Browsing by discipline "allmän mikrobiologi"

Staphylococcus aureus is a commensal bacterium in humans and approximately 30% of healthy people carry it as part of their microbiome, in the nasal cavity and skin, without any harm. However, it is an opportunistic pathogen that causes severe infections in immunocompromised and hospitalized patients. Typical infections caused by S. aureus are wound and skin infections, pneumonia and urinary tract infections in people with a medical implanted device such as for example a catheter. S. aureus has gained resistance to virtually all antibiotics over the years of excessive antibiotic consumption, making treatment nearly impossible in some cases. MRSA, methicillin resistant S. aureus, is a worldwide problem in hospitals and the mortality rate is still rising. One of the most common MRSA lineages is USA300, a community-acquired MRSA, which is notorious not only for its antibiotic resistance but also for its ability to form prolific biofilms. Biofilm production combined with antibiotic resistance complicates treatment of S. aureus even further. A detailed understanding the molecular mechanisms of biofilm formation might bring us closer to a cure for infections caused by MRSA biofilms. The study comprised two parts. First, characterize the phenotype of the mutants under static and dynamic conditions, test the minimal inhibitory concentrations (MIC’s) for antibiotics and verify the gene knockout by real-time RT-PCR. Second, study gene function by transduction to the parental strain USA300-UAS391 EryS and a MRSA strain TCH1516 EryS to study the gene function in a different bacterial background. The methods used were cell culturing for static and dynamic biofilm as well as growth curve, fluorescence microscopy, antibiotic susceptibility testing and real-time RT-PCR. In total seven strains were selected for characterization. The chosen seven knockouts were ΔHAD (HAD-superfamily hydrolase, subfamily IA, variant 1), non-coding region, ΔausA (non-ribosomal peptide synthetase), ΔoppA (Oligopeptide ABC transporter substrate-binding protein), ΔclfB (clumping factor B), ΔampA (cytosol aminopeptidase), and ΔpgsA (CDP-diacylglycerol--glycerol-3-phosphate 3-phosphatidyltransferase). General characterization showed a few changes in biofilm formation for the genes ΔoppA, ΔausA, ΔHAD and ΔpgsA. Especially ΔpgsA is interesting because of increased ciprofloxacin resistance. The real-time RT-PCR showed some altered gene expression patterns, but no connection to poor biofilm formation. With fluorescence microscopy the growth patterns of USA300 transposon mutant strain biofilms could be described. To verify the results of the characterization, further experimentation is needed, such as RNA sequencing and complementation. Also expanding the studies to other gene hits of the screening is recommended.

In addition to Chlamydiaceae, eight novel families have been discovered to belong to the phylum Chlamydiae. The eight families are Parachlamydiaceae, Waddliaceae, Criblamydiaceae, Parilichlamydiaceae, Rhabdochlamydiaceae, Simkaniaceae, Clavichlamydiaceae and Piscichlamydiaceae.These families are phylogenetic relatives to Chlamydiaceae, share the intracellular developmental cycle and are widely distributed in nature and are therefore referred to as environmental Chlamydiae or Chlamydia related bacteria (CRB). CRB have a broad range of potential hosts. All families except Criblamydiaceae cause disease in animals and infect for example fish, arthropods and cattle. Families Parachlamydiaceae, Waddliaceae, Rhabdochlamydiaceae and Simkaniaceae are also shown to cause respiratory disease and adverse pregnancy outcomes in human. Free-living amoebae (FLA) are natural hosts of some CRB. CRB are able to survive and replicate inside of FLA that offers protection and nutrients for CRB. It has been suggested that CRB are transported to new environments inside of FLA. CRB DNA has previously been found on human skin (Hokynar et al. 2016, Hokynar et al. 2018, Tolkki et al. 2018) and in our water distribution system. CRB distributed to our tap- and shower water systems inside of FLA (Thomas and Ashbolt 2011) could be a potential rout of transmission of CRB DNA to human skin. As the diversity