Browsing by Subject "biofilm"

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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.
  • Vanic, Zeljka; Rukavina, Zora; Manner, Suvi; Fallarero, Adyary; Uzelac, Lidija; Kralj, Marijeta; Klaric, Daniela Amidzic; Bogdanov, Anita; Raffai, Timea; Virok, Dezso Peter; Filipovic-Grcic, Jelena; Skalko-Basnet, Natasa (2019)
    Background: Efficient localized cervicovaginal antibacterial therapy, enabling the delivery of antibiotic to the site of action at lower doses while escaping systemic drug effects and reducing the risk of developing microbial resistance, is attracting considerable attention. Liposomes have been shown to allow sustained drug release into vaginal mucosa and improve delivery of antibiotics to bacterial cells and biofilms Azithromycin (AZI), a potent broad-spectrum macrolide antibiotic, has not yet been investigated for localized therapy of cervicovaginal infections, although it is administered orally for the treatment of sexually transmitted diseases. Encapsulation of AZI in liposomes could improve its solubility, antibacterial activity, and allow the prolonged drug release in the cervicovaginal tissue, while avoiding systemic side effects. Purpose: The objective of this study was to develop AZI-liposomes and explore their potentials for treating cervicovaginal infections. Methods: AZI-liposomes that differed in bilayer elasticity/rigidity and surface charge were prepared and evaluated under simulated cervicovaginal conditions to yield optimized liposomes, which were assessed for antibacterial activity against several planktonic and biofilm-forming Escherichia coli strains and intracellular Chlamydia trachomatis, ex vivo AZI vaginal deposition/penetration, and in vitro cytotoxicity toward cervical cells. Results: Negatively charged liposomes with rigid bilayers (CL-3), propylene glycol liposomes (PGL-2) and deformable propylene glycol liposomes (DPGL-2) were efficient against planktonic E. coli ATCC 700928 and K-12. CL-3 was superior for preventing the formation of E. coli ATCC 700928 and K-12 biofilms, with IC50 values (concentrations that inhibit biofilm viability by 50%) up to 8-fold lower than those of the control (free AZI). DPGL-2 was the most promising for eradication of already formed E. coli biofilms and for treating C. trachomatis infections. All AZI-liposomes were biocompatible with cervical cells and improved localization of the drug inside vaginal tissue compared with the control. Conclusion: The performed studies confirm the potentials of AZI-liposomes for localized cervicovaginal therapy.
  • Reigada, Inés; Guarch-Pérez, Clara M.; Patel, Jayendra; Riool, Martijn; Savijoki, Kirsi; Yli-Kauhaluoma, Jari; Zaat, Sebastian A. J.; Fallarero, Adyary (2020)
    Nosocomial diseases represent a huge health and economic burden. A significant portion is associated with the use of medical devices, with 80% of these infections being caused by a bacterial biofilm. The insertion of a foreign material usually elicits inflammation, which can result in hampered antimicrobial capacity of the host immunity due to the effort of immune cells being directed to degrade the material. The ineffective clearance by immune cells is a perfect opportunity for bacteria to attach and form a biofilm. In this study, we analyzed the antibiofilm capacity of three naturally derived biofilm inhibitors when combined with immune cells in order to assess their applicability in implantable titanium devices and low-density polyethylene (LDPE) endotracheal tubes. To this end, we used a system based on the coculture of HL-60 cells differentiated into polymorphonuclear leukocytes (PMNs) and Staphylococcus aureus (laboratory and clinical strains) on titanium, as well as LDPE surfaces. Out of the three inhibitors, the one coded DHA1 showed the highest potential to be incorporated into implantable devices, as it displayed a combined activity with the immune cells, preventing bacterial attachment on the titanium and LDPE. The other two inhibitors seemed to also be good candidates for incorporation into LDPE endotracheal tubes.
  • Castro, H.; Jaakkonen, A.; Hakakorpi, A.; Hakkinen, M.; Isidro, J.; Korkeala, H.; Lindström, M.; Hallanvuo, S. (2019)
    Frontiers in Microbiology Vol. 14, No. 10, p. 1049.
