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

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  • Li, Yanru (2022)
    Phage lysins are enzymes that degrade bacterial cell wall. A wild-type Lactococcus lactis strain LAC460 secretes three phage lysins, LysL, LysP, and LysT, encoded by three different prophages. Unlike common phage lysins, these enzymes do not break down the host's cell wall. Therefore, these lysins can attack other L. lactis strains and behave like bacteriocins, antimicrobial proteins. The binding of a phage lysin to bacterial cell wall requires a specific cell wall binding domain (CBD) in the lysin. However, nothing about the CBDs of LysL, LysP and LysT is known. This study aimed to determine the CBDs of these three lysins and the target specificity of the lysins. Putative CBD regions of the lysins were fused with green fluorescent protein (GFPuv). GFPuv-CBD-LysL and GFPuv-CBD-LysT were ligated into the pASG-IBA4 vector and cloned in Escherichia coli DH5α. After all, only the construction of the GFPuv-CBD- LysL was successful resulting in fluorescent transformants. To analyse the binding of GFPuv- CBD-LysL to cells of different L. lactis strains, the fusion proteins were mixed with the LysL sensitive L. lactis MG1614, LysL resistant L. lactis LM0230, and the LysL producing LAC460 cells. With fluorescence microscope it could be seen that the GFPuv-CBD-LysL decorated the cell surface of L. lactis MG1614 with green fluorescence, but LM0230 and LAC460 cells remained non-fluorescent. The fluorescence of the cells was also measured with a fluorometer, showing strong fluorescence from MG1614, but nothing from the other two strains. This showed that the fusion protein specifically bound to the MG1614 cell surface, but it did not bind to the LysL resistant strain LM0230 or the LysL producer LAC460 cell. In conclusion, the results demonstrate that the C-terminus of LysL contains a specific cell wall binding domain. In addition, the results provide an explanation for how LAC460 can secrete LysL without autolysis, as phage lysins not able to bind onto peptidoglycan are unable to lyse cells.
  • Yassami, Shiva (2022)
    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.
  • Hyvönen, Tinja (2021)
    The spread of antibiotic resistance is a global health threat. Hospitals are a potential source of antibiotic-resistant bacteria and antibiotic resistance genes (ARGs), which may disseminate into the environment via wastewater. Hospital water environments, such as sink traps and shower drains, are known to harbor antibiotic-resistant bacteria, which might spread from the drains to the patients causing nosocomial infections that are hard to treat because of the limited number of treatments available. However, the current understanding of antibiotic resistance in the drains of residences, and how it relates to the situation in hospitals is limited. The aim of this study was to compare the microbial communities and ARGs in the water environments of homes and hospitals. The sink traps and shower drains of three hospital rooms and eighteen homes were sampled for metagenomic sequencing, and bioinformatic tools were used to detect the microbial taxa and ARGs in the metagenomes. The resistomes of hospital environments were distinct from those of homes and exhibited a higher diversity of ARGs. On the other hand, the microbial communities of homes and hospital rooms could not be clearly distinguished, although there were some differences in the abundances of certain taxa. The abundance of ARGs was higher in the hospital shower drains than in the corresponding samples in homes, but there was no statistical difference in the abundance of ARGs between the sink traps of homes and the hospital. Although the study had limitations, such as the low number of hospital samples, it indicates that the water environments of hospitals have a resistome that is distinct from that of homes and highlights the role of hospital sink traps and shower drains as potential hotspots of antibiotic resistance.
  • Suhonen, Anniina (2019)
    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.
  • Niamsap, Thanakorn (2022)
    Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a new respiratory virus capable of transmitting between interspecies and has caused outbreaks in mink farms since April 2020. The infection in mink farms has been a concern due to the overcrowded mink population which allows viral transmission and mutation to rapidly develop. The development of SARS-CoV-2 vaccines for mink is needed to prevent an outbreak in mink farms. In this study, we tested the safety and efficacy of our SARS-CoV-2 vaccine in a mink model. Our vaccine tests reveal no side effects of the vaccine on vaccinated mink, of which 40 mg/dose was found to be the optimal dosage, and was used as a standard for the later infection experiments. Microneutralization tests indicated that the neutralizing antibodies from vaccinated mink can protect cells line against early 2020 dominant variants (Wuhan, and Alpha), but poorly against later dominant strains (Beta, and Delta). The vaccinated mink were further observed the changes in antibody levels by enzyme-linked immunosorbent assay (ELISA), and the results suggested that the antibodies could be detected in sera samples of vaccinated subjects for at least 23 weeks after receiving two dosages of the vaccine. The presence of the virus was monitored in collected saliva samples throughout the infection days to study the effect of the vaccine on preventing the mink from SARS-CoV-2 infection by reverse transcription (RT)PCR. The quantification cycles (Cq) values were similar between vaccinated and non-vaccinated mink of both genders and between different gender, indicating that the vaccine could not help with preventing SARS-CoV-2 infection. Although the vaccine does not protect the vaccinated mink from the infection, the monitoring of clinical signs suggested that it helps the mink by reducing the severity of the disease. In conclusion, the vaccine showed promising results in inducing the mink body to produce neutralizing antibodies against the SARS-CoV-2 and relieved the symptoms of the disease.
