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Pathogenesis in L. monocytogenes is generally well characterised and understood, but the mechanisms of intracellular growth and survival, and how L. monocytogenes changes in the intracellular environment are less well understood. The study of L. monocytogenes strains which display abnormal growth in tissue culture, yet display wild type growth in broth, resulted in the identification of several genes important in pathogenesis. Several of these genes were identified as known virulence factors such as plcA and actA, however several strains were not defective for known virulence genes, yet displayed an abnormal intracellular phenotype. Three such strains were studied in this work, DP-L793 (aig3), DP-L2214 (aig4) and DP-L1107 (plq). These strains were the result of transposon mutagenesis, but only in DP-L2214 was the transposon known to be linked to the virulence phenotype (David Hodgson, personal communication). A transposon tagging approach revealed a region thought to contain the mutation responsible for the Aig3 phenotype; however sequencing of this region did not reveal any base changes in any open reading frame. A point mutation was identified in the putative promoter of the para aminobenzoic acid operon, genes coding for an important enzyme in folate biosynthesis. The expression of the pabA gene was shown to be abolished in broth culture, and after starvation in minimal media, DP-L793 was shown to be folate requiring. The DP-L1107 strain was previously shown to be a threonine auxotroph, and also displayed a small plaque phenotype in tissue culture. The potential link between these two phenotypes was investigated by phage transduction. It was determined that no link exists between the two phenotypes of DPL1107. The aig4 strain, DP-L2214 has the lipoate protein ligase gene lmo0931 disrupted by the transposon Tn917. A second putative copy of this gene was identified by genome sequencing. Attempts were made to perform deletions of both copies of the lipoate protein ligase genes however no deletions were successfully made. Expression of these genes was monitored in broth, and as expected lmo0931 expression was not detected in the DP-L2214 strain, however lmo0764 expression was detected. Yeast-Two-Hybrid assays were used to investigate the interactions between the lipoate protein ligases and potential partners from the lipoate dependent enzyme systems, the pyruvate dehydrogenase complex, the branched chain alpha keto acid dehydrogenase and the glycine cleavage system. Expected interactions were identified for the gene products of both copies of the lipoate protein ligase proteins, a further interaction was noted for the gene product of lmo0764 and a protein of unknown function, which is associated with the gene lmo0931 through proximity. Phylogenetic analysis of the putative lipoate protein ligases, lmo931 and lmo0764 demonstrated that the two genes were divergent.

Paracoccus denitrificans is a facultative anaerobic Gram-negative α- proteobacterium able to shift to denitrification under anaerobic conditions (John & Whatley, 1978; Zumft, 1997). Because of its metabolic versatility, P. denitrificans has been used in this study as a model organism to investigate the role of environmental heterogeneity in maintaining metabolic flexibility in bacterial communities. The hypotheses underlying this study are: - Metabolic flexibility is maintained in situ by environmental heterogeneity and, specifically: - Under constant environmental conditions the metabolic flexibility of the generalist P. denitrificans will be lost by accumulation of mutations in unused genes. Chemostat cultures under constant aerobic conditions revealed how after ~150 generations genetic loci not in use under aerobic conditions (in particular nirS and nosZ) are subjected to a lower selective pressure that leads to a higher genetic polymorphism in the population. The phenotypic analysis of the population resulting from the same chemostat culture showed a lower specific growth rate and a higher yield compared to the ancestor population, suggesting a deactivation of concomitant denitrification and aerobic respiration (Robertson et al., 1988). Furthermore, the resulting population shows a down-regulation of expression of all three denitrification genes tested and a lower production of nitrous oxide (N2O). When cultured in batch cultures for a long period of time under aerobic conditions, P. denitrificans shows a similar adaptative response. Four parallel populations, originated from a common ancestor and propagated aerobically for more than 500 generations undertook some important communal genotypic and phenotypic changes that suggested that P. denitrificans repetitively adapts to constant environmental conditions by losing its characteristic metabolic flexibility. By following the first steps of loss of metabolic flexibility as an adaptative response to novel environmental conditions in a generalist model as P. denitrificans we could empirically witness the important role of the environment on bacterial evolution and speciation.

