Browsing by discipline "Microbiology"
Now showing items 41-60 of 80
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(2013)The aim of this research was to investigate the effects of two different probiotics on intestinal microbiota and secretory immunoglobulin A (sIgA) in healthy, well-trained adults from Canberra, Australia. Master thesis work was performed as a part of a clinical, placebo-controlled double-blind test, which aimed to investigate the ability of probiotics to improve immunity and reduce the risk of respiratory tract infections in above-mentioned group. Also research aimed to find out the possible correlation between health effects, changes in intestinal microbiota and sIgA. A hypothesis of this work is that amounts of probiotic L. acidophilus NCFM, B. lactis Bi-07, B. lactis Bl-04 strains, Bifidobacterium and Lactobacillus species will arise during intervention in groups using probiotics. A number of potentially pathogenic Clostridium cluster XIVab and Enterococcus species will decrease, whereas amounts of secretory IgA will supposedly grow towards the end of intervention. The total number of intestinal bacteria will presumably remain relatively stable. Totally 450 subjects participated in the study and 120 of them gave samples for microbiological and immunological analyses of this work. Intervention was performed in winter season and continued for 150 days. Flow cytometry was used to investigate the total number of intestinal bacteria and quantitative PCR (qPCR) was used to investigate the changes in intestinal microbiota respectively. Sandwich-ELISA was used study changes in sIgA concentrations. The total number of intestinal bacteria was typical for the large intestine and remained stable between time-points. The changes in amounts of sIgA and intestinal bacterial groups weren’t statistically significant in any group. The numbers of bacterial groups studied by qPCRmethod were also normal. Although the results for intestinal microbiota and secretory IgA weren’t statistically significant, a clinical part associated with this research showed that both probiotics reduced the frequency of lower respiratory tract infections and use of medicines. However, B. lactis Bl-04 strain was more effective in preventing upper respiratory tract infections in studied population. Intestinal microbiota and secretory IgA weren’t probably important parameters in the assessment of probiotic effects on respiratory tract infections in healthy, well-trained adults. The reliability of results for this work could be improved by adding the number of subjects and time-points.
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(2019)Elintarvikehyönteisten kulutus on yleistynyt länsimaissa viimeisen viiden vuoden aikana. Tämän tutkimuksen tarkoituksena on esitellä uusin tieteellinen tieto elintarvikehyönteistuotannon riskitekijöistä. Työssä esitellään todellinen kaksitäpläsirkan (Gryllus bimacultus) tuotantoketju kasvatuslaitokselta valmiiksi kuivatuotteeksi. Valmistusketjun omavalvontaan tutustutaan mikrobiologisten ja kemiallisten analyysien näkökulmasta ja lopputuotteen turvallisuutta arvioidaan vastaavilla analyyseillä. Työssä esitellään myös salmonellavapauden osoittaminen kasvatuslaitoksella positiivisen löydöksen jälkeen. Kuivatun lopputuotteen analyysien, kuten raskasmetallien, mykotoksiinien tai tutkittujen patogeenisten mikrobien, osalta ei ilmennyt laatua heikentäviä vaaratekijöitä, vaikkakin tuotteen vesipitoisuuden havaittiin vaihtelevan. Havaintoon liittyen kehitettiin tuotantoketjun omavalvontaa tuotantolaitoksen ilmankosteuden seurannan ja raja-arvojen osalta. Tuontihyönteisistä löydetyn salmonellalöydöksen jälkeen kontaminaatioita ei havaittu kotimaisesta kasvatuslaitoksesta tai tuotetuista hyönteisistä otetuista näytteistä, joten salmonellan leviäminen kasvatuslaitoksen sisällä vaikuttaa epätodennäköiseltä. Kontaminaation lähtöpisteeksi epäillyltä ulkomaiselta kasvatuslaitokselta salmonellaa löytyi jatkotutkimuksissa munitusastiasta, jollaisesta myös alkuperäinen löydös oli tehty. Elintarvikehyönteisten riskinarviointi perustuu yhä kohtuullisen pieneen määrään kokemusta ja tutkimusta. Suuri osa kaksitäpläsirkan elintarviketurvallisuuteen ja prosessointiin liittyvästä tutkimustiedosta on jouduttu johtamaan toisista hyönteisistä, kuten kotisirkasta (Acheta domesticus), saadusta tiedosta. Tarve uudelle tutkimukselle on suuri ja useat perusasiatkin, kuten säilyvyysaika ja olosuhdevaatimukset, perustuvat vähäiseen tietoon. Tuotaessa hyönteisiä ulkomailta on tehtävä kohdennetusti patogeenien analyysejä, jotta voidaan estää kontaminaatioiden mahdollista leviämistä tuotantolaitoksiin.
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(2012)This study was conducted to find suitable methods for the quality control of commercial plant growth-promoting (PGP) microbial products. A commercial bacterial product called TwinN was evaluated for its microbiological quality, physiological features of its bacterial strains, and its ability to promote plant growth. Other bacterial strains which had shown PGP features in earlier studies, were used as reference strains. The microbiological composition of the TwinN bacterial lyophilisate corresponded to the manufacturer's description, as it was composed of bacteria belonging to the genera Azoarcus, Azorhizobium, and Azospirillum. However, the concentrations of some of the bacterial species were lower than reported. If the product was used according to the manufacturer's instructions, the bacterial counts in the plants wouldn't necessarily reach a sufficient level. With the TwinN product, a powdery growth medium was also supplied, which appeared to be highly contaminated with bacteria and moulds. The nature and source of these microbes remained unknown. The bacteria in the TwinN product were isolated as pure cultures, and identified based on the partial sequences of their 16S rRNA genes. Specific plate and broth culturing techniques were used to uncover the potential PGP physiological features of the pure cultures. The bacterial strains were able to produce indole-3-acetic acid, ACC deaminase and siderophore, which suggests that they might also have PGP activity. The ability of the bacteria to promote plant growth was tested in a plant experiment using hydroponic growth pouches. An Azospirillum brasilense strain isolated from the TwinN product was able to increase the dry weight of the shoots of chinese gabbage almost 41 % compared with the uninoculated control plants. Of the five bacterial treatments used, the A. brasilense strain was the only one able to promote plant growth. Chinese cabbage was the only plant species out of six that gave a positive response to the A. brasilense treatment. For some reason, the TwinN product itself didn't show any PGP activity. The plants showed signs of nitrogen deficiency, which indicates that no bacterial nitrogen fixation had taken place. The method used to determine the microbial composition of the TwinN product, was Amplicon sequencing. Combined with the results of traditional culturing techniques, Amplicon sequencing proved to be a useful method for the assessment of the microbiological quality of the product. The physiological tests that could be of use in the quality control of PGP products, are siderophore production test, phosphate solubilization test and the test that measures indole-3-acetic acid production.
