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

Punkkivälitteiset patogeenit (PVP) ovat yleisimpiä zoonoottisten tartuntojen aiheuttajia Suomessa. Tautitaakkaan vaikuttavat erityisesti Borrelia burgdorferi sensu lato -bakteerien (BBSL) ja puutiaisaivokuumeviruksen (TBEV) aiheuttamat tartunnat. Molemmat ovat lisääntyneet Suomessa viime vuosikymmeninä. Tapausten lisääntymisen voi pääosin laskea taudinaiheuttajien päävektorien, puutiaisen (Ixodes ricinus) ja taigapunkin (I. persulcatus), levittäytymisen ansioksi. Ulkoloisina punkit välittävät tauteja useisiin selkärankaisiin ruokinnan yhteydessä. Ankarat talvet ovat rajoittaneet punkkien levittäytymistä pohjoiseen, mutta ilmastonmuutoksen aiheuttamat leudommat talvikelit ovat edistäneet valloitusretkeä uusille alueille. Vaikka sopiva ilmasto on olennaista punkkien esiintyvyydelle, vaikuttaa punkkien isäntälajiston rakenne mitä luultavimmin punkkien ja PVP:ien yleisyyteen paikallisella tasolla. Tässä tutkielmassa punkkeja kerättiin 6 aitausalueelta ja niiden ympäristöstä. Samoilta alueilta määritettiin lisäksi piennisäkkäiden ja hirvieläinten määrät. Koska aitaukset ja niiden ympäristöt luultavasti edustavat samankaltaista ilmastoa, pyrittiin tutkielmassa tutkia, kuinka punkki-isäntiin liittyvät tekijät vaikuttavat punkkien ja PVP:ien esiintyvyyteen. TBEV-positiivisen poolin sekä TBEV-vasta-aineita kantavien piennisäkkäiden perusteella Hangossa varmistettiin mahdollisesti uusi TBEV pesäke. Aitausten, piennisäkäsmäärien ja hirvieläintiheyksien pohjilta luotiin yleistettyjä lineaarisia sekamalleja (GLMM) selittämään punkkien ja PVP:ien esiintyvyyttä tutkimusalueilla. TBEV jätettiin mallinnuksen ulkopuolelle pienen otoskoon takia. Hirvieläintiheydet olivat erittäin merkitsevä punkkien esiintyvyyteen vaikuttava tekijä. BBSL:n yleisyyteen ei yksikään tekijä vaikuttanut merkitsevällä tasolla – mahdollisena syynä piennisäkäspopulaatioiden muutoksen viiveellinen heijastuminen kerättyjen punkkien tartunta-asteeseen.

Emerging research suggests that bacteriophages (phages) may exhibit alternative infection strategies that deviate from the preconceived lytic or lysogenic life cycles. Carrier cell infection is an alternative phage life cycle where complete virus particles are formed and remain within host cells, without cell lysis or integration into the host genome. Phage Φ6 (Φ6), the type member of the double-stranded RNA (dsRNA) virus family Cystoviridae, is a lytic phage that can also establish a carrier cell within its plant pathogenic host, Pseudomonas syringae pathovar (pv.) phaseolicola strain HB10Y (HB10Y). This thesis contributes to current limited knowledge and provides an insight on the underlying mechanisms of the Φ6 carrier cell infection. This study has agricultural and ecological relevance and may contribute to future plant therapeutic options. Synthetic carrier cell lines harboring Φ6 tri-segmented genome or Φ6 genomic constructs in which the coding regions in the S- and/or M- segments were replaced by heterologous sequences from tobacco mosaic virus (TMV) were created using a reverse genetics method. Spontaneous Φ6 carrier cell lines were also isolated from HB10Y after exposure of the host to excess phage. Spontaneous carrier cells were not stable, but rather occasionally released phage into liquid culture. Synthetic carrier cell lines were subjected to secondary phage infection and were found to be less susceptible than wild type (WT) to Φ6 but not Φ8, a more distant member of Cystoviridae. Studies suggest that carrier cell resistance to secondary infection (superinfection exclusion) is exhibited through the Φ6 S-segment gene 8. To test how temperature affects the stability of Φ6 carrier cells, spontaneous carrier cell line culture was incubated at RT and 30°C, and phage productivity was compared. Elevated temperature induced carrier cell stability. Comparison of the growth curves between Φ6 synthetic and spontaneous carrier cell lines and their respective WT strains showed that Φ6 carrier cell infection does not greatly affect host growth.

