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

Norovirukset ovat merkittäviä akuutin gastroenteriitin aiheuttajia maailmassa. Ne aiheuttavat noin 700 miljoonaa infektiota ja 200,000 kuolemaa vuosittain. Tämän vuoksi on noroviruksen mahdollisesta ilmateitse leviämisestä tullut merkittävä kiinnostuksen kohde. Tässä tutkimuksessa kehitettiin aerosolisysteemi, jota käytettiin tutkimaan, miten hiiren noroviruksen (MNV) infektiokyky säilyy aerosoloinnin seurauksena. Koe toteutettiin tuottamalla aerosoleja MNV-virusliuoksesta sumuttimen avulla (nebulizer), jonka jälkeen aerosolit kerättiin ohjaamalla ne suppilon kautta petrimaljalla olevalle DMEM-kasvatusliuokselle sekä RAW 264.7 soluille. Mikroskopoimalla soluja todettiin viruksen onnistuneen infektoimaan solut suoraan aerosoleista. DMEM-kasvatusliuoksesta määritettiin infektiivisten virusten pitoisuus sekä tehtiin RT-qPCR-analyysi. Tulosten perusteella pystyttiin havaitsemaan, että hiiren noroviruksen infektiokyky heikkenee aerosoloinnin seurauksena. Lisäksi havaittiin, että isoa osaa viruksista ei pystytty keräämään, kun aerosolit mahdollisesti tarttuivat letkuihin ja aerosolisysteemin seinämiin. Tulevaisuudessa tarvitaan aerosolilaitteita, jotka käsittelevät viruksia hellemmin, keräävät aerosoleja tehokkaammin, sekä matkivat ihmisen elimistöä paremmin.

Saccharomyces boulardii is a probiotic yeast related to Saccharomyces cerevisiae but with distinct genetic, taxonomic, and metabolic properties. S. cerevisiae has been used extensively in biotechnological applications. Currently, many strains are available, and multiple genetic tools have been developed, which allow the expression of several exogenous proteins of interest with applications in the fields of medicine, biofuels, the food industry, and scientific research, among others. Although S. boulardii has been widely studied due to its probiotic properties against several gastrointestinal tract disorders, very few studies addressed the use of this yeast as a vector for expression of foreign genes of interest with biotechnological applications. I studied the previously constructed S. boulardii SAC12, which secretes the anti-listerial bacteriocin leucocin C originating from Leuconostoc carnosum 4010. The objective was to study if the bacteriocin leucocin C producing S. boulardii could produce leucocin C in beer fermentation and if leucocin C containing beer can be used as marinade to control Listeria monocytogenes in raw chicken breast strips. The results showed that SAC12 has good ability to secrete LecC, and thus it was used to brew anti-listerial beer. According to results, beer could maintain its anti-listerial activity for 38 days. The anti-listerial effect of the beer stored for different times was analyzed through marinating chicken breast strips (spiked with L. monocytogenes) with the beer for overnight. Results indicated a positive impact of anti-listerial beer in reduction of the viable cells of L. monocytogenes by about 1.6 log from (2.2 ± 0.6) × 10⁷ CFU/g (beer from day 24), and 2.2 log from (1.8 ± 0.3) × 10⁵ CFU/g (beer from day 38). To sum up, the S. boulardii SAC12 efficiently secreted the bacteriocin leucocin C. Brewing beer with S. boulardii SAC12 resulted in beer containing leucocin C. Such beer showed anti-listerial effect when used as marinade for chicken breast strips.

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.

Alzheimer’s disease (AD) is the seventh leading cause of death worldwide. One hallmark of AD includes the amyloid beta (Aβ1-42) peptide that accumulates into oligomers, fibrils, and plaques. Aβ1-42 has been shown to be structurally and functionally similar to antimicrobial peptides (AMPs). Publications have reported that Borrelia burgdorferi can be found in the brain of AD patients. B. burgdorferi and B. garinii cause Lyme disease (LD). B. duttonii is responsible for relapsing fever (RF), a disease characterized by recurrent episodes of high fever. The aim of this research was to study whether synthetic Aβ1-42 binds to LD and RF Borrelia sp. and several bacterial molecules important for their virulence, and whether Borrelia sp. have evolved strategies to evade Aβ1-42-mediated killing. Binding of Aβ1-42 to B. burgdorferi, B. garinii and B. duttonii and several microbial molecules was studied by ELISA and immunoblotting. Bacterial culturing and microscopy were used to study survival, agglutination, and phagocytosis of Borrelia sp. in the presence of Aβ1-42 and microglia. In this research, Aβ1-42 was able to bind and agglutinate all of the three studied Borrelia sp. However, Aβ1-42 reduced the survival and increased the phagocytosis of B. duttonii. while B. burgdorferi and B. garinii were unaffected. In addition, potential Aβ1-42 binding molecules were detected from several bacterial species, including FhbA expressed by B. duttonii. In conclusion, this study suggests that some restricted species of bacteria may evade Aβ1-42 entrapment and thus may be involved in the ability of the species to invade the CNS that may trigger neuroinflammation related to AD.

