Browsing by discipline "Mikrobiologi"

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.

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.

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.

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.

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.

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.

Campylobacteriosis, the most common bacterial food-borne disease worldwide, is mainly caused by Campylobacter jejuni and C. coli. Most studies have focused on the genetic diversity of C. jejuni, but little is known about C. coli. The aim of this work was to characterize C. coli from different sources, by evaluating the distribution and/or diversity of certain genetic markers. A total of 145 C. coli isolates from different sources (2 goose, 18 poultry, 35 human and 90 swine) were screened for fucP, ggt, cytC, sialyltransferases genes and CRISPRs. Additionally, the diversity of the LOS loci and of the CRISPRs, were assessed. A frequency of 90.34% was observed for fucP and CRISPRs among C. coli. Conversely, the frequency of GGT phenotype, and cytC, and cst-I genotype was 1.38%, while cst-V genotype was 0.69%. Only one isolate was positive for all markers except fucP; no source association was observed. LOS and CRISPRs exhibited a wide diversity. In conclusion, ggt, fucP, and cytC seem to be lineage related in C. coli, and not host associated. CRISPRs were too discriminatory to be of use in epidemiological investigations. Results suggest a high diversity of the LOS, and there may exist more classes than those previously described.

Syventävät opinnot tehtiin osana HAIVE-yhteistyöprojektia Helsingin yliopiston Eläinlääketieteellisen tiedekunnan peruseläinlääketieteen laitoksen, Haartman-instituutin virologian yksikön, Tampereen teknillisen korkeakoulun Bio- ja ympäristötekniikan laitoksen, Helsingin veden, Tampereen veden, Turun vesilaitoksen, Raision-Naantalin vesilaitoksen, Hämeenlinnan Seudun Vesi Oy:n, TAVASE Oy:n ja Oulun vesilaitoksen kanssa. HAIVE-projektin mykobakteerisouuden tavoitteena oli kehittää nopea detektointimenetelmä mykobakteerien määrittämiseksi vesijohtoverkon vedestä ja biofilmeistä. Määritykseen käytettiin reaaliaikaikaiseen PCR:ään perustuvaa tekniikkaa. Tämän työn tarkoituksena oli kehitetyn menetelmän käyttöönotto ja menetelmän käyttäjäystävällisyyden arviointi. Lisäksi tavoitteena oli kehittää HAIVE-projektin menetelmää erityisesti DNA:n eristysmenetelmän osalta. Biofilmeissä esiintyvien mykobakteerien havaitsemiseksi käytettiin kvantitatiivisen PCR:n (qPCR) lisäksi perinteisiä viljelymenetelmiä. Tutkimuksessa käytetty kirjallisuudesta sovellettu DNA:n eristysmenetelmä osoittautui tehokkaaksi biofilmien mykobakteerimäärien ollessa suuria mutta ongelmalliseksi silloin kun mykobakteerimäärät olivat vähäisiä. qPCR menetelmä osoittautui nopeaksi ja spesifiseksi menetelmäksi. Työssä tutkituista biofilmeistä ei löydetty mykobakteereita. Tulevaisuudessa DNA:n eristysmenetelmää on kehitettävä edelleen, sillä vesijohtoverkon biofilmeissä mykobakteerimäärät ovat todennäköisesti niin vähäisiä, että monivaiheisen DNA:n eristyksen seurauksena qPCR tulos saattaa olla virheellisesti negatiivinen. Tämän työn perusteella qPCR ei korvaa perinteisiä viljelymenetelmiä silloin kun näytteiden mykobakteerimäärät ovat vähäiset.

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.

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.