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Browsing by Subject "ultrasuodatus"

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  • Horsma-Heikkinen, Jenni (2020)
    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.