Browsing by Subject "biofilmi"

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  • Sarekoski, Jenna (Helsingfors universitet, 2018)
    Most bacteria live as biofilms (99%), which is a population of cells attached to a natural or artificial surface and encased in self-produced exopolysaccharide matrix. The extracellular polymeric substances (EPS) in the matrix can vary greatly between species in chemical and physical properties, but primarily it consists of water, polysaccharides, proteins, nucleic acids and absorbed nutrients from the surrounding area. Biofilm formation appears to be a survival strategy of bacteria and the main purpose of the biofilm matrix is to protect the bacteria. In nature, biofilms have been found in variety of different environments, including humans. Bacterial biofilms demonstrate a decreased susceptibility to antimicrobial agents and several mechanisms have been proposed to be involved in this tolerance. One of the reasons why chronic infections develop is that the immune response fails to remove the biofilm. Most of the bacterial infections currently in developed countries are biofilm related and these infections are often recalcitrant and difficult to eradicate with available treatments. In addition to chronic infections, the treatment of acute infections is shadowed by increasing problems with highly resistant bacteria. The presence of dormant persisters in biofilms accounts for their tolerance to antimicrobials and likely are responsible for latent and chronic infections, such as tuberculosis. Persistence is not primarily an active mechanism of antibiotic tolerance, but a dormant state of the bacteria avoiding the mechanism of action of most antibiotics. Persisters form stochastically only in small numbers, and more relevant physiological explanation is related to the stress responses of the cells. Persisters are distinguish phenotypic variants of the normal population and it is not a heritable feature, as no mutations occur. The dormant, persistent state of the bacteria is largely responsible for the multidrug tolerance of recalcitrant infections. Biofilm cause various diseases in humans, as bacteria are able to attach to practically any surface, such as teeth, heart valves, lungs, middle ear, artificial prosthetics and instruments. Biofilms growing on prosthetic joints can cause also serious infections, which are painful for the patient with high risks for complications, expensive and laborious to replace. Biofilm infections are difficult to treat and a huge burden in the healthcare. Many acute infections can be cured with conventional antibiotic therapies, but this is not case with recalcitrant, chronic infections. B. cenocepacia belongs to the B. cepacia complex (Bcc) which consist of 20 closely related and phenotypically similar species. This species was chosen for this study because of its natural tolerance to antibiotics and ability to form biofilms easily. This species causes fatal lung infections in cystic fibrosis patients, and there is no treatment for it other than inadequate combination antibiotic treatment and lung transplant. In this thesis, a promising method was developed and validated for detecting anti-persister activity against B. cenocepacia. The assay is based on measuring the levels of ATP present in the cultures after treatment and it can be used quantify remaining persisters using B. cenocepacia biofilms. Utilizing the method validated, it was confirmed that mitomycin C is an effective anti-persister compound against highly tolerant B. cenocepacia biofilms even at low concentrations. Doxycycline was found to be ineffective against B. cenocepacia biofilms, although the bacteria are susceptible to it in planktonic form, and ciprofloxacin was proved to be effective at very high concentrations.
  • Nissinen, Ulla (Helsingfors universitet, 2017)
    Quartz crystal microbalance (QCM) and surface plasmon resonance (SPR) spectroscopy are methods measuring mass changes on solid surface. During measurement fluid flows over sensor. The aim of this study was to find out if it's possible to culture a biofilm using QCM and SPR methods and compare biofilms with those cultured in test tubes under static fluid conditions. Enrofloxacin antibiotic was tested against biofilm cultured in SPR. Biofilms were imaged electron microscopically. Bacteria used were Staphylococcus pseudintermedius and Corynebacterium auriscanis and a combination of those. Biofilm was successfully cultured by both methods repeatably. S.pseudintermedius formed a biofilm, but C.auriscanis didn't. Together S.pseudintemedius and C.auriscanis formed thicker biofilm than S.pseudintermedius alone. There were difference between biofilms depending on culturing conditions. Biofilm covered the surface quicker and bacterial density was higher under flowing conditions than static fluid. The growth of biofilm was ceased during enrofloxacin feeding, but not destroyed. Growth continued after stopping enrofloxacin feeding. QCM and SPR methods are suitable for culturing biofilms. They measure mass changes on solid surface but tell nothing about the architecture of biofilm. QCM and SPR could be good methods for studying compounds destroying biofilm matrix or trying to find coating materials to prevent bacterial adhesion.