  • Assimakopoulou, Irini Jr (2022)
    The genetic and morphological diversity of viruses and more specifically membrane-containing bacteriophages (phages) with single-stranded DNA (ssDNA) genomes is largely unexplored. It can be difficult to detect evolutionary relationships of viruses using solely sequence-based methods due to their rapid sequence evolution. However, more distant evolutionary connections of viruses have been observed based on structure data. Here we introduce an icosahedral tailless ssDNA phage, Cellulophaga phage phi48:2, isolated from the Baltic Sea that has not been assigned to any virus family or taxa. Phage phi48:2 has been previously linked to the family Finnlakeviridae whose members are icosahedral, internal membrane-containing phages with circular ssDNA genomes. However, the presence of lipids in phi48:2 virion has not been studied. In this study, different buffer conditions were tested for infectivity and stability of phi48:2 allowing us to optimize the purification of the phage particles by rate zonal and equilibrium ultracentrifugation in sucrose. Solvent tests in chloroform and ether, as well as low buoyant density of the virion suggested the presence of lipids in the phi48:2 virion. Analysis of the phi48:2 lipids extracted from highly purified virions by thin-layer chromatography revealed that phi48:2 is a membrane-containing phage and acquires its lipids unselectively from its host bacterium Cellulophaga baltica. Lastly, cryogenic electron microscopy of the purified virions also proposed that lipids form a membrane vesicle under the capsid. Altogether our results show that phi48:2 is an icosahedral membrane-containing phage, thus connecting it further with FLiP, which is the sole member of family Finnlakeviridae. Moreover, FLiP and phi48:2 virions are both ~60 nm in diameter and showed some similarity in their major capsid protein sequences (~21% amino acid identity). To conclude, even though phi48:2 and FLiP share various similarities they cannot be placed within the same family due to the low similarity in their genome sequences. However, for now we can assume they are possible distant relatives. The diversity and abundancy of membrane-containing ssDNA phages is gradually starting to uncover and through their characterization and classification we might consequently understand better their significance in microbial ecology.
  • Huotari, Jaana (2020)
    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.
  • Hietikko, Alli (2019)
    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.
  • Díaz Pérez, Aurora; Díaz Pérez, Aurora (2022)
    The coronavirus disease 19 (COVID-19) pandemic currently poses a challenge to the healthcare system and global public health. The upsurge of new SARS-CoV-2 variants, the uneven vaccine distribution worldwide, and the documented reinfections raise a concern about the protective immunity of COVID-19 recoverees. In this context, reliable methods for the detection of SARS-CoV-2 neutralizing antibodies are needed. Considering the methodological complexity and limitations of traditional virus neutralization tests, surrogate enzyme-linked immunosorbent assays (sELISA) constitute a promising alternative allowing high-throughput testing. However, there is still a need of assessing the specificity and sensibility of these assays so that they can be clinically applied. In this thesis, two goals were pursued; the detection of neutralizing antibodies in COVID-19 recoverees plasma samples using an in-house microneutralization assay and the comparison of these results with those obtained with two sELISA; SARS-CoV-NeutraLISA surrogate neutralization (Euroimmun) and cPass SARS-CoV-2 Neutralization Antibody Detection Kit (GenScript). The SARS-CoV-2 microneutralization assay was performed with VERO E6 cells and the Fin-1 strain of the SARS-CoV-2 virus. The plasma samples were provided by the Helsinki University Hospital and were previously screened with commercial IgG-ELISA targeting the anti-SARS-CoV-2 spike subunit 1 (Euroimmun) and nucleocapsid (Abbott) proteins. A total of 111 samples were tested, 74% of them presented a detectable NAb titer with at least two of the methods. The neutralizing antibody titer obtained with the microneutralization assays resulted in an overall proportion of positives lower than expected. Therefore, the in-house microneutralization assay needs further optimization or a different neutralization assay should be selected instead for future analysis. The combined data from the three tests was used to determine the sensitivity (99%, 83%, 81%) and specificity (72%, 100%, 100%) of cPass, Neutralisa and microneutralization assays respectively. This data suggests the use of cPass (GenScript) in primary screenings, in combination to Neutralisa (Euroimmun) to confirm secondary tests.