Yeasts are a major spoilage threat in carbonated and fermented beverages, causing considerable economic losses for the manufacturers. Dekkera bruxellensis and Zygosaccharomyces bailii are the two most common spoilage yeast in beverages due to their high tolerance towards beverage-related stress factors. For industry, early and reliable detection of contamination is necessary to minimize spoilage potential and maintain product quality. Cultivation on selective/differential media remains the main method for detection of these organisms, with incubation times from 3 to 15 days. Beverage-related stresses may generate sub-population of injured yeast cells and further delay or even prevent the detection in regular media. PCR, flow cytometry and other alternative detection methods also rely on enrichment cultivation to achieve the required sensitivity for the industry. Therefore, reduced incubation time of sample enrichment and improved detection of injured cells is crucial for a more rapid and reliable detection method. Modification of specific compounds in the culture medium composition has been reported to improve recovery of bacteria after stress. As analogue studies have not been performed on spoilage yeast, modification of the culture medium composition offers a possibility to improve the growth of injured and healthy yeast cells. The aim of this study is to reduce cultivation time required for detection of healthy and injured Dekkera bruxellensis and Zygosaccharomyces bailii cells. Initially, conditions for inducing organic acid and heat injury in D. bruxellensis, D. anomala and Z. bailii cells were studied in an artificial beverage containing basic components of soft drinks. Selective and non-selective plate cultivation and fluorescent viability stains were used to assess the level of injury. The organic acid treatments resulted in inconsistent injury of spoilage yeasts, and thus, recovery from organic acid injury could not be screened. The heat treatments resulted in consistent 1-3 log reduction of viable cell counts. Altogether, 46 potential injury-relieving or growth-enhancing supplements were screened for their effects on the growth rate and lag time of heat-treated and untreated cells in non-selective YM broth using high-throughput automated turbidometry. During individual screening, the growth of Z. bailii strains was significantly improved (p<0.05) only by supplementation with three ion sources: calcium chloride, potassium chloride, and magnesium sulphate. Synergistic effects of the three ion sources was optimized for D. bruxellensis and Z. bailii individually using surface response analysis. Optimized D. bruxellensis YM medium showed no consistent impact on healthy or heat-treated D. bruxellensis strains. On the other hand, two out of the three Z. bailii strains showed significant lag time reduction of 63-66% in untreated cells and 34% in heat-treated cells when incubated in optimized Z. bailii YM medium. The lack of differentiation between improvement of growth of untreated and heat-treated cells point to a generalized ionic deficiency in YM medium. In conclusion, the optimized Z. bailii YM medium is a promising candidate for reducing the detection time of the common spoilage yeast, but it would still require validation with additional Z. bailii strains and quality control samples. It would be also interesting to study the benefits of the medium for cultivation of other spoilage yeasts and in the presence of Z. bailii selective compounds. The information about the importance of various salts for growth of Z. bailii may also prove useful in biotechnological applications of this yeast.

In the past 20 years, three known disease emergence events of highly pathogenic coronaviruses have highlighted the importance of monitoring wildlife for the presence of these viruses. Their peculiar characteristics, like high mutation and recombination rate, have increased their potential for species adaptation and interspecies transmission. Understanding the diversity of these viruses in wildlife and increased surveillance might be key to predicting and preventing future spillovers and pandemics. Studies on wildlife coronaviruses commonly focus on the order Chiroptera, mainly in temperate and tropical regions of the Asian continent. Even though animals belonging to this order are considered the main reservoir, the importance of other small terrestrial mammals should not be overlooked. Rodents, for instance, are animals of great interest for many zoonoses, as they often host parasites, bacteria and other groups of viruses that cause diseases in humans. A recent description of several lineages of coronaviruses recovered from rodents from China highlighted and suggested the presence of these viruses in small terrestrial rodents. In this project, we aimed to investigate the presence of coronaviruses in small mammals from France. Samples were collected during spring 2021 in twelve different locations, within two regions of eastern France, Auvergne Rhône-Alpes and Franche Comté. A total of 448 rodents, 13 shrews and 416 bat samples were collected. The samples were screened and coronaviruses sequences were recovered in 20 different samples. Nine Betacoronavirus genus sequences were recovered from rodent colon samples, and one Alpha- and ten Betacoronavirus sequences from bat guano. These results confirmed previous evidence of these viruses’ presence in small mammals from France and provide the first evidence of betacoronaviruses circulating in wild French bats. The study covers two eastern regions that have not been surveilled in previously released studies therefore this highlights the need to increase the efforts in monitoring these viruses and their wildlife host