  • Sarajärvi, Saara (Helsingfors universitet, 2016)
    Lactobacillus rhamnosus GG on yksi eniten tutkituista ja käytetyistä probioottisista maitohappo-bakteereista. Sillä on erinomainen adheesiokyky, mikä on ensisijaisesti sen SpaCBA-piluksen ansiota. Piluksen lisäksi sen pinnalta on löydetty kaksi muuta adhesiivista proteiinia: MabA (modulator of adhesion and biofilm) ja MBF (mucus binding factor). Adhesiivisuuden lisäksi MBF-proteiinin uskotaan vaikuttavan L. rhamnosus GG:n kykyyn muodostaa biofilmejä, joilla voi olla suolistoa suojaavia vaikutuksia. Tämän tutkielman tavoitteena oli kloonata L. rhamnosus GG:n MBF-proteiinia koodaava geeni L. lactis NZ9100 -kantaan ja tutkia sen vaikutusta adheesioon ja biofilmin muodostukseen. Kohdegeenin monistamiseksi L. rhamnosus GG:stä eristetystä genomisesta DNA:sta käytettiin PCR-menetelmää. Geeni liitettiin osaksi kolmea eri kloonausvektoria: pNZ8121, pNZ8122 ja pNZ8124 ja transformaatio suoritettiin elektroporaatiolla. Geenin ilmentymistä L. lactis -soluissa tutkittiin irrottamalla ja denaturoimalla solujen pintaproteiinit ja erottelemalla ne SDS-PAGE-menetelmällä. MBF-proteiinin vaikutusta adheesioon ja biofilmin muodostukseen tutkittiin 96 kaivon kuoppalevyillä. Biofilmin muodostumiskykyä selvitettiin käyttämällä hydrofiilisiä polystyreenipohjaisia kuoppalevyjä. Adheesion tutkimiseksi kuoppalevyjen kaivot oli päällystetty porsaan musiinilla. Kohdegeenin kloonaaminen L. lactis NZ9100 -kantaan onnistui plasmidilla pNZ8124, jossa oli Usp45-proteiinin signaalisekvenssi proteiinin erittämistä varten. Pintaproteiinien tutkimiseen käytetyn SDS-PAGE-menetelmän perusteella näyttäisi siltä, että kohdegeenin sisältäneet L. lactis -solut tuottivat ja erittivät MBF-proteiinia, joka todennäköisesti oli kiinnittynyt LPNTN-motiivillaan isäntäbakteerin soluseinään. MBF-proteiinin määrä solujen pinnalla oli kuitenkin erittäin pieni. Tässä tutkimuksessa MBF-proteiinia tuottavat L. lactis -solut eivät tarttuneet porsaan musiiniin eivätkä alkaneet muodostaa biofilmiä kuoppalevyillä kasvatettaessa. Koska MBF-proteiinin on osoitettu olevan adhesiivinen proteiini, johtui negatiivinen tulos adheesion osalta todennäköisesti siitä, että MBF-proteiinia tuotettiin liian vähän, jotta se olisi tehnyt solut adhesiivisiksi. Biofilmin muodostumisen osalta negatiivinen tulos johtuu myös todennäköisesti MBF-proteiinin pienestä määrästä solujen pinnalla. On myös mahdollista, ettei MBF-proteiini yksin riitä biofilmin muodostukseen, vaan bakteerisolujen on tuotettava muitakin biofilmin muodostukseen osallistuvia pintakomponentteja.
