Enterohaemorrhagic E. coli (EHEC) are important gastrointestinal pathogens of humans. E. coli serotype O157:H7 is the EHEC most commonly associated with human illness. E. coli O157:H7 is carried asymptomatically by cattle which form an important reservoir for the bacterium. E. coli O157:H7 has been found to colonise at the terminal rectum of cattle in preference to other sites in the bovine gastrointestinal tract. The first objective of this work was to characterise the roles of bacterial secreted components responsible for key functions in the modulation of host defences against EHEC. Data presented here reaffirms the role of flagellin in the elicitation of a proinflammatory response in a cultured human epithelial cell line; however, the response of a bovine epithelial cell line to bacterial secreted products was not affected by the presence or absence of flagellin. A role in the modulation of the host response for the StcE protease was also investigated: although its role in interaction with the bovine host was not established, bovine secretory antibodies to StcE were detected in rectal mucosal scrapings from an E. coli O157:H7-challenged calf, suggesting that StcE is expressed and recognised in vivo. The second key objective was to isolate E. coli from the bovine intestinal tract in order to define the colonisation patterns of E. coli within the bovine intestinal tract and relate this to bacterial genotype and to provide bovine E. coli isolates to test for inhibitory activity against E. coli O157:H7 which may yield bacteria with potential as probiotic agents with a view to reducing the prevalence of EHEC in cattle. Genotypic analysis of bovine resident E. coli confirmed that these strains carry a variety of virulence factor-encoding genes; however, certain dominant genotypes were identified and the genomic structure of representative isolates was predicted by genomic microarray. EHEC-related genotypes were found to be positively associated with colonisation at the rectum, whereas non-EHEC genotypes were found to colonise multiple intestinal sites without showing any apparent site-specificity. The third and final objective of this analysis was to carry out genotypic analysis of Scottish EHEC strains in order to predict whether increased incidence of EHEC infection in Scotland may be related to the presence of EHEC strains carrying altered complement of virulence factor-encoding genes. The analysis of EHEC isolated in Scotland revealed that these strains exhibit a genomic profile which is largely typical of EHEC isolated elsewhere, although there were certain differences in the carriage of a certain genomic elements. The results presented here support the proposal that bacteriophages are the key mediators of genetic variability among E. coli isolates.

The growing problem of insecticide resistance is jeopardising current pest control strategies and current insecticide development pipelines are failing to provide new alternatives quickly enough. Metabolomics offers a potential solution to the bottleneck in insecticide target discovery. As a proof of concept, metabolomics data for permethrin exposed Drosophila melanogaster was analysed and interpreted. Changes in the metabolism of amino acids, glycogen, glycolysis, energy, nitrogen, NAD+, purine, pyrimidine, lipids and carnitine were observed along with markers for acidosis, ammonia stress, oxidative stress and detoxification responses. Many of the changed metabolites and pathways had never been linked to permethrin exposure before. A model for the interaction of the observed changes in metabolites was proposed. From the metabolic pathways with the largest changes, candidate genes from tryptophan catabolism were selected to determine if the perturbed pathways had an effect on survival when exposed to permethrin. Using QPCR it was found that all genes in the entire pathway were downregulated by permethrin exposure with the exception of vermilion suggesting an active response to try and limit flux through tryptophan catabolism during permethrin exposure. Knockdown of the tryptophan catabolising genes vermilion, cinnabar and CG6950 in Drosophila using whole fly RNAi resulted in changes in susceptibility to permethrin for both topical and oral routes of exposure. Knockdown of the candidate genes also caused changes in susceptibility when the insecticides fenvalerate, DDT, chlorpyriphos and hydramethylnon were orally administered. These results show that tryptophan catabolism knockdown has an effect on surviving insecticides with a broad range in mode of action. Symptoms that occur in Drosophila during exposure to the different insecticides were also noted. To gain further understanding into the mechanisms affecting survival, tissue specific knockdown was performed revealing tissue and gender specific changes in survival when vermilion, cinnabar and CG6950 are knocked down. Metabolomics was performed on the knockdown strains to determine the efficacy of the knockdowns on tryptophan catabolism and to identify any knock-on effects. The results indicate that tryptophan metabolite induced perturbations to energy metabolism and glycosylation also occur in Drosophila along with apparent changes in the absorption of ectometabolites. As the knockdown of vermilion, cinnabar and CG6950 tended to result in reduced susceptibility to insecticides, they would make poor targets for insecticidal compounds, however, they may be the first examples of genes that are not directly involved in insecticide metabolism or cuticle synthesis that increase insecticide tolerance in Drosophila. As the first metabolomics data set showed evidence for oxidative stress during permethrin exposure, preliminary work was begun for identifying the tissue specificity and timing of oxidative stress in both Dipterans and Lepidopterans using Drosophila and Bombyx mori as models. In Drosophila oxidative stress did not begin immediately suggesting that the insecticide itself is not a cause, however, a rapid increase in oxidative stress occured over a six hour period after a day of oral exposure implicating catabolites of permethrin. Bombyx were highly susceptible to permethrin showing oxidative stress in the Malpighian tubule and silk gland when exposed. This study has shown that metabolomics is highly effective at identifying pathways which modulate survival to insecticide exposure. It has also brought insight into how insecticide induced pathology may cause death. Data has also been generated which could help characterize the putative transaminase CG6950.