    Packaged raw milk contaminated with Yersinia pseudotuberculosis mediated a large yersiniosis outbreak in southern Finland in 2014. The outbreak was traced back to a single dairy farm in southern Finland. Here we explore risk factors leading to the outbreak through epidemiologic investigation of the outbreak farm and through genomic and phenotypic characterization of the farm’s outbreak and non-outbreak associated Y. pseudotuberculosis strains. We show that the outbreak strain persisted on the farm throughout the 7-month study, whereas the non-outbreak strains occurred sporadically. Phylogenomic analysis illustrated that the outbreak strain was related to previously published genomes of wild animal isolates from Finland, implying that wild animals were a potential source of the outbreak strain to the farm. We observed allelic differences between the farm’s outbreak and non-outbreak strains in several genes associated with virulence, stress response and biofilm formation, and found that the outbreak strain formed biofilm in vitro and maintained better growth fitness during cold stress than the non-outbreak strains. Finally, we demonstrate the rapid growth of the outbreak strain in packaged raw milk during refrigerated storage. This study provides insight of the risk factors leading to the Y. pseudotuberculosis outbreak, highlights the importance of pest control to avoid the spread of pathogens from wild to domestic animals, and demonstrates that the cold chain is insufficient as the sole risk management strategy to control Y. pseudotuberculosis risk associated with raw drinking milk.
  • Castro, Hanna; Jaakkonen, Anniina; Hakakorpi, Anna; Hakkinen, Marjaana; Isidro, Joana; Korkeala, Hannu; Lindström, Miia; Hallanvuo, Saija (2019)
    Packaged raw milk contaminated with Yersinia pseudotuberculosis mediated a large yersiniosis outbreak in southern Finland in 2014. The outbreak was traced back to a single dairy farm in southern Finland. Here we explore risk factors leading to the outbreak through epidemiologic investigation of the outbreak farm and through genomic and phenotypic characterization of the farm's outbreak and non-outbreak associated Y. pseudotuberculosis strains. We show that the outbreak strain persisted on the farm throughout the 7-month study, whereas the non-outbreak strains occurred sporadically. Phylogenomic analysis illustrated that the outbreak strain was related to previously published genomes of wild animal isolates from Finland, implying that wild animals were a potential source of the outbreak strain to the farm. We observed allelic differences between the farm's outbreak and non-outbreak strains in several genes associated with virulence, stress response and biofilm formation, and found that the outbreak strain formed biofilm in vitro and maintained better growth fitness during cold stress than the non-outbreak strains. Finally, we demonstrate the rapid growth of the outbreak strain in packaged raw milk during refrigerated storage. This study provides insight of the risk factors leading to the Y. pseudotuberculosis outbreak, highlights the importance of pest control to avoid the spread of pathogens from wild to domestic animals, and demonstrates that the cold chain is insufficient as the sole risk management strategy to control Y. pseudotuberculosis risk associated with raw drinking milk.