  • Miettinen, Ilkka (Helsingfors universitet, 2016)
    Multi-drug tolerance is a phenomenon, in which microorganisms normally susceptible to an antimicrobial agent are able to withstand a treatment via phenotypic alteration. The tolerance is conveyed by a microbial subpopulation that is in a non-replicative and metabolically inactive state also known as persistence. Through this kind of dormancy, the subpopulation may survive an otherwise appropriate course of antimicrobials, since the majority of the drugs target cellular division or metabolism. Upon the reduction of the surrounding antimicrobial concentration the multi-drug tolerant cells - persisters - become resuscitated thus allowing repopulation. As opposed to the more widely acknowledged challenge of antimicrobial resistance, the offspring of the specialist survivor cells are genetically identical to the susceptible majority. Persisters are especially abundant in biofilms, a microbial lifestyle characterized by aggregated microcolonies that are covered in a self-produced slimy matrix known as extracellular polymeric substance (EPS). Partly owning to this protective matrix, biofilms are inherently somewhat tolerant to antimicrobial chemotherapy. Moreover, microbes confined in a biofilm are additionally protected against the components of the host immune system. Conversely, it is assumed that persisters in planktonic, i.e. freely floating state, are easily cleared out by white blood cells. Combined, the immune evasive properties of biofilms and the remarkable multi-drug tolerance of persisters give rise to recalcitrant infections that are immensely difficult to eradicate. The described phenomenon constitutes crucially to the major healthcare challenge of chronic, treatment-resistant infections. Tuberculosis, cystic fibrosis lung disorder, bacterial endocarditis and infections related to indwelling medical devices are only a few examples of such problems. Despite the need for antimicrobials with anti-persister efficacy, no such therapeutics is available and very few are being investigated - one important factor being the lack of relevant drug discovery platforms. Therefore, the aim of this study was to develop an anti-persister assay and to carry out a pilot screening of natural product derived bioactive compounds. Based on the notion that persisters are enriched in bacterial cultures that have reached the stationary phase of growth, a persister model was designed using Staphylococcus aureus ATCC 25923 as the test strain. The bacteria were grown in liquid cultures until they reached the stationary phase and subsequent experimentation was carried out to confirm the tolerant state. After the stationary phase persister model was validated, a small pilot screening of natural products was undertaken in the hope of finding novel anti-persister activity. Mitomycin C, a cytotoxic drug with an existing anti-cancer indication was assigned as the positive control compound because of its previously established anti-persister activity. Since it is common for all of the persister-related diseases that the target microorganisms reside within a protective biofilm, an additional assay based on biofilm regrowth was designed to characterize the hit compounds on a more clinically relevant platform. The persister model culture was shown to be tolerant to conventional antibiotics. The re-induction of metabolic activity by diluting into fresh medium recovered the antimicrobial susceptibility expectedly. A total of 4 compounds were identified as anti-persister hits in the pilot screening campaign. Chromomycin A3, dehydroabietic acid, mithramycin A and oleanolic acid were all able to reduce the viable bacterial count in the stationary phase persister model more than 2 logarithmic units at 100 µM. Mithramycin A was the most potent, reducing the viability over 6 log units. The model compound mitomycin C reduced the viable counts 5.49 (± 0.96) logarithmic units. Out of the 4 hits, dehydroabietic acid was selected for the biofilm relapse assay because of its favourable biocompatibility properties. It reduced regrowth for the treated biofilms by 4 logarithmic.
  • Aho, Riikka (Helsingin yliopisto, 2019)
    Abstract This work was done at R&D Center of Kemira Oy in Espoo. Paper machines are open systems which are impossible to keep sterile. Paper machines offer favourable conditions for microbial growth. Microbiological problems in paper industry have been reported for years and expanding the production facilities and recycling of process waters have increased microbial growth. Microorganisms cause a lot of problems in paper machines and contaminatios can lead to huge economical loses. Biofilms can block filters and nozzles. Biofilms cause production breakdowns. The quality of produced paper is deteriorated, produced paper can have holes and discalourations. Sometimes the paper machine has to be stopped. Biofilms also increase the corrosion of machinery. Biocides are commonly used to control the formation of biofilms. Biocides are dosed with process waters and they are not specific to biofilm forming bacteria. Biofilms tolerate biocides better than planktonic bacteria and the biofilm problem has not been able to solve. There are a lot of different microbes found on paper making processes. Paper machines can contain viruses, bacteria, syanobacteria, molds, yeasts, algae ja protozoa. Paper machines environment and location, temperature, pH, raw materials and other additives have impact on the composition of microbe population. Kemira has a targeted-release biocide product on the market. Polymeric micelles protect biocidal active agents from degradation while it has been delivered to the targeted surfaces. Global patent has been applied for this targeted-release technology. Target of this work was to develop a cheaper version of this product having same targeted effects and better storage stability In this work the stability, efficacy against planktonic cells and against biofilm were tested. qPCR and sequencing were used to test the effect of the biocide to microbe population.