Methicillin-resistant Staphylococcus aureus (MRSA) are isolates of the bacterium Staphylococcus aureus that have acquired genes encoding antibiotic resistance to all penicillins, including methicillin and other narrow-spectrum β- lactamase-resistant penicillin antibiotics. Outbreaks of MRSA occur quite frequently as there is no quick screening test for the presence of MRSA. The aim of this project is to try and develop an antibody based detection test for rapid detection of MRSA, which could help in the prevention of outbreaks. An antigen (UDP-MurNAc-L-Ala-γ-D-Glu-L-Lys(ε-NH2-Gly)5-D-Ala-D-Ala) that resembles the outer surface of the Staphylococcus aureus (contains a Gly5 moiety that is specific for S. aureus) cell wall peptidoglycan has been prepared, and attached to a carrier protein. Sheep antibodies raised against this antigen were screened using ELISA assays. The results showed that antibodies did have specificity for the antigen. Cell walls were prepared from several different bacteria, including two MRSA and one methicillin-sensitive Staphylococcus aureus (MSSA) strain. ELISA assays using these cell walls showed that the antibodies had specificity for cell walls containing (Gly)5 in the order of S aureus (Gly5) > S. simulans (less Gly5) > S. pneumoniae (no Glyn) > E. coli (no Glyn, Gram-negative strain). A particularly high response was observed for one of the two MRSA strains, detectable at 0.1 μg of cell wall. An HPLC-based UV-Vis assay was developed to monitor the activity of peptidoglycan polymerisation enzymes from S. aureus, while preparing polymeric peptidoglycan as an antigen for immunisation. Several intermediates in peptidoglycan polymerisation were detected using S. aureus monofunctional glycosyltransferase (MGT), which allowed us to propose a new hypothesis for the early steps of peptidoglycan transglycosylation.

Antibiotic resistance is a serious problem in human and animal infections. Heavy use of antibiotics in the agricultural environment selects for antibiotic resistance among the microflora of food animals, providing a reservoir of antibiotic-resistant bacteria. These may enter the human population through the food chain or by direct contact, as demonstrated by the recent emergence of livestock-associated MRSA strains. Antibiotic resistance determinants selected in this environment may also be available for transfer to human commensals and pathogenic bacteria. Milk from conventional and organic farms was screened for methicillin-resistant staphylococci to explore the hypothesis that organic farming methods result in reduced selection for antibiotic resistance. A significantly greater number isolates from conventionally farmed milk were resistant to clindamycin, erythromycin, fusidic acid, tetracycline and tobramycin than isolates from organic milk. Isolates from organic milk mostly belonged to S. fleurettii, which does not cause any known pathogenesis or contribute to the mobile methicillin resistance reservoir. Conventional milk harboured a greater number of pathogens, including S. epidermidis and S. sciuri, both of which carried SCCmec. SCCmec elements were diverse, some similar to those found in human-associated staphylococci, and some distinct. The virulence-associated biofilm cluster icaADBC was identified in some S. epidermidis isolates, indicating increased human pathogenesis. A putative new species, S. lactis sp. nov. was isolated from a conventionally farmed herd, reflecting the high diversity of staphylococci in this environment. In pig nasal swabs originating from Thailand, LA-MRSA was identified for the first time. These isolates belonged to the ST9/t337 lineage which has been identified previously in Europe, suggesting importation of colonised pigs as the most likely route of dissemination. These contained apossible new SCCmecvariant, and harboured toxin genes associated with human disease. Isolates were resistant to medically important antibiotics including ciprofloxacin and chloramphenicol. These data support the hypothesis that use of antibiotics in agricultural practice may select for increased resistance in the microbiome of farm animals, and that some of these may be able to cause infections in humans. Early detection of MRSA is the key to effective treatment and control of dissemination; in food production this would allow early identification of contaminated meat or dairy products and prevent these from entering the food chain. The novel application of an isothermal amplification assay, LAMP, was investigated for the sensitive and specific detection of MRSA. Unfortunately, false negative reactions were common due to high variability among SCCmec subtypes, and the possession of non-mec SCC elements by methicillin-sensitive strains precluded this from offering a reliable alternative to existing methods.

