Understanding both the mechanistic basis of virulence and the evolutionary processes under which it can arise, is fundamental if we are to increase our knowledge of disease causing bacteria in an era of ever increasing antibiotic resistance. To date, there has been a substantial effort to understand virulence evolution both theoretically and experimentally. However, comparatively little experimental work has focussed on the evolution of virulence in opportunistic pathogens, and how virulence varies across multiple host organisms. In this thesis, the opportunist Pseudomonas aeruginosa was used to study virulence and its evolution in hosts. The virulence levels of different strains of P. aeruginosa, both laboratory and clinical, were tested in the nematode Caenorhabditis elegans. It was found that virulence of P. aeruginosa was lower in clinical strains isolated from chronic infections, and laboratory strains initially isolated from chronic infections, while a strain isolated from an acute infection was substantially more virulent. Using the C. elegans host, a selection experiment was carried out to test the effect of host density on virulence evolution. This resulted in a varied array of P. aeruginosa virulence phenotypes, but the worm evolved bacteria did not exhibit drastically different phenotypes from the worm negative control experiments. However, the degree of evolution observed across all treatment groups, compared with the wild type, highlighted the changes a bacterial population can undergo in vitro, even from a relatively short period of sub culture. Lastly, P. aeruginosa virulence was tested in alternative host organisms, Galleria mellonella (waxmoth larvae) and Vigna radiate (mung bean seedlings). Virulence varied according to the host, with virulence in G. mellonella being universally high, while no virulence was observed in V. radiate. This work highlights that high or low virulence cannot automatically be assumed in different hosts for opportunistic bacteria such as P. aeruginosa. Furthermore, the results highlight the unpredictable nature of virulence evolution, and how dependent the emergence of virulence is on the interaction between the pathogen and the host. A more complete understanding of the principles underlying bacterial evolution and virulence could allow for the construction of more specific and accurate studies, with experimental conditions carefully engineered to best replicate the clinical conditions of interest.

Oomycetes are a diverse group of organisms with worldwide distribution. The phylum contains a large number of pathogenic species which cause immense damage to both animal and plant host species in cultured, managed and wild ecosystems. As such, the majority of research is focused on host-pathogen interactions and the environmental study of oomycetes is greatly neglected. However, due to their devastating economic and ecological effects it is becoming increasingly clear that oomycete ecology and diversity studies are vital to the monitoring and management of diseases caused by oomycete species. By studying oomycete diversity, we further expand our knowledge of species ecological adaptation, distribution and host ranges, allowing for development of improved biosecurity systems. The diversity of oomycete species within the biogeographically important Falkland Island archipelago was assessed. The Islands contained relatively low oomycete species diversity, with a total of twenty-five species identified, in nine genera; Pythium s. str., Phytopythium, Saprolegnia, Leptolegnia, Newbya, Achlya, Elongisporangium, Globisporangium and Phytophthora. Thirteen of the identified species from the Falkland Islands were putative novel species, within the genera; Saprolegnia, Leptolegnia, Newbya, Pythium s. str., Phytopythium and Elongisporangium. Four of these putative novel species were confirmed as novel taxa through the morphological and molecular analyses presented herein. A fifth species requires further analyses to confirm the taxonomic position. One of the presented species was mildly pathogenic towards Atlantic salmon (Salmo salar) eggs, while another was pathogenic towards ryegrass (Lolium sp.) seedlings. These results expand our knowledge of oomycete taxonomy and provide further knowledge of pathogenic oomycete species.

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This thesis concerns the initial events leading to the induction of the phytoalexin response in the Phaseolus vulgaris L. - Colletotrichum lindemuthianum L. host - pathogen interaction The phytoalexin response is an expression of the resistance of the host to further pathogen invasion. The visible expression of the phytoalexin response is hypersensitive necrosis. One of the initial events leading to the induction of the phytoalexin response is thought to be the induction of the first enzyme leading to the synthesis of phenylpropanoid phytoalexins, namely L-phenylalanine ammonia-lyase (PAL). Standard methods for determination of PAL activities were found to lead to measurements of both phenylalanine amino-transferase (PAT) and PAL activities together. Further, PAT was found to have a higher affinity for L-phenylalanine than PAL. An isotopic assay was devised for the accurate estimation of PAT and PAL activities separately using a specific inhibitor of PAT, L-aspartic acid. These experiments were carried out in a whole (intact) plant system. A rapidly isolated cell and protoplast system wa.s specially devised for isolation of the pathogen cell wall constituents responsible for the induction of the phytoalexin response. Special care was taken to minimise mechanical damage in these systems by optimising methods of purification, viability and intactness. The pathogen cell wall component responsible for the induction of the phytoalexin response was found to be an asialoglycoprotein.

