Browsing by master's degree program "Master's Programme in Microbiology and Microbial Biotechnology"

Lactic acid bacteria have a long history of use in food industry due to their favorable metabolic properties and health benefits for human health. Therefore, they are generally recognized as safe (GRAS) by FDA (U.S Food and Drug Administration) and have QPS (Qualified Presumption of Safety) status granted by EFSA (European Food Safety Authority). Nowadays, antimicrobial resistance (AMR) is a serious global risk and due to the increasing AMRs, more and more microbial infections have become more difficult to treat with antibiotics. AMR has mainly been of concern in relation to pathogenic microbes. However, since fermented foods are favorable environments for AMR gene transfer it should also be considered in the context of beneficial bacteria and their potential to spread AMR genes into pathogenic microbes. The aim of this study was to determine antibiotic susceptibilities of Lactobacillus plantarum, Lactobacillus rhamnosus, Leuconostoc sp. and Weissella sp. strains by E-test method and to detect selected specific antibiotic resistance genes by PCR. In addition, the goal was to define new cut-off values for Weissella strains since, so far, these have not been defined by EFSA. Antibiotic susceptibilities were determined against eight antibiotics: ampicillin, chloramphenicol, clindamycin, erythromycin, gentamicin, kanamycin, streptomycin and tetracycline. The detected AMR genes were blaZ, mecA, cat, lnuA, tetK and tetM. Most of the determined strains were observed to exhibit a notable resistance to kanamycin. Several Leuconostoc sp. and L. rhamnosus strains showed also resistance to chloramphenicol. Interestingly, one L. rhamnosus strain was observed to exhibit multiresistance to chloramphenicol and clindamycin. Moreover, 48% Leuconostoc strains had higher MIC value for streptomycin than the cut-off value defined by EFSA. Any of the selected AMR genes were not detected even though a notable resistance during the phenotypic testing was observed. However, this might be explained by the small amount of detected AMR genes. The results obtained in the present study provided more information about the antibiotic susceptibility and the safety of L. plantarum, L. rhamnosus, Leuconostoc sp. and Weissella sp. strains. Moreover, new cut-off values were proposed for Weissella sp. strains.

Enterotoksigeeninen Escherichia coli (ETEC) -infektio on yksi merkittävimmistä kolibasilloosin aiheuttajista nuorilla eläimillä ja on siksi merkittävä syy E. colin aiheuttamaan ripuliin maailmanlaajuisesti. Välittömiä ratkaisuja tarvittaisiin tämän kriisin ratkaisemiseksi ja erityisesti faagiterapia olisi yksi mahdollinen vaihtoehto ETEC-infektioiden hoitoon. Jumbofageilla on hyvät ominaisuudet mahdolliseen terapiakäyttöön niiden laajan genomin ansiosta, mutta ne ovat vielä varsin huonosti tunnettuja. Tämän tutkielman tavoitteena oli karakterisoida äskettäin eristetty Escherichia coli faagi fPf-Eco01 keskittyen erityisesti faagiterapian kannalta keskeisiin ominaisuuksiin. Tavoitteena oli myös selvittää, soveltuuko faagi terapiakäyttöön. Faagin genomin koko oli 379 kb, mikä luokittelee sen jumbofageihin. fPf-Eco01 faagi on mahdollisesti Asteriusvirus-suvun edustaja kuuluen samalla Caudoviricetes-luokkaan. Genomisekvenssin analysoinnissa ei ilmennyt haitallisia geenejä, jotka voisivat koodata antibioottiresistenssiä tai toksiineja. Sekvenssin perusteella faagin elinkierto ei olisi lysogeeninen, mikä voisi estää faagin käytön faagiterapiassa. Läpäisyelektroni-mikroskopia paljasti, että faagi fPf-Eco01 on kooltaan jumbofagin kokoinen ja sillä on supistuva häntä sekä selkeä häntälevy ja häntäsäikeet. Faagi-partikkelin keskipituus on 227 nm. Faagilla on laaja isäntäkirjo, sillä se infektoi jopa 38 % testatuista kliinisistä E. coli -kannoista. Osa faagin isäntäkantana toimivista kannoista oli laajakirjoisia beetalaktamaasi (ESBL) kantoja, jotka olivat eristetty suomalaisilta sairaalapotilailta. Faagi ei kuitenkaan infektoinut muita ETEC-kantoja, kuin vain sen alkuperäisen eristysisännän. Faagi sieti matalia pH-olosuhteita ja selvisi infektiokykyisenä useissa säilytysliuoksissa, joita voidaan käyttää faagiterapiassa. Näin ollen faagi pysyisi mahdollisesti infektiokykyisenä, mikäli faagihoitoa annettaisiin suun kautta tai faageja annosteltaisiin suonensisäisen nesteen mukana. Faagi fPf-Eco01 vaikuttaa olevan hyvä kandidaatti faagiterapiakäyttöön. Faagin laajan isäntäkirjon vuoksi sitä voitaisiin mahdollisesti käyttää ihmisten E. coli -infektioiden hoidossa. Lisätutkimuksia tarvitaan, jotta voidaan selvittää faagin mahdollista käyttöä ETEC-infektioita vastaan.

