Browsing by Subject " microbiology"

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  • Liu, Liwei (Helsingin yliopisto, 2014)
    Cyanobacteria are one of the most widespread microorganisms on earth, and they are found in almost all ecosystems, from fresh and marine water to terrestrial environments. Cyanobacteria received a great deal of attention as prolific producers of bioactive secondary metabolites. The aim of this study was to screen and characterize novel bioactive natural products, which present biological activity against acute myeloid leukemia (AML) cells, trypsin, and hepatotoxin microcystin/nodularin, from cyanobacteria isolated from different habitats. Of 40 cyanobacteria strains isolated from the Baltic Sea shore and lichen symbiosis were used to look for anti-cancer compounds which could induce apoptosis of AML cells. Half of these cyanobacteria strains contain apoptosis-inducing anti-AML activity, and seven cell extracts contain potent apoptosis-induced activities against AML. Two of apoptogens were confirmed to be new variants of hassallidin and scytophycin. Cyanobacteria produce a plethora of serine protease inhibitors with a broad range of chemical structures. Nostosins, new trypsin inhibitors, were discovered through the analysis of secondary metabolites in methanol extracts of Nostoc sp. FSN. The data showed that they were linear peptides with three residues: Hhpba, L-Ile and L-argininal/argininol. Nostosin A and B inhibited trypsin with IC50 values of 0.35 μM and 55 μM, respectively. Computer docking data indicated that the argininal aldehyde and guanidino groups played the most crucial role in the efficient inhibition of trypsin. The bloom-forming cyanobacterium Nodularia spumigena has been reported to produce several bioactive peptides. In this study, Nodularia spumigena strains from the Baltic Sea were discovered to produce new hybrid peptides named as pseudoaeruginosins. Because of the low abundance in cyanobacteria strains, pseudoaeruginosins were chemically synthesized and the structure was confirmed with LC-MS and NMR. Pseudoaeruginosins contained all the structure blocks of aeruginosin NAL2 except that the Choi was replaced by 4-methylproline (4-mPro), which is a specific subunit of spumigins in N. spumigena. The aldehyde group containing pseudoaeruginosin NS1 exhibited IC50 of 0.19 μM against porcine trypsin. The biosynthesis of pseudoaerugnosins was proposed to be a joint action of aeruginosin and spumigin biosynthesis pathways in N. spumigena. 4-mPro is a rare non-proteinogenic amino acid, which has been found in a small number of bioactive compounds identified from cyanobacteria. A combination of PCR and LC-MS were used to screen 116 cyanobacteria strains for the production of 4-mPro. A total of 12 strains were confirmed to produce 4-mPro, and eleven new cyclic peptides belonging to two groups were identified from two Nostoc spp. XPORK 5A and UK2aImI strains, respectively. Among 11 peptides, the tested five could inhibit the organic anion transporters OATP1B1/B3 to block the induction of apoptosis of hepatocytes by microcystin/nodularin. In this research, a total of two new trypsin inhibitors and five new cyclic peptides with the antitoxic bioactivity against microcystin/nodularin were identified. It was shown that cyanobacteria from both marine and terrestrial environments are a good resource for discovering new bioactive compounds. These findings increased the diversity of bioactive secondary metabolites characterized from cyanobacteria and provide new leads for drug research.