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(2010)Surface (S-) layers, structural entities that surround the cell envelope of various bacteria, are comprised of a porous lattice of identical protein or glycoprotein subunits. Interestingly, the S-layer is able to promote adherence to host epithelial cells in a variety of Lactobacillus species. L. amylovorus DSM 16698, a strain of porcine origin, encodes at least three putative types of S-layer proteins in its genome sequence. In this study the surface structure of L. amylovorus DSM 16698 strain and the adhesion properties of its S-layer proteins to porcine intestinal epithelial IPEC-1 cell line were examined based on preliminary results. In addition, host receptors potentially specific for S-layer proteins were isolated from IPEC-1 cells. Cloned recombinant S-layer proteins rSlpA and rSlpB of DSM 16698 were reassembled onto fluorescent-labeled L. amylovorus cell wall extracts as a means to mimic the native S-layer lattice structure. Adhesion between the reassembled recombinant S-layer complexes and IPEC-1 cells was assessed qualitatively by microscopy and quantitatively by measuring fluorescence intensity. Results from in vitro adhesion assays indicate that the rSlpA and rSlpB proteins both mediated the adherence of the L. amylovorus DSM 16698 strain to porcine intestinal epithelial cells. Antibodymediated adhesion inhibition experiments were also performed, in which the two rSlps were pretreated with their specific anti-rSlp serum, and showed that adhesion between the rSlps and IPEC-1 cells could be inhibited by the antibody treatment. Moreover, by using fluorescent-labeled rSlp-specific antibody, the surface structure of L. amylovorus cells was microscopically examined. With this immunofluorescent technique, the SlpA and SlpB proteins were both observed to localize on the cell surface and exhibit a similar distribution pattern. Putative S-layer host cell receptors were isolated from the interaction between the reassembled rSlp/cell wall complexes and IPEC-1 derived membrane proteins using a SDS-PAGE-based system. Receptor isolation experiments resulted in repeatedly the same protein profile. It has previously been shown that L. amylovorus DSM 16698 attaches to IPEC-1 cells, but the identities of surface-localized components that mediate this microbe-host interaction had yet to be determined. In this present study, S-layer proteins were found to be an important mediator in the interaction between L. amylovorus DSM 16698 and a porcine epithelial cell line. Additionally, it was shown how S-layer proteins are localized on the surface of L. amylovorus DSM 16698 cells.
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(2012)The aim of this work was to collect information about the effects of enzymatic lactose hydrolysis to the structure of yoghurt. The literature part of the study deals with the hydrolysed yoghurt components such as milk, lactic acid bacteria and lactases, lactose intolerance, yoghurt production and the concepts of defining a good structure of yoghurt. The factors influencing the structure of the fat-free and hydrolysed yoghurt is also mentioned. In the experimental part the influences of hydrolysis and the certain lactase to the structure of yoghurt was studied. Purpose of the experimental part of the work was to study on the influence of certain hydrolysis methods and certain lactases on the structure of yoghurt. First of all the optimum solid nonfat content of the hydrolysed and unhydrolysed yoghurt was defined. Three different solid non fat contents in yoghurt were studied and the one showing best viscosity differences between hydrolysed and unhydrolysed yoghurt was chosen for further tests. In the follow-up studies the lactose was hydrolysed using three different methods and four different lactases in a manner, that two methods was made with the same lactase and the third method was made using three different lactases. Both chemical and microbial souring agents were used in the yoghurt production. The samples were examined at the moment of fermenting the yoghurt to seven day old yoghurts. During the acidification of the yoghurts the amounts of lactic acid bacteria, acidification and the titrable acidification rate, tyrosine equivalent-, glucose-, galactose and lactose contents were measured and during the seven days viscosity and whey separation were defined. For the result there was differences in the structure of the hydrolysed and unhydro-lysed yoghurts which could occur because of the lactose hydrolysis. There was also differences in lactases used so both the lactose hydrolysis method and lactase used may affect significantly the structure of the yoghurt.
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(2015)Yersinia enterocolitica is an important zoonotic food-borne pathogen which in Finland is reported to be the cause of hundreds of gastroenteritis cases a year. The species is very heterogenic and comprises of both highly virulent and harmless apathogenic strains. The sources of Y. enterocolitica infection and the reservoirs of pathogenic strains are not yet well-known. In this work, 91 Y. enterocolitica isolates from 39 domestic sheep were characterized using different phenotypic and genotypic methods. Three different biotypes were identified: usually harmless environmental biotype 1A, a common enteropathogenic biotype 2 and a rare pathogenic biotype 5. All biotype 2 and 5 strains carried chromosomal ail, inv, myfA and ystA genes, and plasmid borne virulence genes yadA and virF. Biotype 1A isolates carried several chromosomal virulence genes, Notably also the ail which has a strong correlation with Y. enterocolitica pathogenicity. In pathogenic biotypes, only natural resistance to antimicrobials was detected. In PFGE-analysis, biotype 1A had several genotypes, whereas strains belonging to biotypes 2 or 5 showed only a limited amount of variation. ail and gyrB genes were partially sequenced from a selection of strains, both of which revealed variation between different biotypes. This study indicates that sheep are a reservoir for pathogenic Y. enterocolitica in Finland. Without further analysis the link between sheep and human illness cannot yet be established.