  • Savijoki, Kirsi; Nyman, Tuula A.; Kainulainen, Veera; Miettinen, Ilkka; Siljamäki, Pia; Fallarero, Adyary; Sandholm, Jouko; Satokari, Reetta; Varmanen, Pekka (2019)
    Bacterial biofilms have clear implications in disease and in food applications involving probiotics. Here, we show that switching the carbohydrate source from glucose to fructose increased the biofilm formation and the total surface-antigenicity of a well-known probiotic, Lactobacillus rhamnosus GG. Surfaceomes (all cell surface-associated proteins) of GG cells grown with glucose and fructose in planktonic and biofilm cultures were identified and compared, which indicated carbohydrate source-dependent variations, especially during biofilm growth. The most distinctive differences under these conditions were detected with several surface adhesins (e.g., MBF, SpaC pilus protein and penicillin-binding proteins), enzymes (glycoside hydrolases, PrsA, PrtP, PrtR, and HtrA) and moonlighting proteins (glycolytic, transcription/translation and stress-associated proteins, r-proteins, tRNA synthetases, Clp family proteins, PepC, PepN, and PepA). The abundance of several known adhesins and candidate moonlighters, including enzymes acting on casein-derived peptides (ClpP, PepC, and PepN), increased in the biofilm cells grown on fructose, from which the surface-associated aminopeptidase activity mediated by PepC and PepN was further confirmed by an enzymatic assay. The mucus binding factor (MBF) was found most abundant in fructose grown biofilm cells whereas SpaC adhesin was identified specifically from planktonic cells growing on fructose. An additional indirect ELISA indicated both growth mode- and carbohydrate-dependent differences in abundance of SpaC, whereas the overall adherence of GG assessed with porcine mucus indicated that the carbon source and the growth mode affected mucus adhesion. The adherence of GG cells to mucus was almost completely inhibited by anti-SpaC antibodies regardless of growth mode and/or carbohydrate source, indicating the key role of the SpaCBA pilus in adherence under the tested conditions. Altogether, our results suggest that carbon source and growth mode coordinate mechanisms shaping the proteinaceous composition of GG cell surface, which potentially contributes to resistance, nutrient acquisition and cell-cell interactions under different conditions. In conclusion, the present study shows that different growth regimes and conditions can have a profound impact on the adherent and antigenic features of GG, thereby providing new information on how to gain additional benefits from this probiotic.
  • Nowak, J.; Visnovsky, S. B.; Cruz, C. D.; Fletcher, G. C.; van Vliet, A. H. M.; Hedderley, D.; Butler, R.; Flint, S.; Palmer, J.; Pitman, A. R. (2021)
    Aims To understand the genetics involved in surface attachment and biofilm formation ofListeria monocytogenes. Methods and Results Anin vitroscreen of a Himar1 transposon library ofL. monocytogenesstrain 15G01 identified three transposants that produced significantly different biofilm levels when compared to the wild-type strain; two mutants exhibited enhanced biofilm formation and one produced less biofilm biomass than the wild-type. The mutant 15G01mprF::Himar1, which had a transposon insertion in themprFgene, was selected for further analysis. The mutant produced a more densely populated biofilm on solid surfaces such as stainless steel and polystyrene, as determined using scanning electron and light microscopy. The 15G01mprF::Himar1 mutant remained viable in biofilms, but showed an increase in sensitivity to the cationic antimicrobial gallidermin. The mutant also displayed reduced invasiveness in CaCo-2 intestinal cells, suggesting virulence properties are compromised by the inactivation ofmprF. Conclusions Biofilm formation and gallidermin resistance ofL. monocytogenesis influenced bymprF, but this trait is associated with a compromise in invasiveness. Significance and Impact of the Study The presence of pathogenic microorganisms in the food processing environment can cause a significant problem, especially when these microorganisms are established as biofilms. This study shows that the inactivation of themprFgene results in enhanced biofilm formation and abiotic surface attachment ofL. monocytogenes.
  • 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.
  • Toivanen, Laura (Helsingfors universitet, 2009)
    Listeria monocytogenes is an important food-borne pathogen. It is a gram-positive bacterium, which multiplies in both aerobic and anaerobic conditions at a wide temperature area. It can grow in vacuum and modified atmosphere packages in refrigerated temperatures causing food hygienic risk. Listeria monocytogenes can cause life-threatening infection particularly in individuals who are immunocompromised, pregnant and elderly. The disease is divided into invasive and non-invasive form. The disease manifests typically with septicaemia, meningitis and gastroenteritis in non-invasive form. L. monocytogenes exists widely in the environment such as soil and water. It can find its way from the environment to the food processing plants and cause so called persistent contamination in the plant. The persistent contamination is a sum of many factors. Bacterial adhesion, acid and heat tolerance, the failure of disinfection, the adaptation of bacteria to sanitizing agents, complex processing machines and the existence of compartmentalization has its own role. Listeria monocytogenes strains can be divided into persistent and non-persistent strains. The persistent strains adhere to stainless steel surfaces faster than non-persistent strains. After the adhesion bacteria produce exopolysaccarides around themselves creating a biofilm. There are several methods to investigate biofilms. These methods can be divided into direct and indirect methods. In direct methods the biofilm is observed directly with microscope. The indirect methods are often colorimetric in other words the adherent bacteria are stained and the amount of colour is measured to estimate the amount of biofilm. The aim of the study was to set up a microtiter plate method to examine the adhesion of L. monocytogenes. At the same time the adhesion of persistent and non-persistent L. monocytogenes strains was compared. There were 20 L. monocytogenes strains that were isolated from food processing plants. The test was repeated 2 or 3 times. In the method which was used in this study the strain was incubated in the suspension in the microtiter plate. The adhered cells were stained and then the colour was dissolved. The optical density of the suspension was measured. The higher the optical density of the suspension was, the more there were adhered bacteria. Part of the studied strains had statistically significant difference in the adhesion. However most of the strains did not have a significant difference in adhesion. There was not statistically significant difference in the adhesion of persistent and non-persistent strains. The microtiter plate method proved to be a practical method to examine the biofilm of L. monocytogenes but its repeatability should still be improved. Differences in the adhesion of strains were observed with the method. Many strains can be examined at the same time with the method. The method is also easily modified to different research conditions.