Antibiotic resistance is an increasing, terrible threat to human health, leading to a growing need for alternative therapies. Phage therapy, using bacterial viruses to fight infections, is a promising alternative to antibiotic therapy. However, several obstacles need to be overcome. Regrettably, phage therapy remains inaccessible to many laboratories worldwide due to the need for expensive machinery to establish sensitivity of bacteria to phage. Moreover, shipping phages between laboratories remains challenging. In the current study a device-free bacteriophage typing PhagoGramAssay was developed. In the assay bacteria suspended in soft agar were poured onto a 60-well Terasaki plate containing phages suspended in fibrillated nanocellulose separated from the bacteria by a seal. Phages were released into the bacterial agar layer by puncturing the seal to test for sensitivity observable with the naked eye. Contrast between lysis zone and bacterial lawn was enhanced using 2,3,5-triphenyltetrazolium chloride. Optimized parameters included the amount of bacteria and phage added, volume of phage suspension, agar percentage and thickness and puncturing tool size. In addition, a prototype of such a puncturing tool was developed. The optimized PhagoGramAssay was tested using several bacteria-phage combinations. Moreover, infectivity and stability of phages stored on Terasaki plates was followed over the course of 4 weeks. The optimal bacterial amount added was found to be a 1:300 dilution in soft agar taken from a OD600 = 1 culture. Phage suspensions used in the assay were found to need to have a titer of at least 108 PFU/ml in the original lysate, with 8 µl of 1:10 dilution in fibrillated nanocellulose present in the wells. Optimal agar conditions were found to be 0.4% – 0.5% (w/v) with a thickness of 2 mm – 3 mm. The optimal puncturing tool shape was found to be a slit with a thickness of 0.5 mm. When using these conditions sensitivity could be established for a vast number of bacteria-phage combinations. All phages remained stable and infective over the course of 4 weeks . The newly developed PhagoGramAssay can be further developed into a kit-like phage typing assay that would enable laboratories to test for sensitivity on site whenever a multi-drug resistant bacterial strain is isolated from a patient sample, effectively making phage therapy accessible to laboratories that cannot afford expensive machinery. Additionally, the use of fibrillated nanocellulose should enable laboratories to exchange phages. The final form of such a kit, however, is dependent on manufacturers and investors and may need to be adjusted accordingly.

Coronavirus disease 2019 (COVID-19) is still considered a global pandemic with novel immunoevasive variants constituting a potential threat to life for many susceptible individuals. Despite successful vaccination programmes, which ensued in early 2020, spread of the virus is still an unresolved issue. To address this, innovative prophylactic approaches are being continuously investigated to target the causative agent of COVID-19, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Prevention of infection primarily focuses on the targeting of the receptor-binding domain (RBD) on the spike protein of SARS-CoV-2, which is used to infect host cells presenting angiotensin-converting enzyme 2 (ACE2) on their surface. In 2023, a novel antibody mimetic targeting scaffold, namely the sherpabody platform (SH3; src-homology 3; Recombinant Protein Affinity), was introduced. Accordingly, an intranasally administered, RBD targeting trimeric sherpabody, TriSb92, was demonstrated to prevent infection by SARS-CoV-2 and its recent variants of concern by targeting a conserved region within the spike RBD in vitro and in vivo. This study was performed to further investigate and develop the use of sherpabodies in SARS-CoV-2 prophylaxis. Various homo- and heteromultimeric constructs were assembled and the efficiency of their bacterial production was assessed. Additionally, their functionality, specificity and avidity was analysed. Specifically, the combination of different functionalities within a single molecule – receptor blocking and fusion prevention – was studied. Newly discovered RBD-targeting sherpabodies assembled into multimers were able to neutralize SARS-CoV-2 variants, including the latest Omicron subvariants BA.2.75.2 and XBB.1.5. These multimeric sherpabodies were shown to be easily manufacturable, highly target-specific and multifunctional when desired, making them excellent candidates for intranasally administered SARS-CoV-2 prophylaxis.

Dietamoeba fragilis is a very common human intestinal parasite in the world and also in Finland. D. fragilis causes dientamoebiasis in humans, and symptoms of infection may vary from mild bowel symptoms to prolonged diarrhoea and weight loss. Some of the infected patients are completely asymptomatic. Despite its prevalence, insufficient data exist on the life cycle and host organisms of D. fragilis. Interestingly, D. fragilis has been found in dogs in a few studies. Thus, more research is needed on the role of household pets as part of the parasite’s life cycle and choice of host organism. Comprehensive study on the role of dogs in the life cycle of D. fragilis is warranted. The purpose of this thesis was to design a study on the role of dogs in the D. fragilis parasite life cycle “The presence of Dientamoeba fragilis parasites and multiresistant ESBL-bacteria in dog owners and their pets”, to present research methods and to apply research permit from the ethics committee. The aim of the study is to analyze the prevalence of D. fragilis in dogs in Finland and the role of the dog in the spread of D. fragilis parasite within humans. In addition to D. fragilis, the study combines collection of samples for ESBL bacteria analyses, but this thesis focuses only on the part of study D. fragilis. Two different real-time PCR methods are tested in this project: an in-house method and an Amplidiag® Stool Parasites method, out of which Amplidiag® Stool Parasites method is selected to be used in the study. In the thesis documents for the ethics committee's referral are written and ethical permit to conduct the study from the HUS ethics committee is applied. A favourable opinion will allow a comprehensive study of dogs and humans to be carried out. Ethics permit is need to recruit participants for research on the basis of a laboratory-confirmed D. fragilis -discovery. The committee confirms that the study is ethically acceptable. During the thesis project, the committee gave the study a conditional favourable decision. The implementation of the study will start in the spring of 2023, when the final assent is received.