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(2013)Listeria monocytogenes on elintarvikevälitteinen, opportunistinen patogeeni, joka voi aiheuttaa ihmiselle listerioosin. Kannat sietävät hyvin jääkaappilämpötiloja ja sopeutuvat erilaisiin elinympäristöihin. Elintarvikelaitoksissa listeriat ovat usein pysyviä laitoskantoja, jotka kontaminoivat tuotteita usein pitkällä aikavälillä. Tämän maisterin tutkielman laboratorio-osuus tehtiin syyslukukauden 2012 aikana Eläinlääketieteellisen tiedekunnan elintarvikehygienian ja ympäristöterveyden osastolla, mikrobiologisen elintarviketurvallisuuden huippuyksikössä. Tarkoituksena oli tutkia DEAD-Box RNA helikaasien vaikutusta L. monocytogenes EGD-e –kannan flagellan ilmenemiseen ja kiinnittymiseen. Kirjallisuuden mukaan L. monocytogenes –kantojen kiinnittymiseen vaikuttavat suuresti mahdollisesti adhesiivisina toimivien flagellojen läsnäolo sekä solujen liikkuvuus. L. monocytogenes EGD-e–kannalla on neljä DEAD-Box RNA helikaasigeeniä (lmo0866, lmo1246, lmo1450 ja lmo1722), joista osa vaikuttaa kasvuun ja liikkuvuuteen. Kaksi geeneistä vaikuttaa lisäksi positiivisesti bakteerin selviämiseen kylmän, etanolin ja emäksisen tai hapettavan kasvuympäristön aiheuttamassa stressitilanteessa. L. monocytogenes EGD-e mutanttikannoista ?lmo0866 ja ?lmo1450 on todettu liikkumattomiksi ja ?lmo1722 osittain liikkuvaksi. Nämä mutanttikannat ovat lisäksi kylmänherkkiä DEAD-Box RNA helikaasimutanttikannat ja villityyppi kuvattiin TEM-tekniikalla ja kahden flagellageenin (flhA ja motA) ilmentymistä mitattiin qRT-PCR:lla. Lopuksi kantojen kiinnittymistä tutkittiin mikrotiitterilevymenetelmällä. Kaikki tutkimukset tehtiin kahdessa lämpötilassa (+25 ja +37°C:ssa). Elektronimikroskooppikuvat ja qRT-PCR –tulokset olivat toisiaan tukevia. Mutanttikanta ?lmo0866 ei tuottanut flagelloja lainkaan, kun taas kannalla ?lmo1450 flagellojen tuotto oli heikompaa villityyppiin verrattuna +25°C:ssa. Kannat ?lmo1246 ja ?lmo1722 vastasivat villityyppiä flagellan esiintymisen suhteen. Mutanttikannoista ?lmo0866 ilmensi +25°C:ssa molempia tutkittuja flagellageenejä yli 20-kertaa vähemmän kuin villityyppi. Myös geenin lmo1450 poistaminen johti flagellageenien ilmentymisen vähenemiseen. Mutanttikannan ?lmo1722 osalta ilmentyminen oli alentunut vain toisen tutkitun flagellageenin (motA) osalta. Geenin lmo1246 deleetio ei vaikuttanut flagellageenien ilmentymiseen. Kiinnittymisessä eroja ei havaittu helikaasimutanttien ja villityypin välillä kummassakaan tutkitussa lämpötilassa, joten yksittäiset DEAD-Box RNA helikaasigeenit eivät vaikuta L. monocytogenes EGD-e –kannan kiinnittymiseen. L. monocytogenes –bakteerin kiinnittymiseen polystyreenipinnalle vaikuttavat kirjallisuuden mukaan useat eri tekijät, kuten inkubointilämpötila, -aika, ravinteet ja kanta. Flagellan ja liikkuvuuden merkitys kiinnittymiseen ei ole mahdollisesti niin suuri kuin on aiemmin oletettu.
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(2019)Fecal microbiota transplantation (FMT) is a medical treatment procedure in which feces of a healthy donor are transplanted into a recipient in order to re-establish a healthy gut microbiota. Currently, FMT is used routinely to treat recurrent Clostridioides difficile infections, but it is being studied as a potential treatment for other diseases also resulting from a disbalance in the gut microbiota. FMT provides an excellent platform for studies addressing the determinants of a healthy gut microbiota, as it makes it possible to survey specific microbes involved in the process of re-establishment. In this sense, one particularly interesting group of gut microbes is the bifidobacteria. Although usually associated with commercial probiotics, bifidobacteria are actually one of first major colonizers of the human gut after birth, and they play a key role in the establishment and maintenance of a healthy gut microbiota. The aim of this study was to assess the long-term colonization of donor-derived bifidobacteria in recipients of FMT by using a combination of a culture-dependent method and molecular typing of strains. The culture-dependent method was used for the isolation of bifidobacterial strains from fecal samples of donors and recipients, whereas the molecular typing methods (rep-PCR and whole genome sequencing) were employed to differentiate the isolated strains. This study was based on the premise that in order to be of donor-origin, a bifidobacterial strain must occur in the recipient only after FMT and show close genetic relatedness to a strain isolated from the respective donor. Furthermore, in order to exhibit long-term colonization, a donor-derived strain must be present at later time points of the study’s follow-up period. The results showed that all the recipients had acquired some donor-like bifidobacteria after FMT. In addition, certain donor-derived strains of Bifidobacterium longum had persisted in some recipients throughout the follow-up period, indicating their successful colonization. This finding is of special interest, as extraneous bifidobacteria, such as probiotics, are typically lost without their continuous influx. Additionally, long-term colonization of bifidobacteria has not previously been documented in adults by a culture-dependent method. As efficient colonizers, the strains isolated in this study represent potential candidates for therapeutic agents.
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(2008)The purpose of this work was to identify some of the genes of the catabolic route of L-rhamnose in the yeast Pichia stipitis. There are at least two distinctly different pathways for L-rhamnose catabolism. The one described in bacteria has phosphorylated intermediates and the enzymes and the genes of this route have been described. The pathway described in yeast does not have phosphorylated intermediates. The intermediates and the enzymes of this pathway are known but none of the genes have been identified. The work was started by purifying the L-rhamnose dehydrogenase, which oxidates L-rhamnose to rhamnonic acid-gamma-lactone. NAD is used as a cofactor in this reaction. A DEAE ion exchange column was used for purification. The active fraction was further purified using a non-denaturing PAGE and the active protein identified by zymogram staining. In the last step the protein was separated in a SDS-PAGE, the protein band trypsinated and analysed by MALDI-TOF MS. This resulted in the identification of the corresponding gene, RHA1, which was then, after a codon change, expressed in Saccharomyces cerevisiae. Also C- or N-terminal histidine tags were added but as the activity of the enzyme was lost or strongly reduced these were not used. The kinetic properties of the protein were analysed in the cell extract. Substrate specifity was tested with different sugars; L-rhamnose, L-lyxose and L-mannose were oxidated by the enzyme. Vmax values were 180 nkat/mg, 160 nkat/mg and 72 nkat/mg, respectively. The highest affinity was towards L-rhamnose, the Km value being 0.9 mM. Lower affinities were obtained with L-lyxose, Km 4.3 mM, and L-mannose Km 25 mM. Northern analysis was done to study the transcription of RHA1 with different carbon sources. Transcription was observed only on L-rhamnose suggesting that RHA1 expression is L-rhamnose induced. A RHA1 deletion cassette for P. stipitis was constructed but the cassette had integrated randomly and not targeted to delete the RHA1 gene. Enzyme assays for L-lactaldehyde dehydrogenase were done similarly to L-rhamnose dehydrogenase assays. NAD is used as a cofactor also in this reaction where L-lactaldehyde is oxidised to L-lactate. The observed enzyme activities were very low and the activity was lost during the purification procedures.