  • Teittinen, Anette; Wang, Jianjun; Stromgard, Simon; Soininen, Janne (2017)
    Aim: Elevational biodiversity patterns are understudied in high-latitude aquatic systems, even though these systems are important for detecting very early impacts of climatic changes on Earth. The aim of this study was to examine the elevational trends in species richness and local contribution to beta diversity (LCBD) of three biofilm microbial groups in freshwater ponds and to identify the key mechanisms underlying these patterns. Location: One hundred and forty-six ponds in subarctic Finland and Norway distributed across the tree line along an elevational gradient of 10-1,038 m a.s.l., spanning from forested landscape to barren boulder fields. Time period: July-August 2015. Major taxa studied: Diatoms, cyanobacteria and non-cyanobacteria. Methods: Generalized linear models were used to identify the most important pond variables explaining richness and LCBD. Structural equation models were used to explore the direct and indirect effects of multiscale drivers on richness and LCBD. Results: Diatom and cyanobacteria richness showed unimodal elevational patterns, whereas non-cyanobacteria richness decreased with increasing elevation. The LCBD-elevation relationship was U-shaped for all three microbial groups. Diatom and cyanobacteria richness and LCBD were best explained by local pond variables, especially by pH. Non-cyanobacteria richness and LCBD were related to pond variables, elevation as a proxy for climatic conditions, and normalized difference vegetation index as a proxy for terrestrial productivity. Main conclusions: Aquatic autotrophs were primarily controlled by environmental filtering, whereas heterotrophic bacteria were also affected by terrestrial productivity and elevation. All studied aspects of microbial diversity were directly or indirectly linked to elevation; therefore, climatic changes may greatly alter aquatic microbial assemblages.