and size of the CRB group is large and CRB are very laborious to grow in vitro, it is challenging to detect CRB and to study their pathogenicity. Detection of CRB in clinical and environmental samples is mainly based on PCR methods. A non species-specific PCR method targeting Chlamydiales 16S rRNA (PanChl16S), that in theory amplifies all known CRB, has successfully been used in detection, but post PCR sequencing of the amplicon is required to identify the species. Also, more specific quantitative PCRs have been designed to detect specific families or species of Chlamydiae. However, the volume of clinical specimens available is often limited and allows only few separate analyzes. Due to the challenges identified with detection of CRB, efficient multiplex PCR assays would save time and resources and would be useful tools when detecting CRB DNA. The objective of the work was to explore the possibility of applying multiplexed analyzes to a limited specimen volume effectively. One aim of this thesis was to set up two multiplex PCR assays for detection of seven different CRB and a multiplex PCR for detection of three different FLA. Another aim of the work was to analyze the possibility of CRB to be transported to human skin from our water distribution system inside of FLA. In this thesis we set up two multiplex PCR assays for detection of CRB reference strains P. acanthamoebae, C. sequanensis, S. negevensis, Protochlamydia spp., Rhabdochlamydia spp., W. chondrophila and E. lausannensis. We also set up two PCR assays for detection of three different FLA reference strains: Acanthamoeba spp., Vahlkampfiidae spp., and V. vermiformis. We succeeded in developing two real-time multiplex PCR assays for detection of CRB DNA and two real-time PCR assay for detection of FLA DNA. Variability between replicates for each PCR target was low and the detection limit (100%) for each target ranged from 50-500 control plasmid copies per PCR reaction. The R2-value for each target was ≥0.98 and the reaction efficiency for each target ranged from 82-111%. Samples collected from showerheads (n=18) and water filters (n=2) as well as skin swabs (n=27) were studied with these newly established assays and PanChl16S PCR. The results obtained with the multiplex assays developed in this study were similar to the results obtained with the PanChl16S. CRB DNA was detected in 67% of the showerhead samples, in 100% of the water filter samples and in 31% of the skin swabs. Amoebae DNA was detected in 80% of the showerhead samples. Our results confirm earlier observation that Chlamydiae DNA is frequently observed in human skin swabs and suggest that CRB could be transported to human skin from our water distribution system inside of FLA.

Proteins responsible for homologous recombination are collectively called recombinases. They also have an important role in maintaining genome integrity. Recombinases are found in all three kingdoms of life. The first identified and characterized recombinase was RecA from Escherichia coli. Recombinases exhibit ATP hydrolysis coupled DNA-binding activity and strand exchange activities during homologous recombination. Homologous recombination produces new genetic combinations for evolution and general way to describe the different steps of homologous recombination is a DSBR model. Homologous recombination occurs in eukaryotic and prokaryotic cells during meiosis crossover and horizontal gene transfer, respectively. The homologous recombination machineries are remarkably complex and synergistic and much is not known about detailed mechanisms for a majority of the species. More recently, recombinases have been used in isothermal nucleic acid amplification methods, mainly in recombinase polymerase amplification RPA and strand invasion based amplification SIBA. The aim of this study was to identify, clone, produce and analyze the functionality of novel recombinases from bacteria and viruses. The acitivity for single-stranded DNA binding and strand exchange was studied. The compatibility of the produced recombinases in strand invasion based amplification SIBA method was evaluated. Two recombinases from Enterobacteria phages T2 and RB69 were found to be functional and compatible in three SIBA assays.