  • Rasinkangas, Pia (Helsingin yliopisto, 2016)
    The present thesis studied the biogenesis of the pili of Lactobacillus rhamnosus GG, and examined whether pili influence the in vivo persistence and the ability to modulate faecal microbiota in mice and humans. L. rhamnosus GG is one of the most extensively studied lactic acid bacteria, and it is commonly present in so-called probiotic products. Heterotrimeric proteinaceous pili, which are able to bind for example to mucus, collagen and β-lactoglobulin, have been found and characterised on the surface of L. rhamnosus GG during recent years. Pili are formed by the major pilin SpaA, which forms the shaft of the pilus structure, the mucus-binding tip pilin SpaC and basal pilin SpaB. The pili are synthesised on the cell membrane by the pilin-specific transpeptidase sortase C (SrtC), and attached to the cell wall by housekeeping transpeptidase sortase A (SrtA). As the pilus is the major mucus adhesin of L. rhamnosus GG and, according to various studies, a crucial factor in the L. rhamnosus GG adherence and signalling to the host, it was deemed important to characterise the pilus biogenesis pathway and the functions of the produced pili. Random chemical mutagenesis was used to obtain derivatives of L. rhamnosus GG with variable pilus production capacities to study factors affecting pilus biogenesis. Enrichment schemes for the isolation of the derivatives were devised, and consequently 10 pilus-deficient, 13 highly adherent and one pilus-secreting derivative were characterised. Genotypic and phenotypic characterisation of the derivatives revealed that a functional SrtC is essential for pilus biogenesis, as well as the sufficient expression of the major pilin SpaA. Additionally, the role of the insertion sequence-rich genomic region, containing also the pilus gene cluster, in the adaptation of L. rhamnosus GG to varying environments was revealed, as several derivatives were obtained in which the region was deleted. The adhesion capacity of L. rhamnosus GG was found to be increased either due to a mutation in the tip pilin SpaC or as a result of increased pilin production. One of the highly adherent derivatives with high pilin production harboured a mutation in the SpaA pilin, likely increasing the derivative s capacity to secrete this pilus backbone-forming subunit. In addition, a pilus-secreting derivative was characterised, and noted to harbour a mutated housekeeping sortase SrtA, demonstrating its essential role in the attachment of pili to the cell wall. Bioinformatic analysis revealed that the C-terminal sortase recognition motif of pilins, with the sequence LPxTG, was variable between the proteins recognised by pilin-specific transpeptidase SrtC and housekeeping transpeptidase SrtA. This was found not only for the pilins of L. rhamnosus GG, but also for pilins of other sortase-dependent pili-harbouring Gram-positive bacteria, indicating a role for this motif in the regulation of pilus biogenesis. In a subsequent functional characterisation of L. rhamnosus GG pilins, it was demonstrated that SrtC indeed recognised the shaft pilin SpaA through a conserved triple glycine (TG) motif, and was likely to recognise also the tip pilin SpaC, which harbours the same motif. The basal pilin SpaB, containing a single glycine (SG) motif, was recognised by SrtA, leading to the attachment of pilus to the cell wall. Finally, the in vivo effect of the L. rhamnosus GG pili was evaluated in intervention trials in mice and humans. By performing mouse and human intervention trials with one of the described pilus-deficient derivatives, L. rhamnosus GG-PB12 and the parental strain, it was possible to demonstrate that the presence of pili increased the persistence of L. rhamnosus GG in the gastrointestinal tract and led to significant changes in the faecal microbiota composition, especially in humans. Species richness increased significantly due to the consumption of piliated L. rhamnosus GG in the human trial, indicating potential beneficial effects for the host, as a high species richness of gut microbiota has been associated with stability and resilience towards exogenous species. In conclusion, the research described here provides new insights into pilus biogenesis in L. rhamnosus GG and highlights the importance of the pili in the in vivo adherence capacity and gut microbiota modulation ability of L. rhamnosus GG.
  • Santos-Cortez, R.L.P.; Bhutta, M.F.; Earl, J.P.; Hafrén, Lena; Jennings, M.; Mell, J.C.; Pichichero, M.E.; Ryan, A.F.; Tateossian, Hilda; Ehrlich, G.D. (2020)
    Objective: To review the most recent advances in human and bacterial genomics as applied to pathogenesis and clinical management of otitis media. Data sources: PubMed articles published since the last meeting in June 2015 up to June 2019. Review methods: A panel of experts in human and bacterial genomics of otitis media was formed. Each panel member reviewed the literature in their respective fields and wrote draft reviews. The reviews were shared with all panel members, and a merged draft was created. The panel met at the 20th International Symposium on Recent Advances in Otitis Media in June 2019, discussed the review and refined the content. A final draft was made, circulated, and approved by the panel members. Conclusion: Trans-disciplinary approaches applying pan-omic technologies to identify human susceptibility to otitis media and to understand microbial population dynamics, patho-adaptation and virulence mechanisms are crucial to the development of novel, personalized therapeutics and prevention strategies for otitis media. Implications for practice: In the future otitis media prevention strategies may be augmented by mucosal immunization, combination vaccines targeting multiple pathogens, and modulation of the middle ear microbiome. Both treatment and vaccination may be tailored to an individual's otitis media phenotype as defined by molecular profiles obtained by using rapidly developing techniques in microbial and host genomics. © 2020 Elsevier B.V.