  • Thiruvaiyaru, Aditya (2021)
    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.
  • Olander, Viktor Otto Julius (2022)
    Preserving viral nucleic acids is of outmost importance to capture the viral diversity in metagenomic studies. In my master’s thesis, I compare viromes of genus Culex mosquitos stored in two different virus storage media and empty tubes. The mosquitos were collected from Kalajärvi in Espoo, Finland in the summer of 2020 as larvae and were grown to adults in laboratory conditions. Eight pools of five female mosquitos each were stored in each of the two media as well as empty tubes and the samples were homogenized The homogenates were filtered, and the RNA was extracted from them with TRIzol reagent. RNA was then reverse-transcribed to cDNA and amplified with a whole transcriptome amplification kit. The PCR product was prepared with a library preparation kit for sequencing with Illumina Next Generation Sequencer. The resulting reads were processed with a bioinformatic pipeline for identifying viruses from metagenomic sequence data. The results show a clear difference in virus species distribution by storage media. We identified 34 virus species from at least 13 families. Samples stored in ∑-Virocult had the highest yield of viral reads (70.40% of all reads from the pools) as well as the widest variety of mosquito species (n=26). Samples stored in empty tubes had the second most mosquito species (n=10) but the lowest viral read yield (1.25%). RNAlater stored samples had the least virus species (n=7) but a higher viral read percentage than those stored in empty tubes (3.26%). The results indicate the importance of choice of storage media. Since ∑-Virocult had the highest amount of reads and the widest variety, it might be the most useful storage media for our purposes. However, some viruses were found in other samples but not in ∑-Virocult stored samples, indicating a need of different storages conditions for different viruses. It is also important to be consistent in the use of media as it may affect virome results. More work needs to be done to assess if these results are true for other mosquito species as well.
  • Wood, Steffaney (2020)
    Cyanobacteria of the order Nostocales, including Baltic Sea bloom-forming species Nodularia spumigena, Aphanizomenon flosaquae, Dolichospermum spp., produce resting stages, known as akinetes, under unfavorable conditions. These akinetes can persist in the sediment and germinate if favorable conditions return, simultaneously representing past blooms and possibly contributing to future bloom formation. The present study characterized cyanobacterial akinete survival, germination, and potential toxin production in 40-to-175- year-old brackish water sediment archives in order to understand historical bloom expansion, akinete persistence, and cyanobacteria life cycles in the northern Baltic Sea. Results showed that cyanobacterial akinetes can persist in and germinate from northern Baltic Sea sediment up to 424 and 174 years old, at coastal and open-sea locations respectively. Akinete abundance and viability decreased with age and depth of vertical sediment layers. Increases in sediment organic matter content and akinete abundance largely corresponded with the historical expansion of anthropogenic eutrophication-fueled blooms of cyanobacteria in the northern Baltic Sea, beginning in the mid-twentieth century. The detection of potential hepatotoxin production from akinetes and revived cultures was minimal and restricted to the coastal sediment core. Phylogenetic analysis of culturable cyanobacteria from the coastal sediment core indicated that the majority of strains likely belonged to benthic genera Anabaena. Findings also supported the notion that, in comparison with Nodularia and Aphanizomenon spp. akinetes, Anabaena/Dolichospermum spp. akinetes play a more significant role in their life cycle and bloom initiation strategies. Further research is recommended to accurately quantify akinetes and create a higher rate of toxin gene detection from brackish water sediment samples in order to further describe species-specific benthic archives of cyanobacteria. Overall, measuring cyanobacterial akinete abundance, germination experiments, and genetic methods can be effectively used to determine akinete persistence, viability, and potential toxin production in brackish water sediment samples. This study highlights the prolonged survival of cyanobacterial akinetes in northern Baltic Sea sediment samples, up to 174 years old.
  • Chesnut, Sally (2022)
    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.