Vibrio parahaemolyticus is a seafood-borne pathogenic bacterium which is a major cause of gastroenteritis worldwide. In the present study, the genetic relationships and population structure of isolates originating from clinical and seafood production sources in Thailand were investigated by multilocus sequence typing (MLST). Nucleotide sequence variation of virulence-related genes including haemolysin and TTSS1 genes among Thai and worldwide isolates was also analyzed. The outer membrane proteome of V. parahaemolyticus isolate RIMD2210633 was predicted using bioinformatic approaches, and the outer membrane proteomes of eight isolates from different sources and representing different MLST sequence type characterized using proteomics. The 101 Thai V. parahaemolyticus isolates examined were recovered from clinical samples (n=15), healthy human carriers (n=18), various fresh seafood (n=18), frozen shrimps (n=16), fresh-farmed shrimp tissue (n=18) and shrimp-farm water (n=16). Phylogenetic analysis revealed a high degree of genetic diversity within the V. parahaemolyticus population, although isolates recovered from clinical samples, farmed shrimp and water samples represented five distinct clusters. The majority of clinical isolates were resolved into two genetic clusters and none of these isolates were found to share sequence types (STs) with strains isolated from human carriers, seafood, or water. Similarly, STs representing human carrier isolates differed from those of clinical, seafood and water isolates. The limited genetic diversity of the clinical isolates suggested non-random selection for pathogenic strains, but the absence of such strains in local seafood raises questions about the likely source of infection. Extensive serotypic diversity occurred among isolates representing the same STs and recovered from the same source at the same time point. Furthermore, evidence of interspecies horizontal gene transfer and intragenic recombination was observed at the recA locus in a large proportion of isolates; this has a substantial effect on the apparent phylogenetic relationships of the isolates. Notably, the majority of these recombinational exchanges occurred among clinical and carrier isolates, suggesting that the human intestinal tract is serving as a reservoir that is driving evolutionary change and leading to the emergence of new, potentially pathogenic strains. MLST was also applied to study genetic relationships between V. parahaemolyticus isolates from Thailand (n=101) and those from European countries (n=9). With the exception of the pandemic ST3 which was resolved from two isolates from Thai human carriers, two clinical isolates from England and a clinical isolate from Norway, none of the other European isolates examined in this study shared the same ST with the Thai isolates. This study demonstrated that Thai human carrier isolates are capable of harbouring virulence-related genes including the haemolysin-encoding genes tdhA, tdhS, trh1 and trh2, and the TTSS1-related genes vcrD1, vscC2 and VP1680, that are present in clinical isolates. In particular, the Thai human carrier isolate VP132 shared identical TTSS1-related gene fragments with the pandemic V. parahaemolyticus serotype O3:K6 (RIMD2210633) and related strains (AQ3810, AQ4037, Peru466, AN5034 and K5030) of worldwide distribution. A total of 117 outer membrane proteins (OMPs) were predicted from the genome of V. parahaemolyticus isolate RIMD2210633. A total 73 OMPs proteins were identified from eight V. parahaemolyticus isolates recovered from clinical samples, human carriers, oyster, shrimp tissue and water in Thailand. Of the 117 predicted OMPS, 32 were identified in eight strains by proteomic analysis. OmpU, a non-specific porin protein, represents the most abundantly expressed protein in all eight isolates. OMPs involved in TTSSs (YscW, YscJ, YscC, PopN and VscC2) and iron uptake (IrgA, putative 83 Da decaheme outer membrane cytochrome C, PvuA1, PvuA2, LutA, FhuE, HutA and putative-regulated protein B) were predicted from the genome of V. parahaemolyticus isolate RIMD2210633, but were not recovered from any of the eight Thai isolates. The absence of TTSS and iron uptake related OMPs in the eight representative strains that were grown under in vitro conditions may suggest an important requirement for in vivo growth conditions to induce expression of important virulence factor-related OMPs in V. parahaemolyticus. There was no clear association between OMP profile and the source of isolation, ST or serotype. However, a high degree of variation of OMP profiles was observed in isolates from different sources as well as in the isolates representing the same ST. This study demonstrated the usefulness of a multidisciplinary approach that includes MLST, virulence-related gene DNA sequence analysis, bioinformatic prediction and gel-based proteomic analyses for the study of molecular evolutionary relationships and the epidemiology of V. parahaemolyticus isolates from clinical and seafood production sources. The outcomes of this study highlight the role of human carriers as a reservoir of potentially pathogenic V. parahaemolyticus and this should be considered as one of the possible contamination sources in the surveillance of seafood safety.