  • Barenkamp, Stephen J.; Chonmaitree, Tasnee; Hakansson, Anders P.; Heikkinen, Terho; King, Samantha; Nokso-Koivisto, Johanna; Novotny, Laura A.; Patel, Janak A.; Pettigrew, Melinda; Swords, W. Edward (2017)
    Objective. To perform a comprehensive review of the literature from July 2011 until June 2015 on the virology and bacteriology of otitis media in children. Data Sources. PubMed database of the National Library of Medicine. Review Methods. Two subpanels comprising experts in the virology and bacteriology of otitis media were created. Each panel reviewed the relevant literature in the fields of virology and bacteriology and generated draft reviews. These initial reviews were distributed to all panel members prior to meeting together at the Post-symposium Research Conference of the 18th International Symposium on Recent Advances in Otitis Media, National Harbor, Maryland, in June 2015. A final draft was created, circulated, and approved by all panel members. Conclusions. Excellent progress has been made in the past 4 years in advancing our understanding of the microbiology of otitis media. Numerous advances were made in basic laboratory studies, in animal models of otitis media, in better understanding the epidemiology of disease, and in clinical practice. Implications for Practice. (1) Many viruses cause acute otitis media without bacterial coinfection, and such cases do not require antibiotic treatment. (2) When respiratory syncytial virus, metapneumovirus, and influenza virus peak in the community, practitioners can expect to see an increase in clinical otitis media cases. (3) Biomarkers that predict which children with upper respiratory tract infections will develop otitis media may be available in the future. (4) Compounds that target newly identified bacterial virulence determinants may be available as future treatment options for children with otitis media.
  • Coda, Rossana; Xu, Yan; Moreno, David Sàez; Mojzita, Dominik; Nionelli, Luana; Rizzello, Carlo G.; Katina, Kati (2018)
    This study focused on the performance of the dextran producer Leuconostoc citreum as starter culture during 30 days of wheat flour type I sourdough propagation (back-slopping). As confirmed by RAPD-PCR analysis, the strain dominated throughout the propagation procedure, consisting of daily fermentations at 20 °C. The sourdoughs were characterized by consistent lactic acid bacteria cell density and acidification parameters, reaching pH values of 4.0 and mild titratable acidity. Carbohydrates consumption remained consistent during the propagation procedure, leading to formation of mannitol and almost equimolar amount of lactic and acetic acid. The addition of sucrose enabled the formation of dextran, inducing an increase in viscosity of the sourdough of 2–2.6 fold, as well as oligosaccharides. The transcriptional analysis based on glucosyltransferases genes (GH70) showed the existence in L. citreum FDR241 of at least five different dextransucrases. Among these, only one gene, previously identified as forming only α-(1–6) glycosidic bonds, was significantly upregulated in sourdough fermentation conditions, and the main responsible of dextran formation. A successful application of a starter culture during long sourdough back-slopping procedure will depend on the strain robustness and fermentation conditions. Transcriptional regulation of EPS-synthetizing genes might contribute to increase the efficiency of industrial processes.
  • Paju, Susanna; Oittinen, Juha; Haapala, Henna; Asikainen, Sirkka; Paavonen, Jorma; Pussinen, Pirkko J. (2017)
    In this observational and prospective study, we investigated if microbiological and serological markers of periodontitis associated with conception in 256 non-pregnant women (Mage = 29.2 years; range 19-42 years). Clinical oral and gynecological examinations were performed, major periodontal pathogens in the saliva were detected, and serum and saliva antibodies against major periodontal pathogens were analyzed. The follow-up period for becoming pregnant was 12 months. Porphyromonas gingivalis was significantly (p = 0.032) more frequently detected in the saliva among those who did not become pregnant (8.3%) than among those who became pregnant (2.1%). The median levels of salivary P. gingivalis immunoglobulin A (IgA; p = 0.006) and IgG (p = 0.007) antibodies were higher among those who did not become pregnant compared to those who became pregnant. Hazard ratios (HR) for not becoming pregnant were HR = 3.75 (95% confidence interval [CI] 1.01-13.9; p = 0.048) if the subject was polymerase chain reaction-positive for P. gingivalis with high salivary antibodies against it, and HR = 1.62 (95% CI 1.03-2.54; p = 0.035) if she had high levels of serum P. gingivalis IgA and signs of periodontal infection. P. gingivalis associated with no success in getting pregnant.