  • Virtanen, Maria (Helsingfors universitet, 2017)
    Listeria monocytogenes on yksi yleisimmistä Suomessa esiintyvistä ruokamyrkytyspatogeeneistä. Pastöroimattomat maitotuotteet ovat merkittävä riskielintarvikeryhmä. Raakamaitoon L. monocytogenes voi päätyä esimerkiksi utareen pinnalta huonon lypsyhygienian seurauksena, mutta tartuntareittinä voi myös toimia lypsylaitteistoon tai maitotankkiin muodostuneet biofilmit. Biofilmin avulla bakteerit kiinnittyvät tiukasti alustaansa ja samalla ne suojautuvat mekaanisilta ja kemiallisilta puhdistustoimenpiteiltä. Biofilmit ovat usein erittäin vaikeita saneerata elintarviketuotantolinjoista. Tavoitteenamme oli kartoittaa maidon alkutuotannosta eristettyjen L. monocytogenes –kantojen kiinnittymistä ja biofilminmuodostusta kuoppalevyillä. Valitsimme yhteensä 79 L. monocytogenes -isolaattia (53 eri genotyyppiä), jotka olivat peräisin neljän lypsykarjatilan maidosta, lypsylaitteiston suodattimista ja navettaympäristöstä. Lisäksi tutkittiin referenssikannat ATCC 19115 ja EGD-e. Isolaateille tehtiin kiinnittymiskoe, jossa niitä kasvatettiin kuoppalevyllä vähäravinteisessa elatusaineessa 30 °C:ssa 24 ja 48 tunnin ajan. Tämän jälkeen kuoppalevy pestiin puhtaaksi irtonaisista bakteerisoluista tislatulla vedellä ja pohjaan kiinnittyneet bakteerisolut värjättiin 0,1 % kristallivioletilla. Väri liuotettiin 95 % etanoliin ja kuoppalevyjen absorbanssi mitattiin spektrofotometrin avulla. Kullekin isolaatiolle tehtiin kolme kiinnittymiskoetta ja jokaisessa kokeessa oli mukana kolme teknistä toistoa. Negatiivisena kontrollina käytettiin elatusainetta, johon ei lisätty bakteeria. L. monocytogenes -isolaattien absorbanssimittausten keskiarvo oli 24 tunnin kasvatuksen jälkeen 0,1915 (0,1468 – 0,2477) ja 48 tunnin kasvatuksen jälkeen 0,2266 (0,1694 – 0,3661). Referenssikannan ATCC 19115 mittaustulokset eivät merkitsevästi eronneet muiden isolaattien tuloksista, mutta EGD-e:n absorbanssi oli 48 tunnin mittauksessa keskimäärin suurempi kuin muiden isolaattien. Sivelynäytteistä eristettyjen isolaattien mittaustulokset olivat 48 tunnin jälkeen merkitsevästi suurempia (p<0,01) kuin maitonäytteistä eristettyjen isolaattien. Sivelynäytteistä eristettyjen isolaattien kasvuympäristöt ovat siis sellaisia, joissa runsaasta biofilminmuodostuksesta on hyötyä bakteereille. Maitonäytteiden isolaatit eivät ole kasvuympäristössään hyötyneet tästä ominaisuudesta, ja siksi niiden biofilminmuodostus on heikompaa. Tulosten valossa on perusteltua, että L. monocytogenes -kantojen kasvuympäristön ja biofilminmuodostuskyvyn mahdollista yhteyttä selvittäisiin tarkemmin.
  • 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.
  • Pérez Tanoira, Ramón; Aarnisalo, Antti A.; Eklund, Kari; Han, Xia; Soininen, Antti; Tiainen, Veli-Matti; Esteban, Jaime; Kinnari, Teemu J. (2017)
    Background: Cells of tissues and biofilm forming bacteria compete for the living space on the surface of an implant. We hypothesized the incubation of the implant (titanium, polydimethylsiloxane, and polystyrene surface) with human cells before implantation as a strategy to prevent bacterial adhesion and biofilm formation. Methods: After 24 hours of incubation with human osteogenic sarcoma SaOS-2 cells (1x10(5) cells/mL), the materials were incubated for 4.5 hours or two days with Staphylococcus aureus in serial 1:10 dilutions of 10(8) colony-forming units/mL. The bacterial adherence and biofilm biomass on materials pre-incubated with SaOS-2 cells were compared with our previous results on materials incubated only with bacteria or in simultaneous co-culture of SaOS-2 cells and S. aureus. Fluorescent microscopy and crystal violet stain were used. The number of viable SaOS-2 and bacterial cells present was tested using colorimetric methods (MTT, LDH) and drop plate method, respectively. Results: The pre-treatment with human cells was associated with a reduction of bacterial colonization of the biomaterial at 4.5 hours and 48 hours compared with the non-pre-treated materials. The presence of bacteria decreased the number of viable human cells on all materials. (Supplementary Fig. 1; see online supplementary materials at www.liebertpub.com/sur). Conclusions: These results suggest that the pre-operative incubation of prostheses with host cells could prevent infection of biomaterials.
  • 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.