Growth differentiation factor 15 (GDF15), a member of TGF-β super family is a soluble cytokine that is associated with different pathological conditions including cancer, cardiac and renal failure and obesity. Its high serum levels are linked with symptoms like cachexia/anorexia in cancer patients and can be used as a marker for these diseases. Its crucial role in weight regulation and energy homeostasis has been demonstrated by treating obese mice with GDF15, which results in weight lose along with improved glucose metabolism and increased insulin tolerance. It is now known that GDF15 exerts its metabolic effect by binding to a GDNF receptor -α-Like (GFRAL) receptor along with co-receptor RET. Interestingly, these two receptors co-localize only in the brain stem area of mice and humans indicating involvement of a neuronal circuit in GDF15 mediated effects. Despite its implications in major health disorders, little is known about the interaction of GDF15 with its receptors and how this interaction in turn modulates different cellular signalling and functions. The aim of the thesis was to study the mechanism and factors involved in endocytosis of GDF15. I employed high content imaging and flow cytometry techniques to visualize and analyse the internalization of ligand-receptor complex and investigate the role of actin, dynamin and phosphoinositide 3 kinase in the process. The results suggest that similar to the internalization of other cellular growth factors, the uptake of GDF15 is affected by disruption of the actin cytoskeleton. The role of dynamin is still unclear. I also discovered that the internalization of GDF15 was inefficient even in cells that expressed the receptor GFRAL, with large cell-to-cell variation. By following the intracellular localization of the receptor GFRAL, my results revealed that the receptor GFRAL is not efficiently exported to the plasma membrane and most of the protein is retained in the Golgi compartment of cells. This phenomenon was stronger in murine fibroblast cells, where the receptor was almost exclusively trapped in the secretory compartment, explaining why the uptake of the ligand GDF15 is so inefficient in these cells. The system developed during this project will now be used to analyse different factors involved in the uptake of GDF15 and eventually uncover the possible endocytic pathway. Moreover, the Golgi retention of the receptor opens up new questions to investigate like whether the physiological function of GDF15 is regulated by receptor export signals. This will help deciphering the complex and mysterious interaction of GDF15 with its receptor GFRAL.

The antibiotic resistance of pathogenic bacteria is becoming a major problem in treating bacterial infections and development of new antibiotics is very challenging. In traditional phage therapy the bacteriophages, viruses that infect bacteria, are being used as an optional treatment to eliminate infectious agents. Methicillin resistant Staphylococcus aureus (MRSA) is resistant to several currently used antibiotics and is one of the most common antibiotic resistant bacteria causing infections. Therefore, it is a potential target for the phage therapy. Some of the Staphylococcus aureus strains produce several different enzymes and toxins which can be harmful to patients. Products developed for phage therapy purposes need be free from the material originated from host bacteria. In this study, three different methods were tested for the purification of bacteriophages infecting S. aureus. The main goal was to produce phage lysates with purity and phage concentration suitable for therapeutic purposes using a fast and aseptic procedure upgradable for large volumes. The tested methods were ultrafiltration with filter tubes from two different manufacturers (Sartorius Vivaspin 6 ja Merck Millipore Amicon Ultra 4), polyethylene