  • Oikkonen, Hanna (2022)
    The use of recycled fibers in paper production has increased during recent years. Recycled fibers are a more sustainable alternative compared to virgin fibers made from wood. However, paper mills utilizing recycled fibers have more microbiological problems compared to mills using only virgin fibers. Especially, anaerobic bacteria are harmful for papermaking processes utilizing recycled fibers. Bacteria of the class Clostridia comprise a very diverse group and have many different metabolic properties. Bacteria of class Clostridia can ferment different substrates, for example cellulose and starch, crucial in paper mills utilizing recycled fibers. Fermentation does not only decrease material efficiency, but also the acids produced during fermentation deteriorate papermaking processes. Volatile fatty acids are odorous compounds causing bad odors in the mills and in the final products. Clostridia can also produce, for example, hydrogen which is an explosive gas endangering the safety of the mill employees. Quantitative PCR is a feasible detection method for microbes. Here, a qPCR method was developed for the detection of most abundant bacteria in the class Clostridia in the recycled fiber mills. The designed primers targeted the most harmful bacteria from the genera Clostridium, Ethanoligenens, Fonticella and Ruminococcus identified in the recycled fiber mills. Three primer sets were designed for the target bacterial group. Positive controls of each target bacterial genus was included and close relatives from class Bacilli were used as negative controls. The designed primer sets were compared in efficiency, specificity and performance with process samples collected from paper mills using recycled fibers. One of the primer sets was found the most potential for the qPCR detection method for the diverse target bacterial group. All positive controls were amplified with the designed qPCR assay, whereas the designed primers discriminated well each negative control in vitro. The applicability of the designed qPCR assay was yet confirmed with process samples collected from mills utilizing recycled pulp. Even though the efficiency of the designed primer set was not optimal, the designed assay was determined feasible for the detection of the target group in the recycled fiber mills usually high in bacterial density.
  • Gonzalez Ramos, Victor Manuel (2020)
    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.
  • Sgarabotto, Elena (2022)
    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
  • Andreou, Gregory Michael (2021)
    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.
  • Dirks, Anna (2021)
    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.
  • Mukhtar, Fezan (2022)
    Lactobacilli especially Limosilactobacillus reuteri’s strains inhabit the GI tract of humans with glycerol/diol dehydratase activity metabolizing glycerol and producing a broad-spectrum antimicrobial system called reuterin. It consists of 3-hydroxypropanal (3-HPA), acrolein, and its derivatives. Due to the toxic activity of ubiquitous acrolein, an analytical toolbox to determine acrolein formation by food cultures is needed. We developed assays to estimate microbial formation of acrolein using a colorimetric method based on tryptophan and a fluorescence-based approach with 2-amino-1-methyl benzimidazole (AMBI) as a probe. We compared tryptophan and AMBI-based quantification of reuterin produced by resting cells or during the growth of Lb. reuteri DSM 20016. With 600 mM glycerol, resting cells produced 329 ± 35 mM 3-HPA as quantified by HPLC-RI, and 390 ± 13 mM of 3-HPA/acrolein based on the colorimetric method with 3-HPA as standard. Acrolein (40 ± 11 mM) was detected using an AMBI probe. We also detected 3-HPA and acrolein formation during the exponential growth phase in the presence of 50 mM glycerol in different media. Also, as acrolein induces redox stress, redox potential and sensitivity to reuterin/acrolein of the engineered green fluorescence protein (roGFP2) were determined. Ultimately, the roGFP2 gene was tried to clone in E. coli (JM109) using the pTH1mp constitutive expression vector to establish as a biosensor for acrolein detection. Our results suggest that quantification of acrolein by fluorescence-based approaches and biosensors constitute novel methods to estimate any risk of acrolein formation in presence of glycerol/diol dehydratase-positive microbes and glycerol during food fermentation.
  • Kattilakoski, Matilda (2022)
    Dietary data is essential in creating dietary guidelines and interventions, but the traditional data collection methods can be biased and costly. Wastewater metagenomes present a potential new way to collect dietary data with the utilization of microbial markers. In this study, potential microbial markers for fiber and meat intake were identified from literature. The abundances of these markers and their associations with the corresponding dietary data were analyzed using a previously published global wastewater metagenome dataset covering 58 countries. Majority of these potential markers were detected in the analysed wastewater metagenomes. Of the identified markers, Prevotella and Prevotella copri showed significant associations with whole grain intake, Alistipes and Alistipes putredinis showed significant associations with processed meat intake, and Faecalibacterium showed significant association with red meat intake. In addition, associations between dietary data and both taxonomic and functional annotations of the metagenomes were determined to identify any additional potential markers. Multiple additional species, genera and gene families showed significant associations with red meat and processed meat intakes. Future research should include finer resolution data to validify these results and further investigate the potential of these taxa and genes as markers. In conclusion, microbial markers present a promising way to collect dietary data from wastewater metagenomes.