The developed world is experiencing a steep increase in diseases based on aberrant autoimmune inflammatory responses and allergic conditions. By contrast, such diseases are uncommon in countries endemic for parasitic helminths, suggesting that parasite-derived immunomodulators may protect against their development. One such molecule, ES-62, which is a phosphorylcholine (PC)-containing glycoprotein originally identified from the filarial nematode Acanthocheilonema viteae, has shown therapeutic potential in a range of inflammatory diseases. Although its mechanism of action is not fully understood, ES-62 is known to target dendritic cells (DCs) to subvert T helper cell (TH) differentiation and hence modulate the subsequent host immune response to an anti-inflammatory phenotype. ES-62 requires Toll-like receptor 4 (TLR4) to mediate its effects, and here, we have investigated the mechanisms underpinning ES-62-mediated subversion of TLR4-pro-inflammatory signalling as exemplified by its canonical ligand, bacterial endotoxin (lipopolysaccharide, LPS) that results in the activation of the transcription factor, nuclear factor-κB (NF-κB) and consequent secretion of proinflammatory cytokines. This study investigated the mechanism of ES-62-mediated uncoupling of NF-κB activation by its modulation of TLR4 expression and its associated LPS recognising complex TLR4/Myeloid differentiation factor-2 (MD2), as the internalisation, trafficking and degradation of this receptor complex has previously been shown to regulate its responses to different stimuli. ES-62 and LPS were found to differentially modulate the dynamics and expression of the total cellular pool of TLR4, relative to that of the ‘active’ LPS recognising TLR4/MD2 complex. Specifically, ES-62 acted to antagonise the dynamics of the LPS response, providing a mechanism for its induction of TLR4 hyporesponsiveness. In addition, as preliminary studies demonstrated an interaction between ES-62 and the Platelet activating factor receptor (PAFR), the possibility that ES-62 might signal via TLR4 in concert with one or more co-receptors was investigated but the data did not conclusively support a role for the PAFR as a target of ES-62 action. The dissection of the effects of ES-62 on TLR4 signalling was further pursued by defining its modulation and/or sequestration of transduction elements associated with the TLR4 mediated activation of NF-κB. Of particular interest were PKCα, PKCε and PKCδ members of the protein kinase C family that directly interact with TLR4 and its immediate signalling adaptors to mediate proinflammatory cytokine (TNF-α, IL-6 and IL-12p70) 2 production. Several isoforms including PKCα have previously been shown to be targeted in B cells and mast cells for degradation by ES-62. Here, we utilised pharmacological inhibitors of proteosomal and lysosomal protein degradation pathways to demonstrate that whilst LPS induces the turnover of PKCs through the proteosome, ES-62 directs their degradation through the lysosomes. In conjunction with this, ES-62 and LPS were also found to differentially regulate the expression of tumour necrosis factor receptor associated factor-6 (TRAF6) and c-Casitas B lineage lymphoma (c-Cbl), E3 ubiquitn ligases, which act as scaffolds (via Lysine 63 linked polyubiquitination chains) in the activation and stabilisation of NF-κB. However, it was found that ES-62 did not prevent recruitment of these various TLR4 signal transducers upon subsequent recognition of LPS, suggesting that it acts to attenuate hyper-inflammatory responses without leaving an individual immunocompromised to infection. In addition, by mimicking an inflammatory environment with exogenous granulocyte macrophage-colony stimulating factor (GM-CSF), it was found that the actions of ES-62 had more profound effects on the modulation of the aforementioned signal transducers and its range of targets were expanded to include, myeloid differentiation primary responses gene 88 (MyD88), which transmits the early response signals from all the TLR family receptors, except TLR3.