Aspergillus niger is a filamentous fungus that is known for its ability to degrade plant biomass polysaccharides. A total of 86 sugar transporters have been identified in A. niger, but only 10 of them have been thoroughly characterized. Sugar transporter proteins are crucial for fungi as they enable efficient utilization of sugars in their metabolism and therefore breakdown of plant biomass. Additionally, sugar transporters can be used in various biotechnological applications. L-arabinose is a pentose sugar present in plant biomass and A. niger can utilize it through the pentose catabolic pathway (PCP). Recently, a sugar transporter LatA was identified from A. niger, capable of transporting the PCP intermediate product L-arabitol into fungal cells. L-arabitol is a polyol similar to xylitol and can be used as a low-calorie sweetener in food and beverage industries. Although A. niger LatA has previously been shown to be specific to L-arabitol in vivo, its in vitro functional activity has not yet been described. This study aimed to in vitro characterize two potential L-arabitol transporters from A. niger, LatA and unpublished 9364, using the yeast Saccharomyces cerevisiae. As a platform strain, we used S. cerevisiae IMK1010 that is devoid of all hexose and disaccharide transporters, as well as disaccharide hydrolases. In addition, we used a disaccharide-polyol and a pentose metabolic strain which were generated from the IMK1010 strain. The metabolic strains carried pathways for maltose, saccharose, sorbitol and mannitol, and xylose and arabinose, respectively. This provided a controlled research environment for studying A. niger LatA and 9364 transporters. The sugar specificity of the transporters was tested through two different growth experiments on solid media with all the strains and in liquid media with IMK1010 strains. The tested sugars included D-glucose, D-fructose and D-mannose hexoses, D-xylose and L-arabinose pentoses, maltose and sucrose disaccharides, and D-mannitol and D-sorbitol polyols. In addition, LatA was examined through a disappearance assay, measuring the loss of sugar from the liquid growth medium. Altogether four different combination gene constructs, green fluorescent protein (GFP) gene fusions and plain sugar transporter gene constructs were successfully engineered and 21 different transformant yeast strains produced for this study. GFP gene fusions, were in addition to growth experiments, used to study the localization of the sugar transporters to the cell membrane. In strains containing combination gene constructs encoding sugar transporters and GFP, the sugar transporters were successfully localized to the cell membrane, showing already that the transporters potentially have transport activity in the heterologous expression system. Based on the results, A. niger 9364 transported the tested hexoses and maltose in the growth experiments but did not transport tested pentoses, disaccharides or D-mannitol and D-sorbitol polyols. As expected, A. niger LatA did not transport any of the tested sugars, confirming its specificity to L-arabitol polyol. However, in the disappearance assay LatA unexpectedly did not transport L-arabitol. This might be due to the possible toxicity of the polyols in high concentrations to yeast cells and many of them also serve as regulators of osmotic pressure in cells, which may lower the transport capacity of the sugar transporters. In the future the function of the transporters can be tested in different sugar concentrations and pH in disappearance assay. Alternatively, a L-arabitol metabolic strain could be constructed to investigate sugar specificity using the growth experiment instead of the disappearance assay. The study provided new information of A. niger 9364 and further insights into the sugar specificity of A. niger LatA. These sugar transporters could be used in various biotechnological applications in the future.

In recent years, icosahedral internal membrane-containing single-stranded DNA (ssDNA) phages have been identified. Phages phi18:4 and phi48:2, both infecting Cellulophaga baltica bacteria and isolated from the Baltic Sea, are part of this phage group. Circular ssDNA genomes of these phages are different in length, but they are packed into capsids of uniform size which is unusual among viruses. Initially, the phages were cultivated using their original C. baltica host strains #18 and #48 but during the study, spontaneously induced #18ind virus was observed to originate from the #18 strain and possessed a double-stranded DNA genome. These observations led to an examination of the phage’s host range, ultimately resulting in the use of the #48 strain as a host for both phages. The life cycle strategy of both phages was lytic and efficiently produced viral progeny in the #48 strain. Zymogram analysis was utilized to investigate the presence of peptidoglycan-degrading enzymes in the phage structure, assisting in host cell penetration. Two possible peptidoglycan hydrolyzing enzymes were observed for each phage, although sequence data suggested only one enzyme. The formation of virions was analyzed by exposing the viruses to biochemical conditions. Freezing and thawing, along with guanidine hydrochloride treatment, turned out to separate the lipid membranes and capsid proteins of the virions but require further optimization for a detailed examination of the protein composition of the membrane. This study together with other research findings, clarifies the structure, life cycle, and evolutionary relationship of lipid-containing ssDNA phages in the context of other phages. In the future, phi18:4 and phi48:2 could serve as model systems for further investigations into other discovered Cellulophaga ssDNA phages.

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.

This study aimed to investigate the microbiological quality of the whole and gutted Baltic herring at different seasons by traditional culture-dependent methods combined with the identification of bacterial isolates by MALDI-TOF MS. Additionally, the microbiome of the herring was characterized by culture-independent 16S rRNA gene amplicon sequencing. Bacterial counts were within acceptable limits at all seasons although the H2S-producing bacteria levels were above the recommended level of 5 log10 CFU g-1 at two sampling points. With the culture-dependent methods and the sequencing of the 16S rRNA gene, the microbiome of the whole and gutted herring was dominated by the bacterial class Gammaproteobacteria. Shewanella, Pseudomonas, and Aeromonas were the most frequently isolated genera among the viable population identified with MALDI-TOF MS. With the culture-independent approach, Shewanella followed by Psychrobacter were the most abundant genera. Additionally, a high relative abundance of the phyla Firmicutes and Actinobacteria and, in some samples, Epsilonbacteriaeota represented by the genus Arcobacter, was detected. Variances in the microbiological quality of different herring batches observed in this study could not be attributed to the season. Therefore future research through a longer period was proposed, including data on the environmental factors, such as the fishing location and the water quality, possibly affecting the quality of the herring.

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.