  • Pitkänen, Katriina (Helsingin yliopisto, 2022)
    Bacteria coordinate the virulence factor production and biofilm formation by activating the mechanism of intercellular communication called quorum sensing (QS). The biofilm matrix protects bacteria against variety of stress conditions, including e.g., effects of antibiotics and disinfectants. QS inhibitors (QSI) are molecules that can inhibit the QS mechanism. QQ is a QSI inhibitor that interrupts the cell-to-cell signaling already outside of the bacterial cell. Dairy propionic acid bacteria have known antimicrobial properties but their ability to produce anti-QS compounds have not yet been studied. Previous studies have shown positive results of the anti-QS activity exerted by lactobacilli, but more research is needed to uncover mechanistic details behind their QS-inhibitory effects. The aim of this study was to investigate the anti-QS and QQ activity of propionic bacteria and lactobacilli. The used method was an in-house optimized screening method based on using wild-type and mutant strains of Chromobacterium violaceum. This bioreporter produces a QS-mediated, measurable color pigment. Here, the limit of potential QSI and QQ inhibition was set to 50 % inhibition. Viability of bioreporter was analyzed by resazurin staining. Propionibacterium freudenreichii strains demonstrated both the QSI (64–71%) and QQ (52–60%) activities against the tested C. violaceum reporters. In the case of lactobacilli, Lacticaseibacillus rhamnosus GG, Limosilactobacillus mucosae 2674, Lactobacillus acidophilus 80, Lactobacillus curvatus 453 and Lactobacillus kefiri 3070 inhibited the QS-activation of the wild-type bioreporter strain by 45 to 90 %. There was a great variability in the anti-QS screening assays involving the mutant strain, which may be explained by the possible sensitivity of the compounds to the analysis conditions used or more sensitive response of the mutant strain to the tested agents. In conclusion, the obtained results show that L. acidophilus 80 produced bactericidal compounds. L. reuteri 410 did not have any effect on the QS-indicator production in wild-type bioreporter but was able to inhibit QS-induction in the mutant reporter by 72% (QQ activity). This study showed that P. freudenreichii strains as well as certain lactobacilli have an ability to produce QSI and QQ activity against some gram-negative biofilm pathogens. Pathogens have different QS mechanisms and experiments with C. violaceum strains are only one way to screen for anti-QS compounds. Therefore, validation of the obtained findings requires complementary screening assays against other bacterial bioreporters, involving both gram-negative and gram-positive biofilm bacteria.
  • Puhakka, Heikki (Helsingin yliopisto, 2019)
    The aim of this thesis was to study and develop the process hygiene of the new R1 bottling line at Altia Rajamäki alcoholic beverage plant. The products bottled on the line are mainly low- and non-alcoholic beverages that have limited amount of previously studied knowledge about the efficiency of hygienic practices of their production. In addition, the microbiological quality of the products’ raw materials was studied, and an attempt was made to identify the microbes occurring in the products and on the production line. The literature review deals with the legislation of food industry and the microbiological risks related to various types of beverages and their processing. In the experimental part of the thesis the microbiological hygiene of the bottling process was studied by collecting samples from the surfaces of the bottling line using mainly the microbiological swabbing method. The microbiological analyses of the bottled products were conducted by filtering the sample to a filter paper. Various culture mediums and antibiotics were also tested to identify the bacteria from yeasts and molds. 16S rRNA gene sequencing was used to identify the bacteria frequently occurring in the analyses. DNA isolation and PCR were conducted at the University of Helsinki and the gene sequencing was carried out by the Institute of Biotechnology. Sequence alignment was made using BLAST. The public version of the thesis lacks the confidential information which is provided only for Altia Oyj. Based on the results, the process hygiene of the R1 bottling line is sufficient in case the hygienic practices are followed. No significant microbiological growth was observed in the process hygiene samples. However, endospore producing bacteria were found in the products and these bacteria were presumed to originate from the raw materials of the products or from storage tanks and pipelines of these raw materials. Four bacterial genera, which frequently occurred in the products, were successfully identified. Nevertheless, based on the literature, it was noticed that these bacteria are not able to spoil alcoholic beverages nor to grow in the conditions of bottled products. However, some of these bacteria can substantially form biofilm.