The Baltic Sea is a unique and delicate brackish water ecosystem with high primary productivity driven by oceanic biogeochemical cycles of oxygen, iron, silicon, nitrogen and phosphorus. Elevated anthropogenic nutrient loading into the Baltic ecosystem has resulted in a large-scale increase in destructive cyanobacterial blooms in the open Baltic Sea over the past century. The toxic cyanobacterium Nodularia spumigena is a major component of surface blooms in the open Baltic Sea and continues to bloom even after the depletion of phosphate from the surrounding waters. This phenomenon has been attributed to an ability to scavenge phosphorus from recalcitrant sources. However, the exact phosphorus species that sustain N. spumigena growth in the Baltic Sea remain largely unknown. Here, I employ a comparative genomics approach to determine the evolutionary dynamics of phosphorus scavenging in eight strains of N. spumigena and predict the range of phosphorus sources that may support their growth. Then, I test these predictions by growing six strains of N. spumigena on a number of potentially bioavailable phosphorus sources. Among the phosphorus scavenging genes identified by the genomic analysis, putative pathways for the enzymatic degradation of phytic acid, phosphite, and phosphonates were present and highly conserved in the species. Subsequent growth experiments demonstrated that the organism may grow using phytic acid and phosphite, as well as the phosphonates methylphosphonic acid, ethylphosphonic acid, and nitrilotris(methylenephosphonic acid), as sole phosphorus sources. These results indicate that N. spumigena blooms may be supported by several phosphorus sources previously not known to contribute to eutrophication in the Baltic Sea. While additional growth experiments and further research on the environmental prevalence of these compounds are necessary, the findings presented in this study expand our knowledge of how N. spumigena dominates phytoplankton blooms in a phosphorus-scarce environment and may help to inform future eutrophication mitigation efforts in the Baltic Sea region.

Cryptosporidium, a parasitic eukaryote, is the causative agent of cryptosporidiosis, a diarrheal disease with the potential to be life threatening in immunocompromised people. The number of cases has been rising as diagnostics have improved, suggesting the disease is more common than previously thought. Currently in Finland, samples are only typed to the genus level. The objective of the study was to establish a Cryptosporidium typing protocol for outbreak investigation nationally at the Finnish Institute of Health and Welfare with the long-term goal of preventing outbreaks. Reliable typing methods could also help find new reasons behind the increase in cases, monitor the emergence of new subtypes, and reveal new transmission routes. Typing was set up with samples from the Helsinki and Uusimaa Hospital District laboratory as well as the Finnish Food Authority. DNA was extracted from stool samples, species were identified with real-time PCR of the 18S rRNA gene, and gp60 subtypes were determined with nested PCR as well as Sanger sequencing. The Zymo QuickDNA Fecal/Soil Microbe Miniprep Kit was suitable for extracting troublesome parasite DNA. Out of the successfully subtyped samples, the majority was identified as Cryptosporidium parvum, out of which 60 % belonged to the IIaA15G2R1 subtype. The typing method described in this study was successful in typing 19 of 22 samples and will be utilised by the Finnish Institute of Health and Welfare for outbreak investigation on the national level.

Carbapenemase-producing Enterobacteriaceae (CPE) resist carbapenem class of antibiotics in addition to the other type of antibiotic resistances they usually carry. The blaKPC-3 gene is one of the genes causing the carbapenemase production in bacteria. The aim of this study was to establish the Oxford Nanopore Technologies MinION sequencing method and integrate it with Illumina sequencing into a hybrid assembly for investigating the location of the blaKPC-3 gene in the bacterial genome. We used 14 isolates suspected of plasmid-mediated gene transfer and prepared three libraries using different DNA extraction methods. The contigs from the hybrid assembly sequences were used to annotate the target genes, which were then included in a phylogenetic tree and distance matrix analysis. DNA extraction method had an impact on the amount of data and the length of reads produced in MinION and the Smart DNA prep extraction kit with InnuPure C16 touch extraction device produced the best results. The blaKPC-3 gene was located on plasmids in each isolate, and the similarity of the plasmid sequences indicated that horizontal plasmid-mediated gene transfer has likely occurred between different species. However, the clonal spread cannot be excluded, and further research is needed to confirm these findings.