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(2020)Tämä maisterintutkielma on osa tutkimusohjelmaa, jossa on tarkoitus tunnistaa uusia antibioottien vaikutuskohteita bakteereissa. Tavoitteena oli pystyttää Haartman-instituutissa molekyylibiologinen menetelmä, jolla voitaisiin selvittää faagigeenien tuottamien proteiinien toksisuutta. Menetelmän kehityksessä korostui tarve pystyä analysoimaan tehokkaasti mahdollisimman monta tuntematonta faagigeeniä ja niiden toimintaa. Työssä kehitetty menetelmä perustui bioluminesenssin käyttöön toksisuuden havaitsemiseksi. Ensimmäisessä vaiheessa valmistettiin plasmidi, joka sisälsi luxAB-geenit. Työn toisessa vaiheessa muodostettiin itsemurhavektori, joka sisälsi luxCDE-geenit. Itsemurhavektorin sisältämät luxCDE-geenit integroitiin bakteerin genomiseen deoksiribonukleiinihappoon (gDNA), jonka jälkeen luxAB-geenit sisältävä plasmidi voitiin elektroporoida kyseiseen bakteeriin. Yhdessä nämä muodostivat geeniyhdistelmän luxCDABE, joka pystyi tuottamaan valoa bioluminesenssin avulla. Jos luxAB-plasmidiin kloonattavan geenin tuote on toksinen, eivät plasmidin saaneet transformantit elektroporaation jälkeen säily hengissä, minkä seurauksena transformantit eivät tuota valoa, kun taas, jos geenin tuote ei ole toksinen, tapahtuu valon tuottoa. Toisin sanoen, luminesenssin puuttuessa menetelmä toimisi halutulla tavalla eli indikoisi geenien tuottamien proteiinien toksisuutta. Menetelmän toimivuutta testattiin käyttämällä tunnettuja bakteerikantoja, jotka sisälsivät luxCDE-geenikasetin. Tarkoituksena oli varmistaa luxAB-geenien toimivuus komplementoida minkä tahansa bakteerin luxCDE-geenit. Tarkoitus oli saada aikaan menetelmä, jolla voidaan nopeasti ja tehokkaasti selvittää tuntemattomien faagigeenien toimintaa. Bioluminesenssin tuottama valo erottuu selkeästi ja menetelmä on helposti toistettavissa. Työssä kehitettyä luxAB-geenit sisältävää plasmidia voidaan jatkotutkimuksissa testata ensin tunnetuilla kontrolligeeneillä ja lopuksi käyttää toksisia proteiineja tuottavien faagigeenien tunnistamisessa.
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(2020)Kuluttajille suunnattujen valmiiksi pilkottujen hedelmien ja muiden kasvisten kulutus on kasvanut tasaisesti viime vuosikymmenen aikana. Tuotteet ovat kuitenkin usein suojaavan kuoren poistamisen takia alttiita pilaantumiselle, mikä aiheuttaa ruokahävikin muodossa globaalisti ison hiilijalanjäljen ja taloudellisia tappioita. Jotta valmiiksi pilkottujen tuoreiden kasvisten hyllyikää saadaan kasvatettua, on säilytysolosuhteiden lisäksi mietittävä keinoja estää, hidastaa tai pienentää mikrobien aiheuttaman pilaantumisien vaikutuksia tuotteissa esimerkiksi vaikuttamalla tuotteen mikrobien määrään säilytyksen alkaessa. Monet valmiiksi pilkotut hedelmät ovat myös otollinen kasvualusta patogeeneille, jolloin tuotteiden hyvä tuotantohygienia on lähtökohta sille, että tuotteet ovat turvallisia kuluttajalle. Salico Oy:n Helsingin Kivikon tuotantolaitoksessa tuotantohygienia on ollut mittauksissa erittäin hyvällä tasolla, mutta asiakasvalituksissa toistui valmiiksi leikattujen ananaspalojen osalta ”simamainen” tuoksu, joka viittasi hiivamaisesti kasvavien sienten kasvuun tuotteessa. Tämän vuoksi tutkimuksessa keskityttiin testaamaan ananaksen kuoreen desinfektiomenetelmiä, joiden oletettiin kirjallisuuden perusteella vähentävän juuri hiivamaisesti kasvavien sienten määrää. Lisäksi vesipesun merkitystä mikrobimäärän muutokselle kuorituissa ananaksissa mallinnettiin kahdeksan vuorokauden säilyvyystestauksella. Tutkimuksen desinfektiomenetelmiksi valittiin vesipesu, UV-C-säteilytys kolmella matalalla tehoalueella sekä lyhytkestoinen höyrytys, sillä näistä desinfektiomenetelmistä ei jää jäämiä tuotteeseen, menetelmien toteutus oli suhteellisen helppoa, eivätkä mahdolliset investoinnit tuotantoon olisi kalliita. Ananaspalojen säilyvyystestauksen aika valittiin asiakasvalitusten perusteella parasta ennen -päivämäärän molemmin puolin. Kaikilla tutkimuksen desinfektiokäsittelyillä oli sienien kokonaismäärää pienentävä vaikutus, mutta tilastollinen merkittävyys saavutettiin vain UV-C-säteilytyksellä 0.98 J/cm2-säteilymäärällä. Ananaspalojen vesipesu ja linkous vaikuttivat sienien kokonaismäärään siten, että pestyjen ananaspalojen mikrobipitoisuus oli neljän päivän kohdalla tarkasteltuna ensin 51 % pienempi, mutta kahdeksan päivän kohdalla ja 72 % suurempi verrattuna pesemättömiin ananaspaloihin. Tulosten perusteella UV-C-säteilytys alensi tutkituista desinfektiomenetelmistä sienten kokonaismäärää eniten. UV-C-menetelmä on toteutettavissa teollisessa mittakaavassa. Lisäksi havaittiin, että ananaspalojen vesipesu ja linkoaminen eivät ole kannattavia ananaksen säilyvyyden pidentämiseksi.
