Bacteria resistant to multiple antibiotics pose a number of therapeutic problems. This situation is likely to worsen in the future, as the development of new classes of antibiotics has declined sharply in recent years. Acinetobacter species are an example of antibiotic-resistant bacterial pathogens with increasing clinical significance, particularly with respect to infections in high-risk patients such as those with severe burns. The treatment options for these patients are severely limited, and they often can only be treated with highly toxic antibiotics such as colistin. It is therefore evident that there is a need to investigate alternative approaches to therapy in these cases. This thesis represents some preliminary investigations into the use of bacteriophages for the treatment of Acinetobacter infections of severe burns patients. Bacteriophages are viruses that have bacteria as their host. They cannot attack human cells and indeed they are highly specific for given species of bacteria. The aim of this thesis was to investigate the potential use of bacteriophages in these infections and their possible toxic effects on mammalian (including human skin) cells. Since bacteriophages lytic against Acinetobacter species were not available through conventional culture collections it was necessary to identify other sources. Clinical isolates of Acinetobacter species were obtained from Sussex hospitals and characterised. Several unsuccessful attempts were made to isolate corresponding bacteriophages from the environment and ultimately 10 isolates of Acinetobacter spp. and their corresponding phage were obtained from Laval University, Canada. These bacterial strains were identified and host matched to each phage using a refined screening technique and purified phage preparations were produced for those potentially therapeutic phages. The results of this study formed the basis for the selection of a small number of phage to take forward for cytotoxicity studies. Very little scientific evidence has been published on the cytotoxic potential of bacteriophage or their bacterial lysis products, although phages have been routinely used for therapeutic purposes over many years. The establishment of phages as a mainstream treatment choice for bacterial infections in the West will require stringent testing and proof that phage preparations are not harmful. Human dermal fibroblast (HDF) and keratinocytes (HDK) were isolated from human biopsies. A 3T3 mouse fibroblast cell line, HDF and HDK with 3T3 feeder layers were exposed to dilutions of purified phage. The cytotoxic impact and effect on cell proliferation was measured using a range of assays. No statistically significant difference could be detected between controls and phage with the Trypan blue method (3T3 cells). Hoechst propidium iodide stain experiments remained inconclusive (3T3 cells). Lactate dehydrogenase (LDH) and MTS tetrazolium compound reduction (MTS) results showed no evidence of statistically significant cytotoxic effect of phage on 3T3 cells, HDF or HDK. Some data suggested phage may have some beneficial effect on cell survival of HDK and proliferation of HDF and 3T3 cells compared to controls. Endotoxin levels present in phage lysates and purified phage preparations were compared to those found in bacterial suspensions subjected to lysis by other methods including autoclaving, bead beating, sonication and high concentrations of antibiotics. There was no evidence to suggest that bacteria lysed by phage liberated significantly more endotoxins than the other methods of cell disruption. A variety of endotoxin preparations and phage lysates were incubated with HDF and cytotoxicity together with cell proliferation were measured using LDH and MTS assays. Changes in interleukin levels of the same samples were monitored, measuring IL-1β, IL-6, IL-8 and TNF-α levels. IL-1β or TNF-α could not be detected in any samples. Highly concentrated phage gave rise to significantly higher IL-6 outputs than any other samples. Ten-fold dilutions of the same phage preparation were not statistically different to any other samples, including controls. This suggested components introduced during the phage purification process, rather than the phage itself may have contributed to higher IL-6 readings. Purified and crude phage lysate gave rise to significantly higher IL-8 outputs than all other samples. Colony formation assays utilising a V79 hamster cell line were used to indicate the cytotoxicity of purified phage in different diluents and provided comparison with endotoxin containing preparations used in other experiments. There was no statistically significant difference in terms of colony numbers or colony size (where measured) between phage samples and controls. This thesis has therefore demonstrated that because of the lack of cytotoxic effect of phage on mammalian cells in culture, there is potential for therapeutic applications of bacteriophage.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:500775 |
| Date | January 2009 |
| Creators | Henein, Alexandra Elisabeth |
| Publisher | University of Brighton |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://research.brighton.ac.uk/en/studentTheses/9726f331-e5dc-4b67-a282-44cac1fe978f |
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