  • Vartema, Linda (Helsingin yliopisto, 2020)
    Bacterial surface layers (S-layers) usually consist of a single protein or glycoprotein species and they form the outermost layer of many bacteria. Three S-layer protein genes have been found in Lactobacillus brevis ATCC 14869, but the expression of only two of them, slpB and slpD, has been described in planktonic conditions. Preliminary results have suggested that the third gene, slpC, may be expressed in biofilm cells. The aim of this thesis was to study the expression of S-layer protein genes slpB, slpC and slpD in the different growth modes of L. brevis ATCC 14869. Surface proteins were detached both chemically for SDS-PAGE analysis and enzymatically for surfaceome analysis by LC-MS/MS and label-free quantification (LFQ). Messenger RNA transcript levels of the S-layer protein genes were analyzed by reverse transcriptase droplet digital PCR (RT-ddPCR). The expression of all three S-layer protein genes slpB, slpC and slpD was confirmed in the study. In the surfaceome analysis, SlpD was the most abundant protein detected on the cell surface, representing more than a third of all identified proteins, whereas SlpB and SlpC were less abundant. The S-layer proteins were detected by gel electrophoresis. Transcripts of all three genes were observed; slpD transcript amounts were the highest, whereas slpB and slpC transcript amounts were lower. The S-layer proteins and their transcripts were present in equal amounts in biofilm and planktonic cells. In this study, SlpC expression is demonstrated for the first time in L. brevis ATCC 14869. The expression of slpB, slpC and slpD was at the same level in biofilm and planktonic cells.
  • Iivonen, Lasse (Helsingin yliopisto, 2017)
    Suussa elää sadoittain eri bakteerilajeja osana suun normaalia mikrobistoa. Suussa tavataan sekä aerobisiin että anaerobisiin bakteereihin kuuluvia lajeja, joista yleisimmät kuuluvat Streptococcus-, Prevotella-, Veillonella- ja Fusobacterium-sukuihin. Eräät suun bakteerilajeista aiheuttavat toisinaan infektioita. Suuhun joudutaan kirurgisten toimenpiteiden yhteydessä asentamaan erilaisia vierasesineitä, jotka suurentavat infektion kehittymisen riskiä. Muualla elimistössä vierasesineinfektioista on löydetty tiettyjä bakteerilajeja, joiden mikrobilääkehoito on osoittautunut hankalaksi eivätkä yleisimmät suun infektioissa käytetyt mikrobilääkkeet kata näitä bakteereja. Vierasesineinfektioista aiemmin tavattuja mikrobilajeja ovat muun muassa Staphylococcus aureus ja Pseudomonas aeruginosa. Tutkimuksessa selvitettiin, löytyykö suun alueen vierasesineinfektioista tyypillisiä vierasesineinfektioissa aiemmin tavattuja bakteereja vai suun normaalin mikrobiston bakteereja. Lisäksi selvitettiin, mitä mikrobilääkkeitä näiden infektioiden hoidossa on käytetty, ja onko käytetty mikrobilääke kattanut todetut mikrobit. Tutkimus oli retrospektiivinen. Tutkimuksessa mukana olleille potilaille oli tehty Helsingin yliopistollisen keskussairaalan (HYKS) Suu- ja leukasairauksien klinikassa Kirurgisessa sairaalassa leikkaussalissa toimenpide luun kiinnitysmateriaalin poisto (EFB40) 1.1.2014-30.6.2015 välisenä aikana. Potilaita oli yhteensä 86. Bakteeriviljelynäytteitä oli otettu 26 potilaalta. Suurimmasta osasta viljelynäytteitä oli löydetty suun normaaliin mikrobistoon kuuluvia bakteerilajeja. Osasta näytteistä löytyi kuitenkin vierasesineinfektioille tyypillisiä ja muita vaikeahoitoisia mikrobeja. Potilaiden saama mikrobilääkehoito osoittautui hyvin vaihtelevaksi, eivätkä käytetyt mikrobilääkkeet kaikkien potilaiden kohdalla kattaneet todettuja löydöksiä. Tutkimuksen perusteella on suositeltavaa ottaa bakteeriviljelynäyte kaikista suun vierasesineinfektioista kohdennetun ja toisaalta riittävän kattavan mikrobilääkehoidon valitsemiseksi.