Antibiotic-resistant bacteria are an increasing threat to global health, caused by the excessive use of antibiotics and the lack of new antimicrobial agents being introduced to the market. New approaches to prevent and cure bacterial infections are needed to halt the growing crisis. One of the most promising alternatives is phage therapy which utilizes bacteriophages to target and kill pathogens with specificity. Pseudomonas aeruginosa is a common opportunistic pathogen that is intrinsically resistant to antibiotics, making it one of the most heavily studied targets of phage therapy. In this study, I characterized four P. aeruginosa phages, fHo-Pae01, PA1P1, PA8P1 and PA11P1, and evaluate their potency in therapeutic applications. Bioinformatic analysis of the genomes revealed the phages to be genetically highly similar and belonging to the Pbunavirus genus of the Myoviridae family. No genes encoding harmful toxins, antibiotic-resistance, or lysogeny were predicted. On the other hand, many of the predicted genes had unknown functions. The host ranges of the phages were assessed using 47 clinical P. aeruginosa strains and predicted host receptor binding tail proteins were compared. Some correlation between the host ranges and mutations in the tail proteins were observed but this alone was not sufficient to explain the differences in the host ranges. The recently isolated vB_PaeM_fHoPae01 (fHo-Pae01) phage was further characterized by a one-step growth curve and imaged with a promising atomic force microscopy method that had not been used before in the Skurnik group. Though the imaging results failed to provide any further knowledge of the phage, the 70-minute-long latent period of infection could be determined from the growth curve. Anion- exchange chromatography was found inefficient in purifying the fHo-Pae01 phage, so alternative methods such as endotoxin columns should be used when purifying these phages for patient use. In conclusion, all four phages appeared to be safe for therapeutic use based on current knowledge, and PA1P1 and PA11P1 were the most promising candidates due to their broad host ranges.

Semliki Forest virus (SFV) is an enveloped virus with positive-sense single-stranded RNA genome that encodes nine proteins, of which four non-structural proteins, nsP1-4, form the replication/transcription complex (RTC) along with several host proteins, which play an important role in the replication of the virus. To establish the interactome of SFV RTC, a promiscuous biotin ligase capable of biotinylating proximal endogenous proteins in the presence of exogenous biotin was genetically fused to nsP3. After establishing the stability, kinetics and functionality of this virus, BHK-21 cells were infected with this mutant SFV at multiplicity of infection of 50 plaque forming units per cell. At an early time point of 2.5 hours post infection, 50 μM biotin was added to medium for 15 minutes. Cells were lysed, and biotinylated proteins were enriched with streptavidin beads, and analyzed through tandem mass-spectrometry. We were able to identify several key host protein interactions, some of which were already established before, but also a several new ones. Many of the host proteins detected were involved with the formation of stress granules, including G3BP’s, or contained a SH3-binding domain (SRC homology 3) like CD2AP, SH3KBP1 and BIN1, and some of them also had RNA binding motifs. In future, we wish to study the role of these identified host proteins in the replication of SFV through gene silencing as well as their co-localization with the RTC and nsP3 with the help of Immunofluorescence.

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.

Understanding the biomes and niches within forest ecosystems is key to maintaining and predicting micro-organism led processes, such as, nutrient recycling and disease proliferation. Insect-vectored fungi occupy the tree bark biosphere as incidental associates. Also, more selective transmission of fungi is seen via the beetle’s specialised structure called the mycangium. Mites carried by these insects, have also been described to vector fungi. Within these fungi are mycoviruses that express cryptic, beneficial, or detrimental effects to the host. The positive and negative effects on fungal host phenotypes encourage investigations into unknown virospheres. A study into the distribution of mycoviruses within bark-beetle vectored fungi in Finnish forests has yet to be carried out. The master’s thesis work continued an evaluation of viromes from 52 forest, bark-beetle vectored, fungal isolates transformed into 4 RNA libraries via high throughput sequencing platforming, using Illumina chemistry. Scots pine, Pinus sylvestris, and Norway spruce, Picea abies, logs were sampled. A further 31 fungal isolates were screened, via RT-PCR, for 22 putative viral sequences recovered from the RNA libraries. Patterns in viral sequence host range, co-infectivity and similarities between viral sequences were investigated. The viral sequences described in this study were unique to the databases searched against and could be looked at when maintaining the Finnish forest ecosystem. It was shown that positive-sense ssRNA viruses could play a major role in the virome of bark-beetle vectored fungi as 77.3 % of viral sequences described were classified as so. Mitovirus infections were most frequent across the two forests and, the interspecies-infective Ophiostomatoid mitovirus 2 strain was seen to infect at least four species, across two fungal genera. The description of Kuraishia capsulata narna-like virus 1 showing RNA dependent RNA polymerases (RdRp) across 2 genomes segments, supports current growing evidence, which in turn could contribute to the new classification of viruses within the Narnaviridae family.

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.

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.

Finding and developing new antimicrobial compounds against clinically important antimicrobial drug resistant bacterial pathogens is necessary to counter the threats to global health, food security and development caused by these organisms. One potential source for leads for novel antimicrobial agents are bacteriophages, whose genomes hold vast numbers of genes encoding for proteins that are able to inhibit bacteria in yet uncharacterized ways. Characterization of these proteins and their functions is likely to aid the discovery of new antimicrobial drugs. This study aimed to optimize the heterologous production of three bacteria-inhibiting proteins from bacteriophage φR1-RT for the characterization of the proteins and their interactions with the bacterial host cell. Expression plasmids were successfully constructed for the heterologous production of the proteins in both Lactococcus lactis and Escherichia coli -based expression systems. The L. lactis expression system utilized a tightly regulated nisin-controlled promoter and featured a lactococcal SSusp45 secretion leader to target the produced protein to extracellular secretion. The E. coli expression system used a tightly regulated arabinose-inducible promoter to control the expression of the bacteriotoxic proteins. Despite the successful construction of the expression plasmids, the bacteriophage φR1-RT proteins were not able to be produced in quantities suitable for protein purification in either of the expression systems used in this study. The lack of protein expression is likely due to either codon bias or the harmful effects of the bacteriotoxic proteins that build up inside the bacterial cells. Codon optimized genes or a eukaryotic expression system could be tried to produce enough protein for purification and further characterization.