  • Koort, Joanna; Avall-Jaaskelainen, Silja (2021)
    The COVID-19 (coronavirus disease 2019) pandemic has forced universities to find new ways to conduct learning and teaching, as traditional face-to-face teaching has been prevented or restricted to an absolute minimum in many instances. Therefore, we redesigned and taught second-year veterinary student microbiology laboratory exercises (labs) with a hybrid learning approach. For this, a novel 'remote partner' model was implemented in which students present on-site in the laboratory worked synchronously pairwise with their remote partner present online. A student feedback survey revealed that in this remote partner model, both on-site and online participation in the labs were experienced as being useful in improving their laboratory skills. The students' overall performance in hands-on microbiological laboratory skills and safe working practices was similar in the hybrid learning approach (the 2021 class) and in the traditional on-site participation approach (the 2018-20 classes). This study suggests that the remote partner model is an effective way to acquire microbiological laboratory skills. This learning approach can be used in the non-pandemic future and/or also be applied to other fields.Microbiological laboratory skills can be developed using a hybrid model combining synchronous on-site and online learning for students working in pairs.
  • Mousavi, Seyed Abdollah (Unigrafia, 2016)
    ABSTRACT Studies of the taxonomy of bacteria were initiated in the last quarter of the 19th century when bacteria were classified in six genera placed in four tribes based on their morphological appearance. Since then the taxonomy of bacteria has been revolutionized several times. At present, 30 phyla belong to the domain Bacteria , which includes over 9600 species. Unlike many eukaryotes, bacteria lack complex morphological characters and practically phylogenetically informative fossils. It is partly due to these reasons that bacterial taxonomy is complicated. Due to the improvement of methods to obtain sequence level characters plus new methods for their analyses, the taxonomy of bacteria has also been improved. However, there is still no official classification of prokaryotes. Biological nitrogen fixation (BNF) is a process in which bacteria reduce inert nitrogen gas to biologically useful ammonia. The symbiotic interaction between rhizobia and legumes (Fabaceae or Leguminosae) is important both in natural systems and in agriculture. Rhizobia is a general name for a group of bacteria that can enter symbiosis with legumes. Until 1982, all these were classified into one single bacterial genus, Rhizobium. The number of rhizobial genera increased to 17 by the year 2011, from which five genera, Rhizobium, Allorhizobium, Agrobacterium, Ensifer (syn. Sinorhizobium), and Shinella were accommodated in the family Rhizobiaceae. The genus Agrobacterium, a group of mostly pathogenic bacteria, was placed among the beneficial nitrogen-fixing bacteria (rhizobia) in the family Rhizobiaceae. That resulted in several taxonomic issues regarding the family Rhizobiaceae. The main nomenclatural issue regarding the genus Agrobacterium resulted from transferring this genus to the genus Rhizobium. Moreover, the phylogenetic position of the former nitrogen-fixing Rhizobium galegae complex was not clear. This group of bacteria was in previous studies clustered with either Agrobacterium or Rhizobium or placed in a lineage separately from other genera of the family Rhizobiaceae. During the last decade, the number of the rhizobial species increased dramatically, especially in the genus Rhizobium. However, Rhizobium is an inappropriate genus name for some of the species assigned to the genus. To resolve some of the major taxonomic uncertainties of the family Rhizobiaceae, two separate multilocus sequencing analyses (MLSA) were performed. In the first study, an MLSA of 114 rhizobial strains was performed by using six housekeeping genes (atpD, glnA, glnII, recA, rpoB, and thrC). The first MLSA study was focusing on the phylogeny of the taxa belonging to the former Rhizobium galegae complex and the genus Agrobacterium. In the second MLSA, a total of 100 strains representing 81 species of the family Rhizobiaceae were studied using four housekeeping genes namely 16S rRNA, atpD, recA, and rpoB. Based on these