No description available.

No description available.

Campylobacter jejuni is the main cause of gastroenteritis in developed countries and the exact nature of the organism’s virulence continues to be the subject of much research. Initially the main area of focus for this project was to establish whether the glycosylation of iron uptake proteins is necessary for their function. We know that protein glycosylation is essential for interaction with host cells and that bacteria also require functioning iron uptake systems in order to colonise their host. Mutants in pglB, pglI and pglK were utilised in growth assays and compared with mutants in all known iron uptake systems. The haem uptake system was investigated and a haem biosynthesis gene hemE targeted, which will be used to establish whether exogenous haem can be used for metabolism. The C. jejuni haem oxygenase ChuZ has been expressed, purified and its viability assessed by absorbance spectrophotometry, with a view to solving the structure through X-ray crystallography. With knowledge of the structure it is anticipated that it will be possible to clarify the exact function of the protein. The catechol noradrenaline has been shown to enhance growth in iron-restricted conditions through the receptor CfrA. This study has investigated the effects of glycosylation on iron uptake via CfrA. The gluconeogenic enzyme GAPDH has been shown to localise to the outer membrane where it interacts with host transferrins. Whilst the effects of glycosylation on the uptake of iron from lactoferrin have been investigated, work has also begun on constructing a gapA mutant which will be used to investigate the interaction with lactoferrin and transferrin. This study has revealed that glycosylation of iron uptake proteins was not necessary for their function. Levels of natural competence were also studied and it was found that mutants in pglB and pglK were compromised with respect to their ability to naturally transform.

Despite being the principal bacterial cause of gastroenteritis world wide, the epidemiology of Campylobacter jejuni is poorly understood. This is largely because the proportion of human disease caused by different sources of infection is unknown. In this study a diverse strain collection was selected comprising of 91C. jejuni isolates from diverse animal and environmental ecological niches as well as clinical isolates from patients representing a range of disease outcomes. Whole genome comparisons were performed by DNA microarray analysis with the dual aim of identifying genetic markers specific to strains from different ecological niches and identifying novel virulence determinants. A new phylogenomic technique for the analysis of DNA microarray data was developed combining Bayesian algorithms to model phylogeny based on whole genome data with parsimony based algorithms to identify the key genes contributing to the clade formations. This method revealed a previously undetected C. jejuni population structure comprising two main clades, a "chicken clade" and a "nonchicken clade". These statistically supported clades differentiated strains from distinct ecological niches with 94% of strains isolated from chickens and 41% of clinical isolates contained within the "chicken clade". C. jejuni isolates from ovine, bovine and sand isolates also formed distinct clades within the "non-chicken clade". Key genes contributing to the distinction of strains from one ecological niche from another were identified. In particular a putative glycosylation islet cj 1321-cj 1326 was found to be present in strains within the "chicken clade" but absent or divergent from strains in the "non-chicken clade', a result validated by peR screening. This locus represents a potential genetic marker of C. jejuni strains of avian origin. The DNA microarray data analysis method described in this study may be used to study other bacterial pathogens facilitating the identification of bacterial phylogenies and genetic markers associated with specific phenotypes.

No description available.

This investigation set out to study the epidemiology and distribution of non- capsulate H. influenzae in Morecambe Bay. All H. influenzae isolates from patients in hospital and the community were collected over a 2 year period. These were tested using traditional culture, biochemical and molecular techniques. Data collected included medical history, smoking history, demographic and socio-economic details for 762 patients. The distribution of housing types in Morecambe Bay was different from the rest of the UK. Housing types for patients in the study were not representative of the local population profile. Most infections were found in patients living in urban areas. Less affluent patients were most likely to have a history of smoking, especially patients aged over 50 years. Patients presenting with more than 1 episode of infection during the study were more likely to have smoked. Some patients who presented with more than 2 episodes of infection were co-Ionised by non-capsulate H. influenzae with the same sequence type (ST) for up to 2 years, some acquired different STs and some STs evolved into closely related STs. There was no significant change in the rate of antibiotic resistance across Morecambe Bay between 2001 and 2005, although severity of chest disease did have a significant effect on ampicillin resistance. Age did not have a significant impact on ampicillin resistance but more elderly patients were infected by multiple antibiotic resistant bacteria. The ICEHin1056 element did offer an !I explanation for ampicillin resistance involving the bla gene. ICE negative strains may have acquired resistance from other bacteria colonising the airway. Different age populations were found in the 3 hospital areas. There was no significant difference in numbers of male and female patients presenting with eye or chest infections but significantly more ear infections were found in males. The findings of this study suggest that most non-capsulate H. influenzae chest infections were found in the less affluent elderly population.

The human upper respiratory tract is colonised by a diverse commensal flora that is tolerated by the host without inducing an aberrant inflammatory response. N. meningitidis is an opportunistic pathogen that asymptomatically colonises the nasopharyngeal epithelium of older children and adults, but infrequently causes invasive disease. N. lactamica is a closely related commensal that colonises the nasopharyngeal epithelium of young children without causing disease. Both species can colonise for prolonged periods without being cleared. We have Investigated the development of mucosal immunity to N. lactamica and compared the response with N. meningitidis to further understand how each maintains a commensal relationship with the host.