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(2011)The human gastrointestinal tract (GIT) contains a complex microbiota which starts to develop after birth. Various factors such as age, health, diet and medication affect the composition of the GIT microbiota. The number and types of bacteria are different in each part of the GIT, but most of the bacteria are anaerobic. In faeces the number of bacterial cells is as high as 1011-1012 cfu/ml. The normal intestinal microbiota is essential for intestinal development, protein and carbohydrate metabolisms, and protection against pathogens. Sulphate-reducing bacteria (SRB) are typically anaerobic bacteria which use sulphate as a terminal electron acceptor to produce sulphide in their metabolism. Sulphate-reducing bacteria are widespread in all ecosystems including fresh water and marine sediments but are also present in the GIT. Most of SRB species are Gram-negative and they can use more than hundred compounds as electron donors. Dissimilatory sulphite reduction (dsrAB) gene is essential in sulphate reduction. dsrAB-gene encodes the enzyme called dissimilatory sulphite reductase, which is a key enzyme in the reduction of sulphite to sulphide. Recent findings suggest that SRB may have a role in human diseases, e.g. in periodontitis and inflammatory bowel disease (IBD). Connection between these disorders and SRB may be due to the highly toxic hydrogen sulphide. The aim of this study was to develop PCR-DGGE and qPCR methods for monitoring of sulphate-reducing bacteria from human faecal microbiota. In this study we used dsrAB-gene specific primers, which were used successfully in previous environmental microbiology studies. Previously published dsrAB-specific primers were used for PCR-DGGE. However, besides positive controls, two negative controls also amplified regardless of the modifications on temperature, amplification times, primers and MgCl2 concentration. In qPCR, specific and sensitive amplification was attained by using dsrA-gene specific primers. When the samples from paediatric patients with IBD (Crohn’s disease and ulcerative colitis) and healthy children were amplified, no differences were found between different disease groups. However there was a statistically significant difference (P <0.05) between the paediatric patients with Crohn’s disease who were on remission and those patients who’s disease was active (number of SRB; active<remission).
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(2018)Nanomateriaalit ovat hyvin pienikokoisia materiaaleja, joiden koon tuomat erityisominaisuudet ovat teollisuuden kannalta hyödyllisiä. Nanomateriaalit reagoivat eri tavalla myös biologisen materiaalin kanssa, joten samat teollisuuden kannalta suotuisat ominaisuudet on otettava huomioon toksikologisesta näkökulmasta. Nanotitaanidioksidia ja nanosinkkioksidia käytetään aurinkovoiteissa, kun taas nanohopeaa käytetään haavahoidossa ja vaatteissa. Näille materiaaleille voi täten altistua ihon kautta. Ihon yksi tärkeimpiä tehtäviä on suojata meitä ulkoisilta haitoilta ja iholla on omat mekanisminsa tätä varten. Iholla on myös oma mikrobiyhteisönsä, joiden poikkeavuudet ovat yhteydessä erilaisiin ihosairauksiin. Ihon mikrobien sekä nanomateriaalien välistä yhteyttä ei ole tutkittu kattavasti ja tässä maisterintutkielmassa tutkittiin tätä yhteyttä. Karvattomia, SKH-1-kannan hiiriä altistettiin nanotitaanidioksidille, nanosinkkioksidille tai nanohopealle. Osa hiiristä lopetettiin vuorokauden sekä osa seitsemän päivää altistuksen jälkeen, ja hiiristä kerättiin ihonäytteitä mikrobi-DNA:n ja hiiri-isännän RNA-eristystä varten. Hiiri-isännän ihopalojen RNA:n eristystä optimoitiin erilaisilla helmihakkausputkilla ja RNA:n laatu tarkastettiin. RNA:sta tutkittiin metallotioniinien ja tulehdusta edistävien sytokiinien geenien ilmentymistä kvantitatiivisella PCR:llä. Mikrobi-DNA eristettiin ihopaloista ja puikkonäytteistä, ja eristystä optimoitiin kolmella eri menetelmällä. DNA:sta monistettiin 16S rRNA-geeni, sekvensointiin Suomen molekyylilääketieteen instituutissa ja käsiteltiin R-ohjelmistolla. Myös sekvenssidataa käsiteltiin kolmella tavalla ennen kuin saatiin varsinaiset tulokset. Hiiren ihon geenien ilmentymisessä ei havaittu erityistä muutosta. Sen sijaan ihon bakteerien kokoonpanossa havaittiin muutos. Bakteereita tarkastellessa vuorokauden titaanidioksidi- ja sinkkioksidiryhmät erosivat hieman naiiviryhmästä, ja seitsemän päivän hopearyhmä erosi naiiviryhmästä selkeästi. Erityisesti Corynebacterium-bakteerien osuus nousi hopea-altistuksen myötä, kun taas osan bakteerien suhteellinen osuus laski. Tämän tutkimuksen menetelmäoptimoinnin avulla saavutettuja tuloksia voidaan hyödyntää tulevaisuuden ihon mikrobiomitutkimuksissa ja tarvittaessa tutkimuksia voi tarkentaa esimerkiksi käyttämällä tautimalleja.