  • Hassan, Ghada (Helsingfors universitet, 2016)
    The aim of this study was to synthesize antimicrobial and anti-biofilm agents using abietic (AA) and dehydroabietic acids (DHAA). Bacterial biofilms are formed when bacteria cells cluster together within a self-produced extracellular matrix. This lifestyle makes bacteria highly resilient to different environmental stresses and conventional antibiotics when compared to single-cell bacteria. Currently, there are no approved anti-biofilm agents as drugs and only a few number of compounds can selectively target biofilms and eradicate them at low concentrations. Potent drugs targeting them are needed. AA and DHAA are abietane-type diterpenoids found in the resin of conifer trees. Antibacterial effects of resin acids have been widely studied, specifically against methicillin-resistant Staphylococcus aureus strain (MRSA). Through the combination of DHAA with different amino acids, Manner et al. (2015) discovered a new class of hybrid compounds that target both planktonic and biofilm bacteria in Staphylococcus aureus. The study group also discovered two of the most potent abietane-type anti-biofilm agents reported so far in literature. This thesis followed the work of the research group by designing and synthesizing additional AA and DHAA derivatives to target bacterial biofilms. Rings A, B and C of the diterpenoid core were modified and 24 derivatives were successfully synthesized. Amino acids were attached to the compounds either before or after ring modification. Standard structural elucidation techniques were used to confirm the structure of the synthesized compounds.
  • Ahola-Iivarinen, Elina (Helsingfors universitet, 2016)
    In the literature review of this study, the focus was on biofilms that certain microbes produce, and their potential use in food industry. Biofilms consist of microbial cells and extracellular products, e.g., polysaccharides. Pullulan as an exopolysaccharide has many industrial applications and the aim of this study was to explore a new potential alpha-glucan, dextran, and especially its ability to form a stand-alone film. Pullulan and dextran were separately mixed in de-ionized water. The dynamic viscosities of dextran and pullulan solutions were determined. Film formation of dextran was not successful, not even with sorbitol as a plasticizer. The optical properties, water vapor and oxygen permeabilities and tensile strengths of pullulan films were studied. Additionally, Whatman42-filter material was coated with or immersed in dextran solution. Hence the changes in tensile strength and permeability values between a well-known material and dextran treated material could be detected. Pullulan films had low haze values (2.1–3.9%) and they were transparent to UVA-, UVB- and visible light. The tensile strength values of pullulan films were 47–53 MPa. For filter paper, the corresponding values were 10 MPa and application of dextran coating increased it to 15–19 MPa. All polysaccharide solutions exhibited Newtonian behavior and their relative viscosities were <10 mPa, 5% pullulan with viscosity around 20 mPa as an exception. Pullulan solutions had higher viscosities than dextran solutions. The air permeabilities were 10–50 ml/min for pullulan films, 10 ml/min for dextran-sorbitol film, 200 ml/min for dextran film and 200–500 ml/min for Whatman42 material. The oxygen permeability values for pullulan films were <0,1 cm3·μm m-2·d-1·kPa-1. Based on results in this study, pullulan films are impermeable to oxygen. As the films tolerated water vapor poorly, pullulan might be a potential component in packages made of composite materials, as individual packaging material in dry environment or possibly chemically modified to obtain better resistance to water vapor. Our results show that without additional modifications dextran does not form a continuous self-supporting films in these conditions.