Salmonella is a major cause of enteric illness in both humans and animals. The ability of Salmonella isolates to cause disease in animals and humans encompasses a spectrum of host specificity and disease severity. In terms of evolution Salmonella isolates have remained relatively similar in terms of genetic content. It would therefore seem paradoxical that the diversification of many ecological niches and the invocation of very different disease symptoms by Salmonella have been possible. This infers great importance on differences in gene content between Salmonella isolates. Consultation of genomic sequence data from various Salmonella isolates has confirmed that a large proportion of strain-specific DNA is laterally transferred. More specifically a large proportion of this laterally transferred DNA was found to be bacteriophage-associated. The assertion could therefore be made that infections of isolates by temperate phages may play a significant role in Salmonella evolution and differentiation. Indeed, it has been suggested that a variable assortment of prophages provides a transferable repertoire of pathogenic determinants in Salmonella. The temperate phages present in Salmonella enterica serovar Typhimurium isolates have been most extensively investigated to date. However, how variable is the excisable prophage content between different Salmonella species, subspecies, serovars and individual isolates? In the current study the temperate phages present in 102 Salmonella isolates from a wide variety of species, subspecies and serovars were characterised by various methods. To assess the diversity of excisable temperate phages present in Salmonella isolates phages were induced with mitomycin C and viewed by electron microscopy. Temperate phages were identified in 81.4 % of 102 Salmonella isolates and the most commonly isolated phages belonged to the Myoviridae morphology family. The host ranges of the induced phages varied greatly. Sixty-five lysates contained phages that could infect one other Salmonella isolate from a panel of 24 indicator isolates. Six different methods of phage DNA extraction were tested. The amount of phage DNA extracted from lysates varied considerably depending on the Salmonella isolate from which the lysate was made. Fifty-three Hind III restriction digest profiles were obtained from eighty-six lysates and thirty-seven of these profiles were found to be unique. Phages with identical restriction profiles were identified and one of these phages was associated with clinical Salmonella isolates. Finally PCR profiling of the phage DNA and chromosomal DNA obtained from Salmonella isolates was used to assess the diversity of functional and defective phages present in the isolates. Genes associated with the ST64B phage were most commonly identified in the phage DNA extracted from lysates and in the chromosomal DNA from Salmonella isolates. Great diversity was observed in the temperate phage content of Salmonella isolates for all of the characterisation methods utilised. PCR profiling was determined to be the most sensitive method to identify temperate phages in Salmonella isolates. Assessment of the inducible and non-inducible prophage content of isolates by PCR profiling was also shown to have potential for the characterisation of Salmonella strains.

Food-borne disease continues to be a major cause of human morbidity and mortality. During the past few decades, Campylobacter jejuni has ascended to become the greatest cause of bacterial enteric disease worldwide. Anecdotal evidence suggests the majority of human campylobacteriosis in industrialised countries is caused by the consumption of undercooked chicken. Campylobacter continues to frustrate current control strategies throughout the food chain and in 2001 was responsible for over 56, 000 cases of food poisoning in the U.K. alone. The work presented in this thesis examined the potential of host-specific bacteriophage as a novel measure to control the population of Campylobacter in poultry production. Several surveys in this thesis revealed that campylobacters and their bacteriophage permeate the entire poultry meat supply chain, from chickens in the broiler house to packaged retail products. Characterisation of the bacteriophage recovered from such sources showed that retail poultry isolates exhibited greater similarities in host range than those originating from broiler houses, implying poultry processing selected for a subpopulation of phage. Additionally, broiler chickens harbouring bacteriophage in their gastrointestinal tract generally contained fewer campylobacters. All of the phage isolates studied belonged to the Myoviridae virus family as they possessed dsDNA genomes encapsulated in an icosahedral head with a rigid, contractile tail. Fragments of the phage genomes exhibited significant sequence homology with a number