results, we proposed delineation of two new genera, Neorhizobium gen. nov. and Pararhizobium gen. nov., and 16 new species combinations, Neorhizobium galegae comb. nov., Neorhizobium huautlense comb. nov., Neorhizobium alkalisoli comb. nov., Agrobacterium nepotum comb. nov., Agrobacterium pusense comb. nov., Agrobacterium skierniewicense comb. nov., Allorhizobium vitis comb. nov., Allorhizobium taibaishanense comb. nov., Allorhizobium paknamense comb. nov., Allorhizobium oryzae comb. nov., Allorhizobium pseudoryzae comb. nov., Allorhizobium borbori comb. nov., Pararhizobium giardinii comb. nov., Pararhizobium capsulatum comb. nov., Pararhizobium herbae comb. nov., and Pararhizobium sphaerophysae comb. nov. (Paper I and II). A total of 159 bacterial strains were isolated from the nodules of the Chinese specimens of the plant genus Glycyrrhiza L. The results of the study showed that 29 true symbiotic strains belong to the genus Mesorhizobium. To estimate the phylogenetic position of the 29 isolates an MLSA was performed for 59 mesorhizobial strains by using three housekeeping genes 16S rRNA, recA, and rpoB. Moreover, the phylogeny of three symbiotic genes (nodA, nodC, and nifH) of these 59 mesorhizobial strains was investigated. The results of MLSA showed that 21 test strains belong to the species M. tianshanense, M. gobiense, M. temperatum, M. muleiense, M. amorphae, M. alhagi, and M. camelthorni, whereas eight test strains might belong to a novel species of Mesorhizobium. The results of the analyses of accessory genes in this study showed that the mesorhizobial strains isolated from the plant genus Glycyrrhiza have probably acquired some genetic material from other rhizobia co-evolving with Glycyrrhiza and other legumes (Paper III).
  • Ruuskanen, Arttu (Helsingin yliopisto, 2018)
    Tutkimuksen tarkoitus: HUS-sairaanhoitopiirin alueella hoidetaan vuosittain satoja suun alueen hammasperäisiä märkäpaiseita. Paiseiden ensisijainen hoito on hammaslääketieteellinen infektiofokukseen kohdistuva hoito, mutta sen tukena käytetään usein mikrobilääkkeitä. Mikrobilääke aloitetaan hammaslääketieteellisen hoidon yhteydessä empiirisesti, koska paisemärästä otettu viljelyvastaus valmistuu vasta myöhemmin. Siksi on tärkeää, että käytettävissä on tietoa suun paiseiden mikrobiologiasta ja mikrobien alueellisesta herkkyystilanteesta. HUS-alueella paiseiden bakteeriviljelytutkimukset tehdään HUSLAB:ssa. Tämän tutkimuksen tarkoituksena oli selvittää neljän vuoden ajalta hammasperäisissä paiseissa todetut bakteerilöydökset sekä löydösten mikrobilääkeherkkyys. Tutkimuksessa saatua tietoa voidaan käyttää empiirisen lääkehoidon valinnan tukena suun alueen paiseissa. Materiaalit ja menetelmät: Tutkimusaineistona oli HUSLAB:ssa vuosina 2012-2015 aikana tutkitut suun märkäpaiseiden bakteeriviljelytulokset. Viljelylöydöksistä tutkittiin saadut mikrobilöydökset ja löydösten mikrobilääkeherkkyydet. Näytteitä oli yhteensä n=1239. Tulokset: Yleisin löydös oli anaerobi gram-negatiivinen sauvasekafloora (739 näytettä). Tämän jälkeen yleisimmät löydökset olivat alfahemolyyttisiin streptokokkeihin kuuluvat Streptococcus anginosus- ryhmän streptokokit (267 näytettä) ja muut Viridans-ryhmän streptokokit (233 näytettä) sekä anaerobeihin gram-positiivisiin kokkeihin kuuluva Parvimonas micra-ryhmä (261 näytettää). Myös Candida albicans oli varsin yleinen löydös (189 näytettä). Tutkimuksessa havaittiin, että anaerobi gram-negatiivinen sauvasekafloora oli merkittävässä osassa löydöksiä resistentti usein empiirisessä hoidossa käytettävälle penisilliinille, mutta herkkä metronidatsolille. Sen sijaan Viridans-ryhmän streptokokeista valtaosa oli penisilliinille herkkiä. Sen sijaan Viridans-ryhmän streptokokkien herkkyys klindamysiinille on HUS-sairaanhoitopiirin alueella jonkin verran heikentynyt. Johtopäätökset: Suun alueen paiseissa on tyypillisesti sekä aerobeja että anaerobeja bakteereja sisältävää sekaflooraa, jonka löydökset tutkituin osin ovat pääsääntöisesti herkkiä penisilliinin ja metronidatsolin yhdistelmälle. Vain harvoin näytteissä todetaan esim. Staphylococcus aureusta, johon kyseinen lääkeyhdistelmä ei tehoa. Tutkimuksen tulokset tukevat toukokuussa 2017 julkaistun päivitetyn Hammasperäiset äkilliset infektiot ja mikrobilääkkeet Käypä hoito -suosituksen suosittelemia ensisijaisia mikrobilääkkeitä akuuttien suun alueen märkäpaiseiden hoidossa.
