Human disease due to food-borne pathogens remains a medical problem worldwide. Campylobacter jejuni is the most common cause of bacterial food-borne diarrhoeal disease, but the mechanisms by which it causes disease remain unclear. This bacterium is also the most common antecedent to the peripheral neuropathies Guillain-Barre syndrome (GBS) and Miller-Fisher syndrome (MFS). The main aim of this project was to compare cytotoxicity of isolates of C. jejuni towards mammalian cells in culture, to purify any cytotoxic activity and to compare strains by plasmid and proteomic analysis. Using the API Campy identification kit, 37 of the 39 strains were identified as C. jejuni jejuni serotype 1 or 2 with identification (ID) scores > 98.3%; and two strains were identified as C. jejuni jejuni 1 with ID scores of 88.3%. GOT (Gamma Glutamyl Transferase) test was the most important test to differentiate between C. jejuni jejuni 1 and 2 which showed negative and positive results, respectively. The cytotoxic activity against Vero and Caco-2 cells of some of the typed strains of C. jejuni was tested. Cytotoxic activity was clearly demonstrated using Vero and Caco-2 cells and these activities peaked after incubation of the cell culture with sample for 16h. Comparison of wild-type strains 81-176 and 11168 with mutants of these strains lacking cytolethal distending toxin (CDT) or putative haemolysin or phospholipase toxins indicated that the toxicity observed was not due to any of these proteins, as cytotoxicity was unaffected by their absence. Some isolates (e.g. C0L12) showed low cytotoxicity while others, especially C. jejuni 81-176, consistently produced high cytotoxin activity which was heat-labile and lethal to tissue culture cells. Cytotoxicity comparisons of inner and outer membrane preparations with a cytoplasmic fraction of C. jejuni strain 81-176 and C0L12 indicated that greatest activity was found in the inner membrane fraction. Proteins in the membrane preparation could not be easily fractionated and cell-free extracts of strains with low (COL12) and high (81-176) cytotoxic effects were chosen for further purification and characterisation studies. Although some differences were detected after DEAE- Sepharose fractionation and two-dimensional electrophoretic (2-DE) separation of proteins, no individual proteins could be clearly related to cytotoxicity. However, proteomic analysis did reveal some interesting properties of the C. jejuni strains. Of the proteins characterised by mass spectrometry after 2-DE, oxidoreductases were the most frequently identified in the virulent strain 81-176, but not in the type strain 11168. There was no apparent correlation between toxin production and clinical symptoms in patients from whom the strains were isolated. The study indicated that not all C. jejuni strains tested produced cytotoxin (s), but that cytotoxin producers seemed to be predominantly human strains. The presence of virB11 and tetO genes carried by plasmids was examined by PCR in the 39 C. jejuni isolates together with strains 81-176 and NCTC 11168. Detection rates for the virB11 and tetO genes in the clinical isolates were 28.2% and 12.82%, respectively.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:754301 |
| Date | January 2006 |
| Creators | Mohammadzadeh, Abdolmajid |
| Publisher | University of Glasgow |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://theses.gla.ac.uk/9057/ |
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