glycol (PEG) precipitation and ion exchange chromatography. Three different bacteriophage strains were used. One was isolated from a commercial Russian phage therapy product (vB_SauM_fRuSau02) and the other two from feces of pigs (vB_SauS_fPf-Sau02 and vB_SauS_fPfSau03). Host bacteria strains for the first bacteriophage were S. aureus strains TB4 and 13KP originally isolated from human infections. Two host strains for the latter two phages were MRSA strains isolated from healthy pigs. Purification of the phage lysates was evaluated by measurement of enterotoxins produced by S. aureus bacteria, measurement of free double stranded DNA (dsDNA), and by cytotoxicity test in cell cultures. All evaluation methods were commercially available tests. To determine how much of the bacteriophages were lost in the process, the phage concentrations of the lysates were determined before and after the purification and recovery rates were calculated for the viruses. After two separate ultrafiltrations, the recovery rates of the bacteriophages were mainly good, but there was a lot of variation in the results. The lowest recovery rate calculated was 5%, the highest 57%, and the mean of all the rates 24%. In this study the ion exchange chromatography was combined with ultrafiltration which was used in pre-cleaning of the lysates and changing the phages in a buffer suitable for the chromatography. The recovery rates from the ion exchange chromatography varied between 14-26% but the results may be affected by the ultrafiltration steps performed before and after, since a lot of variation was seen in ultrafiltration processes. PEG precipitation was performed for one phage lysate only in order to compare the laboriousness of the method and the rates of the recovery to the other methods used. The rate of recovery from the PEG precipitation was 9,5% which was fairly low. The purity of this lysate was not evaluated since the method was estimated to be too laborious compared to the other methods. Ultrafiltration turned out to be an efficient method in the removal of small protein molecules, such as enterotoxins from bacteriophage lysates. With two sequential ultrafiltrations 96-99% of the enterotoxins in the lysates were removed. The removal of the free dsDNA was also successful but there was variation between the phage lysates. Approximately 67-93% of the free dsDNA was removed but it is possible that some of the measured DNA originated from lysed bacteriophages as their genome also consists of dsDNA. Ion exchange chromatography produced extremely well purified phage products. The fractions had no enterotoxins left or the amount was below the detection limit of the test (<0,5-1 ng/g). Ion exchange chromatography was able to remove 96-99% of the free dsDNA of the lysates. It is possible that some of the DNA left in the lysates originated from the bacteriophages lysed during the process or in storage after that. When comparing how simple and fast the methods were, the ultrafiltration turned out to be superior. It can be used in fast production of bacteriophage products for the treatment of S. aureus infections. The purification achieved with the ultrafiltration should be adequate for a topical use of the product. When higher purity products are required, e.g. for administrating the product intravenously, ion exchange chromatography might be a safer option.