  • Gilbert-Girard, Shella; Savijoki, Kirsi; Yli-Kauhaluoma, Jari; Fallarero, Adyary (2020)
    In an effort to find new repurposed antibacterial compounds, we performed the screening of an FDA-approved compounds library against Staphylococcus aureus American Type Culture Collection (ATCC) 25923. Compounds were evaluated for their capacity to prevent both planktonic growth and biofilm formation as well as to disrupt pre-formed biofilms. One of the identified initial hits was fingolimod (FTY720), an immunomodulator approved for the treatment of multiple sclerosis, which was then selected for follow-up studies. Fingolimod displayed a potent activity against S. aureus and S. epidermidis with a minimum inhibitory concentration (MIC) within the range of 12-15 mu M at which concentration killing of all the bacteria was confirmed. A time-kill kinetic study revealed that fingolimod started to drastically reduce the viable bacterial count within two hours and we showed that no resistance developed against this compound for up to 20 days. Fingolimod also displayed a high activity against Acinetobacter baumannii (MIC 25 mu M) as well as a modest activity against Escherichia coli and Pseudomonas aeruginosa. In addition, fingolimod inhibited quorum sensing in Chromobacterium violaceum and might therefore target this signaling pathway in certain Gram-negative bacteria. In conclusion, we present the identification of fingolimod from a compound library and its evaluation as a potential repurposed antibacterial compound.
  • Reigada, Inés; Pérez-Tanoira, Ramon; Patel, Jayendra; Savijoki, Kirsi; Yli-Kauhaluoma, Jari; Kinnari, Teemu; Fallarero, Adyary (2020)
    Biofilm-mediated infection is a major cause of bone prosthesis failure. The lack of molecules able to act in biofilms has driven research aimed at identifying new anti-biofilm agents via chemical screens. However, to be able to accommodate a large number of compounds, the testing conditions of these screenings end up being typically far from the clinical scenario. In this study, we assess the potential applicability of three previously discovered anti-biofilm compounds to be part of implanted medical devices by testing them on in vitro systems that more closely resemble the clinical scenario. To that end, we used a competition model based on the co-culture of SaOS-2 mammalian cells and Staphylococcus aureus (collection and clinical strains) on a titanium surface, as well as titanium pre-conditioned with high serum protein concentration. Additionally, we studied whether these compounds enhance the previously proven protective effect of pre-incubating titanium with SaOS-2 cells. Out of the three, DHA1 was the one with the highest potential, showing a preventive effect on bacterial adherence in all tested conditions, making it the most promising agent for incorporation into bone implants. This study emphasizes and demonstrates the importance of using meaningful experimental models, where potential antimicrobials ought to be tested for the protection of biomaterials in translational applications.
  • 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.
  • Hjerppe, Jenni; Rodas, Sampo; Korvala, Johanna; Pesonen, Paula; Kaisanlahti, Anna; Özcan, Mutlu; Suojanen, Juho; Reunanen, Justus (2021)
    The aim of this study was to evaluate the surface roughness of fixed prosthodontic materials after polishing or roughening with a stainless steel curette or ultrasonic scaler and to examine the effect of these on Streptococcus mutans adhesion and biofilm accumulation. Thirty specimens (10 x 10 x 3 mm(3)) of zirconia (Zr), pressed lithium disilicate (LDS-Press), milled lithium disilicate glazed (LDS-Glaze), titanium grade V (Ti) and cobalt-chromium (CoCr) were divided into three groups (n = 10) according to surface treatment: polished (C), roughened with stainless steel curette (SC), roughened with ultrasonic scaler (US). Surface roughness values (Sa, Sq) were measured with a spinning disc confocal microscope, and contact angles and surface free energy (SFE) were measured with a contact angle meter. The specimens were covered with sterilized human saliva and immersed into Streptococcus mutans suspensions for bacterial adhesion. The biofilm was allowed to form for 24 h. Sa values were in the range of 0.008-0.139 mu m depending on the material and surface treatment. Curette and ultrasonic scaling increased the surface roughness in LDS-Glaze (p <0.05), Ti (p <0.01) and CoCr (p <0.001), however, surface roughness did not affect bacterial adhesion. Zr C and US had a higher bacterial adhesion percentage compared to LDS-Glaze C and US (p = 0.03). There were no differences between study materials in terms of biofilm accumulation.