Magister Scientiae - MSc / The organisms in this study were chosen due to their associations with foods and their potential as food borne pathogens. Food borne diseases are an import public health problem in most countries. Bacteria of the genera Campylobacter, Salmonella and Listeria can be transported by poultry and poultry products to humans. Gastroenteritis, typhoid fever, diarrhea, dysentery may originate from the infection. This study was undertaken to determine the incidence of pathogens in a poultry processing plant using polymerase chain reaction and conventional tests and to determine the formation and survival of biofilm cells of food pathogens in trisodium phosphate. / South Africa

Many microorganisms, and in particular fungi, are able to grow over a wide pH range. Thus, these microorganisms must possess some regulatory mechanism or system that senses the environmental pH signal and ensures that gene expression of certain molecules is tailored to the pH of the environment (Penalva and Arst, 2002). In Aspergillus species and several other fungi, pH regulation is mediated by seven genes viz. palA, palB, palC, palF, palH, palI and the global pH regulatory gene, pacC (MacAbe et al, 1996; Negrete-Urtasun, 1999; Denison, 2000). The activated form of the PacC protein activates genes that are required at alkaline pH, e.g. genes coding for alkaline phosphatases, and represses certain genes that are functional at acidic pH, e.g. genes encoding acid phosphatases (Negrete-Urtasun, 1999). PacC (and its homologues) also positively regulates genes involved in penicillin biosynthesis, e.g. the isopenicillin N synthase gene, ipnA, in A. nidulans (Penalva and Arst, 2002). It has also been hypothesised that pacC may negatively regulate aflatoxin biosynthesis, a carcinogenic secondary metabolite in several species of Aspergillus (Keller et al, 1997). To elucidate the role of pacC a novel method of post-transcriptional gene silencing known as RNA interference was utilized. This method involved the cloning of a partial pacC gene fragment first in the forward and then the reverse orientations in a fungal expression cassette to create an RNA interference (RNAi) vector. The unique structure of this vector would allow the cloned fragments to be expressed and the resulting RNA to immediately form a double stranded stem-loop structure or short hairpin RNA (shRNA; McDonald et al, 2005). The formation of this shRNA, in turn, would be responsible for activating the endogenous RNA degradation complexes that would lead to mRNA degradation and subsequent gene silencing (Liu et al, 2003; Kadotoni et al, 2003; McDonald et al, 2005). The results presented here have shown that confirmed pacC RNAi mutants produced aflatoxins irrespective of environmental pH (i.e. the mutants produce aflatoxins under acidic and alkaline conditions). Thus, pacC is essential for pH regulation of aflatoxin production in A. flavus. There are numerous other biological (e.g. presence of oxylipins, lipooxygenases) and non-biological factors (pH, carbon source etc.) which affect maize colonisation and aflatoxin production by A. flavus (Burrow et al, 1996; Wilson et al, 2001; Calvo et al; 2002; Tsitsigiannis et al, 2006). However, all the genetic mechanisms involved have as yet not been identified. It has been shown by Caracuel et al (2003) that pacC acts as a negative virulence regulator in plants and these workers have hypothesised that PacC prevents expression of genes that are important for infection and virulence of the pathogen. Therefore the physiological effects that pacC silencing had on the growth, conidiation and pathogenicity of A. flavus mutants were also investigated. The results of this study showed that pacC does not play a significant role in primary growth and development but does affect conidial production. SEM results showed that mutants have many “open ended” phialides and poorly developed conidiophores. This would suggest that pacC activation of conidial production genes is also required. Furthermore, pacC RNAi silencing severely impaired the ability of the A. flavus mutants to infect and cause damage on maize. The results obtained here are similar to that of pacC null mutants in A. nidulans, C. albicans and F. oxysporum which also exhibited low pathogenicity (Davis et al, 2000; Fonzi, W.A, 2002; Caracuel et al, 2003; Bignell et al, 2005 and Cornet et al, 2005). This study indicates that pathogenicity of A. flavus on maize is directly related to the structural integrity of conidia, which in turn is greatly influenced by PacC. This gene is a global transcriptional regulator and may either repress or activate one or many genes in each of the above pathways (Penalva and Arst, 2002). Studies on the genetic mechanisms of pacC regulation on these pathways are needed to elucidate the mechanisms of activation or repression of these genes.

No description available.

The purpose of this study is to investigate the rate of natural fermentation of solutions at different temperatures. Whatever microorganisms that chanced to be present in the air and that chanced to fall into the inoculating medium are the ones which brought about fermentation when transferred to the fermentable solution.

No description available.

No description available.