Antibioottiresistenssi yleistyy infektioita aiheuttavien bakteereiden keskuudessa. Infektioita hoitaessa joudutaan siten miettimään uusia tapoja infektioiden parantamiseksi. Yhden vaihtoehdon tarjoaa bakteriofagiterapia. Siinä infektiota sairastavalle annetaan bakteriofageja, jotka infektoivat bakteereita, lisääntyvät niissä ja tuhoavat ne. Kirjallisuudessa on runsaasti lupaavia esimerkkejä tästä terapiamuodosta. Kuitenkin bakteriofagituotteiden on oltava tarpeeksi puhtaita, erityisesti siinä tilanteessa, jos niitä annetaan suonensisäisesti. Gramnegatiivisten bakteereiden tärkeä rakenneosa on lipopolysakkaridi eli LPS, joka ihmisen verenkiertoon päästessään voi olla vaarallinen. Siksi, mikäli bakteriofagit on tuotettu gramnegatiivisissa bakteereissa, LPS:t on kyettävä poistamaan näytteistä. Tässä Pro Gradu-työssä tutkittiin viidessä eri koejärjestelyssä, kuinka erilaiset tekniikat ja niiden yhdistelmät vaikuttivat bakteriofagien ja LPS:n määrään näytteissä. Lisäksi arvioitiin SDS-PAGE-geelissä näytteiden proteiinikoostumusta. Näytteistä mitattiin myös dsDNA-pitoisuus. Tutkitut tekniikat olivat ultrafiltraatio, anioninvaihtokromatografia, oktanoliuutto ja kaupalliset endotoksiininpoistotuotteet EndoTrap- ja Pierce high endotoxin removal-pylväät. Koejärjestelyssä 1 suodatettua bakteriofagilysaattia puhdistettiin ensiksi oktanoliuutolla, sitten ultrafiltraatiolla, anioninvaihtokromatografialla ja lopuksi uudella ultrafiltraatiolla. Endotoksiinipitoisuus laski eniten kromatografiassa mutta toisen ultrafiltraation jälkeen se pysyi varsin samana. Tiitteri pieneni vain oktanoliuutossa ja kromatografiassa (jossa osittain laimenemisen takia), kun ultrafiltraatioissa muutos edeltävään tiitteriin oli varsin pieni. Lopuksi näytteissä oli keskimäärin n. 15 EU (endotoksiiniyksikköä)/10^9 pfu (plaque forming unit). Toisessa koejärjestelyssä aloitettiin ultrafiltraatiolla ja sen jälkeen näytteet puhdistettiin joko EndoTrap- tai Pierce-pylväällä. Kaikki vaiheet poistivat endotoksiineita. Ultrafiltraatio pienensi hieman tiittereitä mutta EndoTrap-pylväs ei. Pierce-pylväässä tiitterin aleneminen oli huomattavaa, jolloin EndoTrap-pylvään jälkeen näytteissä oli keskimäärin 0,0574 EU/10^9 pfu ja Pierce-pylvään jälkeen 33700 EU/10^9 pfu eli suhde oli jopa enemmän kuin lysaatissa. Kolmannessa koejärjestelyssä aloitettiin ultrafiltraatiolla, jonka jälkeen oli kromatografia ja uusi ultrafiltraatio. Ensimmäisessä ultrafiltraatiossa tiitterit pienenivät jonkin verran mutta kromatografian ja toisen ultrafiltraation jälkeen se oli varsin sama, kuin ensimmäisen ultrafiltraation jälkeen. Endotoksiinipitoisuus pieneni taas eniten kromatografiassa ja se oli varsin sama toisen ultrafiltraation jälkeen, jolloin puhdistetuissa näytteissä oli keskimäärin 57,1 EU/10^9 pfu. Neljännessä koejärjestelyssä lysaattia ultrafiltroitiin ensiksi, sen jälkeen ruiskutettiin kromatografialaitteeseen, ultrafiltroitiin uudestaan ja sen jälkeen puhdistettiin joko EndoTrap- tai Pierce-pylväällä. 1. ultrafiltraatio pienensi taas hieman tiitteriä mutta se oli varsin sama EndoTrap-pylväällä puhdistetussa näytteessä. Pierce-pylvään kanssa tiitteri pieneni taas huomattavasti. Endotoksiinipitoisuudet pienenivät taas kaikissa vaiheissa, jolloin EndoTrap-pylväällä puhdistetussa näytteissä oli keskimäärin 0,0839 EU/10^9 pfu ja Pierce-pylväällä 5480 EU/10^9 pfu. Viides koejärjestely oli muuten sama, kuin koejärjestelyn 2 Pierce-pylväs järjestely mutta korkeamman NaCl-pitoisuuden eluutiopuskurilla ja suuremmalla näytetilavuudella. Tällöin tiitteri ei pienentynyt yhtä paljon, endotoksiinipitoisuus oli lopussa vain hieman korkeampi kuin koejärjestelyssä 2 ja loppuun asti puhdistetussa näytteessä oli keskimäärin 71,21 EU/10^9 pfu. SDS-PAGE-tutkimuksessa ja dsDNA-mittauksissa näkyi, että ultrafiltraatio yksin riittää poistamaan muut bakteeriperäiset proteiinit ja dsDNA:n. Tosin koejärjestelyssä 1 käytetty oktanoli saattaa vaikuttaa dsDNA:n puhdistumiseen ultrafiltraatiossa. Töissä onnistuttiin tuottamaan näytteitä, joissa sekä riitti bakteriofageja, että endotoksiinipitoisuus oli pudonnut huomattavasti. Kun huomioidaan käytännöllisyys ja nopeus, parhaaksi tavaksi puhdistaa LPS:t valikoitui ultrafiltraatio yhdistettynä EndoTrap-pylvääseen. Johtuen bakteriofagien ja LPS:ien monimuotoisuudesta, ei toisella bakteriofagilla välttämättä päästä samoihin