of genes involved in DNA replication from phage T4. Studies of the attachment and replication of the phage isolates in vitro suggested that adsorption to the host cell was efficient but the burst size was low (˂10 virions per cell). Campylobacter jejuni was found to produce membrane vesicles but these did not significantly affect bacteriophage replication in vitro. A series of trials using 'phage therapy' in broiler chickens revealed that Campylobacter colonisation can be reduced by ≥log[subscript]10 8.0 cfu g[superscript]-1 caecal contents by dosing with specific bacteriophage. However, both the timing and extent of the reduction in Campylobacter colonisation showed considerable variation. Additionally, the ability of bacteriophage to infect their host in vitro was not a reliable indicator of their efficacy in vivo. The direct application of bacteriophage to the surface of chicken skin artificially contaminated with Campylobacter led to a significant reduction in the number of recoverable host cells. Host resistance to bacteriophage infection was not detected in either the in vivo trials or when recovering Campylobacter cells from chicken skin treated with phage. The work presented in this thesis demonstrates that bacteriophage have considerable potential in the control of Campylobacter in poultry production. They already appear to constitute a limiting factor in Campylobacter colonisation of the chicken gastrointestinal tract and can be detected with their host on retail products. However, further research is required to fully realise their potential and optimising the timing, level and type of bacteriophage used in dosing will be important for their efficacy in vivo.

The enteric bacterium Serratia marcescens is an opportunistic human pathogen. The strain studied here makes the red pigment prodigiosin (Pig) and the ß-lactam antibiotic (5R)-carbapen-2-em-3 carboxylic acid. Mutants were isolated which were affected for pigment production. Approximately 20% of these mutants were also concomitantly deficient for the production of antibiotic. These mutants were presumed to be defective in the rap (regulation of antibiotic and pigment) gene. This study set out to investigate the rap gene which had been cloned by direct cosmid complementation of a Rap mutant from a cosmid library (pNRT300). Sequence analysis of the rap gene revealed a predicted product showing strong homology to S1yA, classified by Libby et al., (1994) as a virulence determinant in Salmonella. Homologues of the rap gene were detected in several genera including Salmonella, Yersinia, Enterobacter and species of the enteric plant pathogen Erwinia. The Erwinia horEc gene (homologue of rap) was cloned and encoded a product which was highly homologous to both the SlyA and Rap proteins. The gene arrangement around the rap locus in Erwinia was identical to that in Serratia in that rap and horEc were both situated upstream of two genes encoding homologues of the lipoprotein Pcp and a gene encoding a protein of unknown function from Yersinia enterocolitica. This observation led to the search for the Yersinia homologue of rap (horte) which was subsequently cloned and sequenced. This gene too encoded a protein highly homologous to Rap and HorE. Data base searches revealed that these proteins shared a significant level of homology with a number of bacterial protein regulators involved in exoenzyme production, virulence in plant and human pathogens, multiple antibiotic resistance and xenobiotic catabolism. The findings of this study cast serious doubt on the conclusions of Libby et al., (1994) and in a recent report which was published whilst this thesis was being compiled, Ludwig et al., (1995) reclassified S1yA as a regulatory protein capable of activating cryptic haemolysin genes in Escherichia coll. Marker exchange mutants (horEc:k: anR) of the Envinia carotovora subspecies carotovora were found to be affected in the production of a carbapenem antibiotic and showed decreased levels of production of multiple exoenzyme virulence factors. Transcriptional fusion data revealed that the horEc mutation affected the transcription of carA a carbapenem biosynthetic gene. Antibiotic and exoenzymes are known to be regulated by a small molecule dependent regulatory system analogous to the Lux system controlling bioluminescence in Photobacterium fischeri. The results of regulatory studies in which autoinducer was added exogenously, or carR was added in trans imply a role for HorE in this pheromone-signalling system. The functional expression of prodigiosin in a Erwinia carotovora subspecies carotovora was found to be dependent on autoinducer and the gene product of horEc. Some interesting observations were also made regarding differential patterns of prodigiosin gene expression within bacterial colonies. These patterning effects were strikingly strain-specific.