  • Cheng, Jing (Helsingin yliopisto, 2016)
    The interactions between a host and his/her microbiota have co-evolved over time and they exert profound effects on each other. Intestinal microbiota has been linked with a number of diseases, such as irritable bowel syndrome; it is considered to be a major etiopathological factor since it can alter intestinal homeostasis. However, the role of intestinal microbiota, especially commensals, is unclear in celiac disease. To date, most efforts for detecting potential microbial changes affected by celiac disease have focused on adult individuals and have examined fecal materials, although it is known that early life is the critical period for the microbiota to colonize and establish their niche in the human intestine. At this time in healthy individuals, there is continuing cross-talk with the host e.g. via the immune system, leading to the establishment of homeostasis in both metabolic and immunological programming. Since the intestinal epithelium is the main interface for host- microbe interactions, the role of mucosa-associated microbiota may be distinct from that of fecal microbiota, but both the normal fluctuations in intestinal microbiota and the composition of duodenal mucosa-associated microbiota are still not fully clarified. The aims of thesis were to characterize the development and stability of intestinal microbiota in healthy young children and to compare the microbial features between children and adults. Furthermore, the aim was to investigate host-microbe interactions in celiac disease by studying duodenal mucosa-associated microbial signatures and mucosal gene expression in healthy children and their counterparts with celiac disease. The microbiota profiles were characterized by using the human intestinal tract chip (HITChip), which is a bacterial phylogenetic microarray. The amounts of Bifidobacterium spp. in children and adults were verified with real time qPCR. The levels of mucosal gene expressions were quantified with reverse transcriptase quantitative PCR. The results showed that intestinal microbiota is not fully matured at the age of five in children. A common core microbiota, including several butyrate-producing bacteria, was identified in children and it was developing towards core microbiota found in adults. The different progression pattern of major bacterial taxa may reflect the physiological development steps in children. Moreover, differences were observed between healthy- and celiac disease- associated microbial signatures. The differences may reflect changes in epithelial integrity associated with the disease. On the other hand, the studies on both microbiota and mucosal gene expression indicated that the persistently enhanced Th1 type immune responsiveness in subjects with celiac disease after treating with gluten-free diet might result from the increased expression of TLR9, which recognizes unmethylated CpG motifs in bacterial DNA via the direct stimulation of immune cells and/or intestinal epithelial cells. The results of this thesis project suggest that specific symbiotic and dysbiotic microbial signatures may provide potential functional diagnostic or therapeutic targets for promoting healthy/natural microbiota development. Long-term studies in a controlled environment with an adequate number of participants will be necessary to decode the disturbed microbial signatures. These trials should be combined with systematic pathological surveillance to reveal how the changes in the microbiota influence the onset of disease.