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(2020)Nautojen (Bos taurus) sorkka-alueen ihotulehdus (DD) on ympäri maailman levinnyt sairaus, jota on myös havaittu muilla sorkkaeläimillä. Nykytiedon mukaan nautojen DD on bakteeri-, ei virus- tai sienitauti. Se aiheuttaa haavoja, vaurioita ja känsiä. DD leviää helposti ja aiheuttaa eläimelle myös kipua, heikentää sen yleiskuntoa ja hyvinvointia sekä aiheuttaa tuotantotappioita. Keski-Euroopassa ja Yhdysvalloissa DD:n on havaittu leviävän lähes epidemialuonteisesti, kun taas Pohjoismaissa sitä on tavattu yksittäisissä karjoissa. Taudin etiologiasta, esiintyvyydestä ja parhaasta mahdollisesta hoitomuodosta on eri teorioita. Nykytiedon ja tieteellisen kirjallisuuden mukaan merkittävimpinä DD:n aiheuttajabakteereina pidetään eri Treponema-bakteerilajeja. Uusimpien tutkimusten perusteella taudin vakavuuteen vaikuttaa eri Treponema-lajien yhteisvaikutus keskenään tai muiden bakteereiden kanssa. Tämän tutkielman tavoite oli pystyttää bakteerien 16S rRNA-geenin rinnakkaissekvensointiin perustuva metataksonomiatyövuo bakteereiden tunnistamiseksi. Tutkielmassa vertailtiin kahta eri DNA-eristysmenetelmää, kahta sekvensointityövuota ja kahta bioinformatiikan analyysimenetelmää. Näytemateriaalina oli suomalaisten nautojen sorkka-alueen ihosta otetut biopsianäytteet (n=10). Viisi näytteistä oli terveestä M0-naudasta ja viisi M2-naudasta. Tuloksena havaittiin pieniä määriä Spirochaetaceae-bakteeriperheen sekvenssejä kahdesta M0 -diagnosoduista naudasta. Kaikista M2 - diagnoosin naudoista havaittiin runsaasti Spirochaetaceae - sekvenssejä. Lisäselvityksiä Treponema-bakteerien luotettavasta lajitason tunnistamisesta sekä niiden roolista taudissa tarvitaan. Bovine (Bos taurus) digital dermatitis (DD) of the foot is a widespead disease around the world, and it has also been diagnosed with several other hoofed ruminants. According to current knowledge, bovine digital dermatitis is a bacterial disease, not a viral or fungal disease. It causes ulcers, lesions, and raised calluses. DD spreads easily, and it can cause pain to the animal, weaken its overall health and well-being, as well as cause loss of production. DD has been almost epidemic in Central Europe, as well as in the United States of America, whereas in Scandinavia it has only been encountered in individual herds. According to current knowledge and the scientific literature, the most important bacteria responsible for DD are Treponema species. Recent studies suggest that the severity of the disease is affected by the interaction of different Treponema species with each other or with other bacteria. The aim of this thesis was to set up a metataxonomic pipeline for the parallel sequencing of the 16S rRNA bacterial gene, in order to identify the bacteria. Two different DNA extraction methods, two different sequencing pipelines, and two different bioinformatics pipelines were evaluated in this thesis. Sample material was biopsy samples of the skin of the Finnish bovine hoof (n=10). Five of the samples were from healthy M0 cattle and five from M2 cattle (acute active ulcerative lesions). As a result, small amounts of Spirochaetaceae bacterial family sequences were detected in two M0 diagnostic cattle . Abundant Spirochaetaceae sequences were found in all bovine diagnosed with M2. Further studies on the reliable species identification of Treponema bacteria and their role in the disease are needed.
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(2016)Lantibiotics are a subgroup of bacteriocins, produced by Gram-positive bacteria to inhibit the growth of closely related strains. They are used as food preservatives e.g. nisin, and some are in clinical trials, e.g. duramycin A and microbisporicin. Cinnamycin is a 19 amino acid lantibiotic that inhibits the growth of Gram-positive rods. Recent work suggests that cyanobacteria might be able to make variants of cinnamycin. Here I determined the product of a cinnamycin biosynthetic pathway present in the genomes of a benthic cyanobacteria. The genome mining analysis demonstrated that three cyanobacterial strains and seven actinobacterial strains contained the genes responsible for the production of cinnamycin. Cinnamycin variants were detected from cyanobacteria Oscillatoria sp. PCC 10802 and actinobacteria Streptomyces roseoverticillatus DSM 40845, respectively. Oscillatoria sp. PCC 10802 produced a cinnamycin variant named oscillamycin, with mass of 1966.86 Da. Stable nitrogen (15N) and sulphur (34S) isotope labeling of the cyanobacterium indicated that the oscillamycin contains 3 sulfur atoms and 23 nitrogen atoms. However, the mass of oscillamycin was 16 units bigger than the bioinformatic predictions. LC-MS analysis suggested that the oscillamycin contains a hydroxyl-proline in addition to hydroxyl aspartic acid. The oscillamycin gene cluster was cloned and successfully expressed in Escherichia coli BL21. Small amounts of oscillamycin (0.25 µg) were purified from Oscillatoria sp. PCC 10802 and showed tentative antimicrobial activity against Bacillus subtilis HAMBI 251. This study demonstrated that cyanobacteria and actinobacteria share a lantibiotic gene cluster and that the lantibiotic produced differed in just four amino acids. The phylogenetic analysis suggested that the cinnamycin gene cluster was transferred from actinobacteria to cyanobacteria by an ancient horizontal gene transfer event. This study expands the chemical diversity of cinnamycin variants. This is the first report of a lantibiotic from cyanobacteria suggesting that cyanobacteria might be a novel source of antibiotics, which could be useful in addressing the antibiotic resistance issue.
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(2016)Nisin and silver have antimicrobial effects on bacteria growing on meat. Nisin is a bacteriocin peptide produced by lactic acid bacteria Lactococcus lactis. It is known to have a wide killing effect against many gram-positive bacteria. Food industry utilizes nisin as a food preservative. The antimicrobial effect of silver is known over the couple of decades. It has a killing effect against many gram-positive and gram-negative bacteria. In food industry silver can be used in packaging materials to prevent bacterial growth. In this study our aim was to evaluate the use of nisin and silver on the meat spoilage microbiota and the rate of spoilage in vacuum and modified atmosphere (80 % oxygen and 20 % carbon dioxide) packed meat. The antimicrobial effect of nisin and silver was evaluated by microbial counts and sensory evaluations. B. thermosphacta, Enterobacteriaceae, lactic acid bacteria and Pseudomonas sp. bacteria were interest of. Pyrosequencing was used to specify the bacterial taxonomy in spoiled meat samples. Nisin showed to have an antimicrobial effect only against lactic acid bacteria in vacuum packed meats. The growth rate of lactic acid bacteria was 2 logarithmic scales lower in nisin threated samples than in control samples. In modified atmosphere packed meat nisin didn´t show to have effect on bacterial growth. Nisin didn´t have antimicrobial effect on other bacteria. Silver treatment didn´t show to have effect on bacterial growth in both packing methods. Vacuum packed meat was evaluated of being spoiled after 56 days with nisin treatment and control samples spoiled couple of weeks earlier. Silver didn´t prolong the shelf life of meats compered to control samples. Based on the sequence analyse, total of 29 bacterial genus and 11 family where identified. Leuconostoc, Lactogbacillus and Serratia being the most abundant genus in vacuum packed meats in nisin series. Serratia and Pseudomoas sp. where spoilage bacteria associated in modified atmosphere packed meats. Lactic acid bacteria species belonging to genus Lactobacillus and Lactococcus where dominating in vacuum packed meats in silver series. Spoilage bacteria in modified atmosphere packed meat in silver series was mainly Leuconostos sp. The shelf life of vacuum packed meat was evidently longer in nisin treated samples and that’s why the allowance to use nisin in meat should be reconsidered. Silver didn’t have the effect to prolong the shelf life of meats on either packing methods even though its usage is allowed in food packaging materials.
