at 25 °C. The amount of synthesized dextran ranged from 1.1 to 1.7 % w/w (of the wet weight of the whole sample matrix). The rheological properties of BSG were modified via LAB fermentation and dextran synthesis, resulting in more viscous texture, and its applicability in food systems was thus potentially enhanced.

Common sexually transmitted pathogens like human papillomavirus (HPV) and Chlamydia trachomatis have profound effects on sexual health of women ranging from acute genital tract infections to cancer and infertility. These infections are often asymptomatic, increasing the risk of being left untreated and thus increasing the probability of complications. Most cervical cancer cases are caused by persistent HPV infection and would be preventable by timely HPV detection. Cervical cancer screening using HPV nucleic acid detection has proven efficient and is used in many European countries, including Finland. Chlamydia trachomatis and Neisseria gonorrhoeae infections can lead to upper genital tract infection and increased risk of ectopic pregnancy and infertility. C. trachomatis infections are also associated to increased risk of epithelial ovarian cancer. Due to lack of national screening program and often asymptomatic nature of these infections, many of them are left untreated. In order to streamline cervical cancer screening and to include C. trachomatis and N. gonorrhoeae analysis in the same package with HPV, a liquid-based cytology sample medium, BD SurePath™ was validated for use with cobas® 4800 HPV and CT/NG nucleic acid detection tests. Work presented here shows that HPV and CT/NG tests and cytological analysis can all be done from a single sample in an efficient, reliable and cost-beneficial way. Based on the results, Vita Laboratoriot will be able to offer the package analysis to their customers.

The COVID-19 pandemic has brought into discussion the role of airborne transmission in infectious diseases. Many studies on enveloped viruses such as influenza suggest that respiratory viruses can be transmitted with large or small droplets formed when the patients talk, breathe, sneeze or cough. This comes under the category of direct contact. These droplets may also be transmitted indirectly as fomites through contact with contaminated surfaces. It has been difficult to prove that aerosols' transmission as the methods to capture virus in the air are not very sensitive. SARS-CoV-2 is a novel coronavirus affecting millions of people since 2019, and it has been challenging to contain the spread of this virus. Hence it is of vital importance to understand the transmission of the virus through aerosol and droplets. In this study, aerosol samples were collected from patients in the Surgical Hospital in Helsinki and patients at home in quarantine using various bioaerosols sampling devices like Biospot, Dekati, Button, and Andersen samplers, and passive sampling techniques to capture aerosols and droplets in the air. Such samples were subjected to cell culture on TMPRSS2 expressing Vero E6 cells to check for infectious viruses and RT-PCR using the N-gene targeting method to detect the presence of SARS-CoV-2 RNA in the samples. Out of the 32 saliva samples collected, 19 samples were tested positive by RT-PCR, but cell culture was not always positive. Bioaerosol samples collected using Dekati, Button, and Biospot samplers were negative by PCR. However, Andersen samplers showed positive results along with various passive aerosol samples collected on MEM, indicating aerosols' production of small sizes that can be transmitted air in the air to far distances and settling due to gravity. A relation between saliva samples and symptom days indicates the decrease in saliva viruses' infectivity with the prolonged infection as seen from the RT-PCR. From these findings, it can be concluded that SARS-CoV-2 can be spread by airborne and fomite transmission, and more so by patients with symptoms day 2-7 who are proven to be more infectious. Additionally, it was inferred that the Six Stage Andersen impactor would be the most efficient for aerosol sampling. Further studies are still needed to understand the characteristics of the spread and extent of infection caused by the variants of SARS-CoV-2.