  • Hoornstra, Douwe (Helsingin yliopisto, 2014)
    Bacillus cereus is a ubiquitous food poisoning bacterium, but only producers of the emetic toxin, cereulide can be life threatening. Therefore a fast and reliable method is needed for identifying strains that produce the toxin. In this thesis the previously developed sperm bioassay was refined into a tool for the detection of microbially produced mitochondria toxic substances. This refined tool, Sperm Combi Assay effectively detected not only cereulide, but also other mitochondrial toxic substances and yielded data for classifying the substances into 3 different classes. The cereulide class (includes also valinomycin and enniatin) was characterised by a spotted mitochondrial sheath when stained with the membrane potential responsive dye JC-1. The gramicidin class (includes nigericin, calcimycin, monensin, narasin, salinomycin and antimycin A) was characterised by gradual fading of the dye during loss of the membrane potential. The ionomycin type (includes staurosporine, oligomycin) was characterised by loss of sperm motility with no changes of the mitochondrial membrane potential. The novel Sperm Combi Assay was put to the test to clarify the cause of illness connected to consumption of food supplements (seaweed capsules). The Sperm Combi Assay revealed highly toxic contaminants in the capsules. The test revealed that the poisonous substance was in the seaweed inside the capsule, not involving the capsule shell. The currently available official methods had failed to disclose this information. The test further helped to identify bacteria responsible for producing the toxic substances in the seaweed. The effects shown by the Sperm Combi Assay were similar to the gramicidin type of response. Next the Sperm Combi Assay was used to track potential sources of cereulide producers in food. We found cereulide producing B. cereus from potato tuber originating from the consumer market and had not undergone industrial treatment. We describe in this thesis how the initial plate count procedure can be improved to recognise the cereulide producing B. cereus already on a plate. Cereulide producers have been reported in industrial foods and rice by many researchers. Our report appears to be the first one where potato crops were screened for cereulide producers by methods that were effective for detecting cereulide producing B. cereus. This development is important because the potato is a major food crop in the European diet. Not only potatoes but many other plants such as trees in the forest contain cereulide producing B. cereus as an endophyte. The forest industry makes food packaging material from wood pulp. To trace the behaviour cereulide producing B. cereus contained in wood, we used the same protocol which was useful for finding the cereulide producing B. cereus in potato. We found that, of the spores of cereulide producing B. cereus, ca. 5 % may be retained in the paper product. The Sperm Combi Assay was used to track the distribution of cereulide, potentially released into the pulp slurry by cereulide producing B. cereus. We found that ca. 10 % present in the pulp slurry was recovered in the paper product. We measured if cereulide contained in paper could migrate from the paper product into packaged food and found almost all cereulide leached into simulated fatty foods, but less than the detection limit into warm or cold drinks. Food waste contains valuable nutrients and should be recycled into agricultural production. The question arises about the fate of cereulide producing B. cereus contained in food. We studied the behaviour of cereulide producing B. cereus spores throughout four seasons in an industrial scale biowaste processing plant that involved a heating step of 24 min at 137 °C followed by anaerobic digestion. We traced cereulide producing B. cereus in the digested biowaste with the same culture methods and the Sperm Combi Assay which proved operative in potato and with non culture based methods (PCR). We showed that the liquid biofertilizer contained ca. 1000 total B. cereus per ml of which 10 % were cesB (indicator of cereulide synthase) positive. This persistence of cereulide producing B. cereus under harsh conditions demonstrates that it is not possible to eradicate cereulide producers from the food cycle. Instead, we have to learn to design our food processing protocols so as to not give cereulide producers a chance to accumulate the cereulide toxin in foods. In this study we used porcine sperm cells as a tool to detect cereulide (their sensitivity is high), with an EC100 (equal to lethal dose) 0.3 ng ml-1. The question is if sperm sensitivity to cereulide reflects the sensitivity of other kinds of cells and tissues, for instance in the human body. We assessed this question by comparing the sensitivity of primary cells, cell lines and pancreatic islets towards cereulide. We found that the EC100 values for loss of mitochondrial membrane potential (24 h exposure) to human peripheral blood mononuclear cells and keratinocytes, porcine kidney epithelial cells and murine fibroblasts and pancreatic islets (containing the beta cells, which produce insulin) ranged from 0.4 to 2 ng of cereulide per ml. The results show that different kinds of cells were similarly vulnerable to mitochondrial toxicity by cereulide. These results strongly indicate that the Sperm Combi Assay is useful not only for tracking cereulide in foods and environmental samples, but can also be useful in assessing human health risk by materials contaminated by cereulide. We also showed that the external concentration of potassium (300µM increased to 850-950 µM) influences the speed of the potassium efflux, measured in HaCaT and PBMC cells. We introduced this Sperm Combi Assay to deal with a bacterium and/or toxin that can hardly be eliminated in foods. This assay is a fast and reliable method to detect mitochondria toxic substances as well as the reliable plating method for screening the producing organisms. Because it is not possible to eliminate cereulide producing B. cereus, it should be kept under control by avoiding conditions permissive for cereulide production.