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(2018)Human gut microbiota is an important topic for many different disciplines. Various factors, e.g. antimicrobial drugs and diet, affect the development and balance of gut microbiota and its interactions with the host. Plant based carbohydrates that transit unabsorbed and undigested through the upper parts of gastrointestinal tract are an important source of energy for the colon bacteria. Some of colon bacteria produce short chained fatty acids (e.g. acetate, propionate, butyrate) from these carbohydrates. SCFAs provide a source of energy and regulate the cell growth and metabolism. The changes in the diversity and abundance of the SCFA producing bacteria have been linked to many gut related diseases. Studying gut microbiota with today’s analytical methods is still challenging. In this work the effects of dietary fibers on gut microbiota were monitored with a static, single vessel batch model. A batch model is typically developed for the quick high-throughput screening of samples. Fiber samples were processed in various ways to increase their solubility and thus fermentability. In this work butyrate producing bacteria, Akkermansia muciniphila and bifidobacteria were targeted. Enumeration was performed with selective growth media and quantitative PCR. Bacterial population was characterized by 16S rRNA based sequencing. To quantitate only viable bacterial cells from the sample matrix by qPCR, samples were treated with propidium monoazide (PMA), which after light activation inhibits the amplification of double-stranded DNA from dead and lysed cells. Since acidic SCFAs accumulate in the sample suspension, pH decreases clearly during the incubation in the static model. This leads to conditions which do not resemble the ones in the colon. Two different buffer solutions, pH adjustment and shorter incubation time were tested to overcome this challenge. The numbers of A. muciniphila and some of the butyrate producers decreased in acidic environment and the proportion of acid-tolerant bacteria was clearly increased and dominated the bacterial population. The optimization of PMA treatment for fecal suspension samples proved to be challenging due to the highly variable composition of sample matrixes. Dietary fibers were observed to cause different changes in bacterial population: the most soluble fibers caused greater decrease of pH and thus greater proportion of acid tolerant bacteria in the population.
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(2020)Mikrobikantakokoelmissa säilötään mikrobeja muun muassa tutkimuksen, opetuksen ja teollisuuden tarpeisiin. Kantakokoelmien tulisi taata mikrobien säilyminen kymmeniä vuosia eläväisinä ja geneettisesti ja toiminnallisesti muuttumattomina. Ekologisen sopeutumisensa takia pintasienijuurisienet kasvavat laboratoriossa usein hitaasti ilman isäntäkasvia ja selviytyvät heikosti erilaisissa lyhyen ja pitkän ajan säilytykseen käytettävissä menetelmissä. Pintasienijuurisieniä on syväjäädytetty, mutta ne selviytyvät usein huonosti perinteisellä menetelmällä, jossa rihmastoa syväjäädytetään glyserolin vesiliuoksessa. Pintasienijuurisieniä ja muita herkkiä sieniä varten on kehitetty menetelmiä, joissa sienet pakastetaan suoraan kasvualustassaan. Tässä työssä tutkittiin perliitin, tuliperäisen mineraalin, soveltuvuutta pintasienijuurisienten syväpakastuksessa. Perliitti toimii rihmaston kantajamateriaalina ja pienentää jäätymisvaiheessa syntyvää lämpötilannousua, mikä saattaa lisätä sienten selviytymistä pakastusprosessista. Tutkittavina sienikantoina oli 20 pintasienijuurisienikantaa ja vertailukantoina endofyyttinen Phialocephala fortinii ja kuusi lahottajasienikantaa. Säilöntäkäsittelyinä oli viisi erilaista säilöntäkäsittelyä: perliitti -140°C, perliitti +4°C, glyseroli -140°C, glyseroli +4°C ja vesi +4°C. Perliittikäsittelyissä tutkittavat sienirihmastot kasvatettiin ja syväpakastettiin kolmen kuukauden ajan, tai säilytettiin +4°C lämpötilassa perliittiä sisältävissä kryoputkissa, sulatettiin ja siirrostettiin kasvamaan rihmastoa maljoille. Selviytymistä arvioitiin katsomalla silmämääräisesti, kasvoiko maljalle sienirihmastoa, ja mittaamalla maljalle kasvaneen rihmaston pituus vähintään kolmena mittauspäivänä. Lisäksi pintasienijuurisienten ja Phialocephala fortinii -sienen toiminnallisten ominaisuuksien säilymistä tutkittiin havainnoimalla sienen kykyä muodostaa sienijuurta männyntaimen kanssa sekä tutkimalla muutaman hydrolyyttisen ravintoyhdisteitä pilkkovan entsyymin ja lakkaasientsyymin tuottoa sienissä säilöntäkäsittelyn jälkeen. Rihmaston kasvun ja sienten entsyymintuoton tuloksia analysoitiin tilastollisilla testeillä. Glyserolimenetelmissä rihmastot syväpakastettiin tai säilytettiin +4°C:ssa agarpaloissa glyserolin vesiliuoksessa, ja vesimenetelmässä rihmastot säilytettiin agarpaloissa +4°C steriilissä MilliQ-vedessä. Osa tutkittavista sienistä onnistuttiin syväpakastamaan perliittimenetelmällä nestetypen höyryfaasissa: sienet kasvoivat pakastuksen jälkeen maljoilla. Nämä sienet olivat Amphinema byssoides (kuopikka), jonka selviytyminen oli 100 %, n = 5, Suillus bovinus, SBL 1, (nummitatti), jonka selviytyminen oli 100 %, n = 3, ja Tomentellopsis echinospora (keltamujukka), jonka selviytyminen oli 100 %, n = 5. Useimpia tutkimukseen valituista pintasienijuurisienikannoista ei kuitenkaan pystytty siirtämään nestetypen höyryfaasiin, sillä näiden kantojen rihmastot eivät kasvaneet perliittiä sisältävissä kryoputkissa. Tämän kokeen tulokset osoittavat, että syväpakastetut pintasienijuurisienikannat kestävät perliitti -140°C-käsittelyn säilyttäen toiminnalliset ominaisuutensa, mutta sienten kasvua perliitissä pitää tutkia lisää ja kasvualustaa kehittää pintasienijuurisienille paremmin soveltuvaksi. Tällä menetelmällä säilytettyinä sienten voidaan olettaa säilyvän useita kymmeniä vuosia.