Phage therapy is the use of bacteriophages to treat bacterial infections. Once a prominent method of antibacterial therapy, phage therapy became almost forgotten following the discovery of antibiotics in 1928. The rising instance of antibiotic resistant bacteria in the last number of years has resulted in resurgence in interest in phage therapy. Research into delivery methods for bacteriophages has been very limited. Up to very recently, phages had only been delivered parentrally using crude and purified phage stocks and orally for gastrointestinal infections. In order for phage therapy to become suitable for use in mainstream medicine, suitable dosage forms and phage delivery platforms must be developed. This research project endeavoured to successfully formulate a model bacteriophage, T4 phage, into useful polymeric delivery systems, beginning with an alginate based microparticulate system. Bacteriophages were successfully stabilized within a Ca-alginate-trehalose system and m icroparticles were fully characterised. The release profi le of these m icroparticles suggest that they could be used to successfully treat gastrointestinal infections. The present study also devised a phage stable soluble microneedle system. It was found that increasing the trehalose concentration within the microneedles, improved rnicroneedle strength, as did the addition of a Poly (methyl vinayl ether-eo-maleic acid) backing layer. A poly(carbonate) hollow rnicroneedle system was used to successfully deliver phages both in vitro and in vivo using a rat model. This study was the first time that bacteriophages have been delivered transdermally in vivo. Aside from the development of bacteriophage delivery systems, novel work in the application of Phage-Antibiotic synergy (PAS) to E.co/i biofilrns was completed with extremely successful results. It was found that administering T4 bacteriophage in combination with the antibiotic Cefotaxime improved biofilrn eradication, compared to treatment with bacteriophage or antibiotic alone. This is the first time that PAS has been applied to a biofilm model.

Peripheral blood monocytes represent the rapid response component of the mononuclear phagocyte host defence. This thesis has examined the fate of highly purified human monocytes following challenge with a range of Gram positive and Gram negative bacterial pathogens. Exposure of monocytes to high bacterial loads resulted in a rapid loss of cell viability and decreased innate responses, including reduced generation of ROS and decreased rates of phagocytosis. Monocyte cell death following exposure to bacteria in many circumstances was due to apoptosis. Exposure of monocytes to high numbers of Escherichia coli and Klebsiella pneumoniae however, resulted in a death process with evidence of caspase-I activation and extracellular trap formation. Reducing the bacterial load with antibiotics converted the death process to apoptosis. Exposure of the monocytes to Neisseria meningitidis showed a delayed death response compared with other bacterial pathogens investigated. Delayed cell death was associated with preservation of intracellular ATP and maintenance of innate anti-microbial function. Delayed induction of cell death however, resulted in prolonged pro-inflammatory cytokine production. Prolonged exposure of monocytes to Neisseria meningitidis eventually induced apoptosis that occurred via the intrinsic (mitochondrial) pathway and was independent of lysosomal mediated apoptosis. Apoptosis was associated with the alternative splicing of certain proapoptotic Bcl-2 proteins and the down regulation of Mcl-1. I also investigated the utility of using differentiated THP-I cells as a model of monocytes and macrophages. Prolonged exposure of differentiated THP-I cells to Neisseria meningitidis resulted in the induction of apoptosis with, in contrast to primary monocytes, relative preservation ofMcl-I, and up-regulation of the pro-apoptotic Mcl-l isoform, Mcl-I Exon-1. Overall this thesis has shown that monocytes are capable of activating a range of cell death programmes and are more closely related to neutrophils with regard to their susceptibility to cell death and the features of cell death they exhibit. The thesis has also shown that failure to engage a death process following exposure to Neisseria meningitidis results in prolonged cytokine release that may have implications in the pathogenesis of meningococcal disease.

The human pathogen S. aureus is able to cause a large range of diseases, from minor skin diseases to life threatening sepsis. This versatility is governed by an extensive repertoire of virulence determinants and an acute ability to respond to the host environment in order to adapt, survive and proliferate. The problem of S. aureus is exacerbated by the rapid spread of antibiotic resistance, leading to such agents of public concern as methicillin resistant S. aureus (MRSA). There is therefore a desperate need to develop new therapeutic agents against S. aureus. Harnessing human innate defenses may provide a novel mechanism to combat the scourge of MRS A and other antibiotic resistant strains. Human skin fatty acids have been shown to have highly anti staphylococcal activity. In particular the major skin fatty acid cis-6-hexadecenoic acid (C-6-H) is able to rapidly kill S. aureus, by an unknown mechanism. Also C-6-H at concentrations, which do not kill the bacteria, is able to inhibit the production of major virulence determinants. This project aimed to determine the molecular response of S. aureus to C-6-H, a major facet of the human innate defence. Initial experiments showed that exposure to sub-MIC levels of C-6-H resulted in the induction of a resistance mechanism. In order to begin to determine the molecular basis for the induced resistance and the inhibition of virulence determinant production by C-6-H transcriptome and proteome studies were carried out. The transcriptome revealed an altered level of transcription of over 500 genes, in response to sub-MIC C-6-H, which are involved in virulence, amino acid biosynthesis, energy metabolism, stress response, purine and pyrimidine metabolism, cell wall and cell envelope dynamics and several regulatory systems. Interestingly, the expression of the toxins (hla, hlb, hlgBC) was highly reduced in the presence of C-6-H, whereas the expression of genes involved in host defence evasion (cap, sspAB, kalA) were increased. The proteome studies also showed a decrease in the production of several toxins. Members of the SaeRS regulon were reduced in expression, in response to C-6-H and several were confirmed by qRT-PCR. The use of specific mutants revealed the effect of C-6-H on toxin production is likely mediated directly or indirectly via SaeRS. The global effects ofC-6-H on S. aureus physiology are discussed.