  • 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.
  • Hiltunen, Anna (Helsingfors universitet, 2015)
    Periodontitis is a globally significant disease which destroys the attachment tissues and alveolar bone of teeth, eventually leading to tooth loss. Biofilms, the most intrinsic lifestyle of bacteria, play a pivotal role in the occurrence of this disease. Periodontal biofilms can be treated with topically administered chlorhexidine and strain-specific antibiotics. However, these antimicrobials do not offer solutions for periodontal attachment tissue and alveolar bone loss. Some therapeutical alternatives for these conventional treatments have been investigated. In numerous studies, periodontitis is treated successfully (increased attachment and/or alveolar bone levels) with topically and systemically administered bisphosphonates. Furthermore, a topically administered bone graft substitute (bioactive glass) has shown to improve periodontal parameters. In addition, bioactive glass has known antimicrobial and anti-biofilm effects. Moreover, a few bisphosphonates have shown antimicrobial activity against some bacterial strains. Hence, both bisphosphonates and bioactive glass are promising materials for dental applications, also raising interest in their combination. Indeed, it could be hypothesized that this combination product could simultaneously treat both the underlying cause (biofilms) and consequences (alveolar bone and attachment tissue loss) of periodontitis. Open research questions remain for the combination product. Is the anti-biofilm effect enhanced when bioactive glass is combined with bisphosphonates? Moreover, do bisphosphonates have intrinsic anti-biofilm properties? These questions are investigated in this thesis, which is a continuation of a recent doctoral dissertation. In this dissertation, a clodronate-bioactive glass combination product was studied by applying it into periodontal pockets. However, anti-biofilm effects were not assessed. In this thesis, a close examination is carried out on these effects, utilizing relevant biofilm models. The aims of this work were to investigate anti-biofilm effects of bisphosphonates (alendronate, clodronate, etidronate, risedronate and zoledronate) (i) alone, administered as solutions and (ii) combined with bioactive glass S53P4. Optimization of the used assay methods (96-well plate assay, Static Biofilm method) was performed. The anti-biofilm effects of bisphosphonate solutions were screened in the 96-well plate assay using a model organism Staphylococcus aureus Newman and a periodontopathogen Aggregatibacter actinomycetemcomitans ATCC 33384. After this, experiments were conducted with bisphosphonate-bioactive glass combinations. The experiments were performed with a single-specie (A. actinomycetemcomitans ATCC 33384) dental biofilm model based on the Static Biofilm method. The model mimics conditions encountered by periodontal bacteria in the oral cavity. In this part, bisphosphonate particle sizes were measured to determine a suitable control material. In addition to bacterial experiments, pH measurements were carried out to gain an insight to a possible anti-biofilm mechanism. Bisphosphonates administered as stand-alone compounds did not have an effect on either the Gram-positive model organism (S. aureus Newman) or the Gram-negative periodontopathogen (A. actinomycetemcomitans ATCC 33384). In contrast, most combinations of bisphosphonate-bioactive glass revealed a statistically significant increase in anti-biofilm effect on A. actinomycetemcomitans ATCC 33384. The combinations were compared to a control composed of inert glass and bioactive glass. In these assay conditions, the risedronate-bioactive glass-combination was the most effective (significant statistical difference, p < 0.05). Other combinations also reduced biofilms (significant statistical differences, p < 0.05), with the exception of clodronate-bioactive glass, where the change was not statistically significant. The most effective combinations (containing risedronate and etidronate) subjected the biofilms to a period of low pH. Conversely, the least effective combination (clodronate-bioactive glass) rapidly became alkaline, similarly to the control compounds (inert glass and bioactive glass). Thus, anti-biofilm efficacy could be connected to lowered pH. This observation is supported by recent literature where A. actinomycetemcomitans has been deemed highly sensitive to acidity. However, establishing the anti-biofilm rank order of bisphosphonate-bioactive glass combinations would benefit from experiments with equal bisphosphonate particle sizes.