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(2018)Inorganic phosphate (Pi) is the only readily utilizable form of phosphorus for toxic diazotrophic cyanobacterium Nodularia spumigena (N. spumigena). Pi is one of the limiting nutrients in the Baltic Sea where surprisingly N. spumigena are highly abundant especially during the summer. This indicates that N. spumigena possibly has an alternative pathway to fulfill its phosphorus requirement. The Baltic Sea, like most aquatic environments, is enriched with organic phosphorus compounds among which phosphonates may constitute a significant fraction. Interestingly, the Baltic Sea N. spumigena strains UHCC 0039 and CCY9414 have been found to carry phosphonate degrading gene cluster (phnC-M) implying that these cyanobacteria could assimilate phosphonates as a phosphorus source. However, the significance of the presence of phn gene cluster in N. spumigena for phosphonate utilization has not been investigated in detail. Here, I aimed to understand how N. spumigena copes with Pi limitation and utilizes phosphonates in laboratory conditions using biochemical assays, PCR-based methods and bioinformatics tools. This would aid in finding a suitable marker for Pi deficiency in cyanobacterial blooms in the Baltic Sea. In this study, bioinformatics and PCR screening showed that phn gene cluster was conserved in the Baltic Sea N. spumigena strains. The studied N. spumigena strains UHCC 0039 and UHCC 0060 were found to utilize naturally produced low molecular weight phosphonates, methylphosphonate (MPn), ethylphosphonate (EPn) and 2-aminoethylphosphonate (2APn). Among these phosphonates, MPn seemed to be the most preferred phosphorus source. Alkaline phosphatase activity, an indicator of Pi limitation, was found to be elevated in the media with Pi and 2APn questioning its suitability as a marker for phosphorus limitation. In addition, growth on MPn released methane indicating that massive blooms of N. spumigena might contribute to an elevated methane supersaturation in the Baltic Sea. Reverse transcriptase quantitative PCR (RT-qPCR) in N. spumigena strains did not show expected upregulation of high-affinity phosphate transporter pstS in Pi limitation. It demonstrated an induction of phosphonate transporter gene phnD in media lacking Pi and supplemented by 2APn. The phosphonate lyase gene phnJ was however, upregulated only in the presence of MPn suggesting that phnJ gene could be used as a marker for phosphonate bioavailability. The findings from this study suggest that the presence of phn gene cluster could provide N. spumigena a competitive advantage in Pi-limited cyanobacterial blooms in the Baltic Sea. The molecular detection methods designed in this study thus could be used in future to monitor the expression of genes induced during Pi limitation and the presence of phosphonates, and the method could be further optimized for screening natural water samples.
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(2017)Phycobilins are the main light harvesting pigments in picocyanobacteria. Chlorophyll-a is the main photosynthetic pigment in cyanobacteria as in all phytoplankton. In cyanobacteria, most of chl-a is positioned within the non-fluorescing photosystem I (PSI). Cyanobacteria phycobiliproteins are the main photosynthetic pigments in photosystem II (PSII). Phycobilins fluorescence can be used to help assess the presence and monitoring of cyanobacteria. The fluorescence intensity depends on the examined cyanobacteria group, pigment concentration and phytoplankton growth phase. In this research I studied, using flow-through fluorometers, where the phycoerythrin (PE) fluorescence is originating from and its variation in the Baltic Sea. PE fluorescence signal measured with flow-through fluorometers was also compared with other optical measurements. This study was performed in summer 2016 as part of Alg@line and JERICO-Next projects. Flow-through fluorometers (TriOS and Chelsea) were installed to M/S Finnmaid ship, which trafficked regularly on its route Helsinki–Travemünde. The automated flow-through sensors onboard M/S Finnpartner collected continuous data during 25.5–31.8.2016. Along the route Travemünde-Helsinki, a refrigerated sampler collected water samples once a week from 3 stations. Water samples acted as a reference samples for PE fluorescence signal analysis. Water samples were separated by filtration into three size fractions (total < 2 µm, and < 0.2 µm) and an excitation-emission spectrum was measured. The number of picocyanobacteria/ml, their surface area/ml and biovolume/ml was calculated using epifluorescence microscope. The number of PE-containing picocyanobacteria cells/ml and size was determined by flow cytometer and number of larger PE-containing phytoplankton cells, their size and taxonomy was determined using FlowCam. Most of the PE-fluorescence measured during summer 2016 was originating from pico-fraction. There was not a clear connection between flow-through PE fluorometers and other optical measurements. PE signal originating from fluorometers did not correlate with total fluorescence signal measured with spectrofluorometer. A reason for this can be that the sample has suffered preservation and transport due to the elapsed time. Some of the optical measurements correlated well with each other, and some did not. Excitation-emission spectrum measured from pico-fraction correlated with picocyanobacteria surface area/ml calculated with epifluorescence microscope. This can be explained by the fact that picocyanobacteria pigments are mainly located in the cell membrane. Number of cells/ml calculated with flow cytometer was much lower than the number/ml calculated with epifluorescence microscope. Sample could have been too dense when multiple cells has been interpreted as one larger cell. The program used for the grouping of cells could have also left low PE fluorescence value containing cells without counts. PE fluorescence originating from over 2 µm size fraction measured with spectrofluorometer and fluorescence originating from over 3 µm fraction pictured with FlowCam was not observed similar incidence of various stations in the summer of 2016. PE fluorometers alone are not sufficient for monitoring picocyanobacteria cells containing phycoerythrin in the Baltic Sea but PE fluorometers can be used as support to other methods.
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