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.

Inoculation experiments were carried out to determine the relation between bark moisture level of certain host species and their susceptibility to facultative parasites. In these experiments, cuttings of 1-to 3-year old host material and the mycelial mat of the pathogens contained in an agar cylinder were used. In the first instance, fungi that were known or found in association with bark lesions were considered: these were Cryptodiaporthe salicella (Fr.) Petrak on Salix scouleriana Barratt (Scouler willow), Dactylosporium sp. and Fusarium sp. on Acer macrophyllum Pursh. (broadleaf maple, Libertella sp. on Cornus stolonifera (Michx.) var. occidentalis (T. and G.) C. L. Hitchc. (western dogwood), Melanconis sp. on Alnus rubra Bong. (red alder). The results demonstrated that fungi normally associated with lesions of living host material proved to be pathogenic when the relative turgidity of the host bark was lowered from the field level of above 80 per cent to the range of 69 to 77 per cent. Secondly, an attempt was made to determine if correlation existed between bark moisture level and canker development by fungi not known, and not found to occur in association with lesions of some hosts. The following fungi and hosts were considered: C. salicella on red alder (Alnus rubra Bong), trembling aspen (Populus tremuloides Michx.), bitter cherry (Prunus emarginata Dougl.), black cottonwood (Populus trichocarpa Torrey and Gray), western dogwood (Cornus stolonifera var. occidentalis), and on broadleaf maple (Acer macrophyllum Pursh.); Fusarium sp. on red alder, bitter cherry, western dogwood, and on Scouler willow; Libertella sp. on red alder, bitter cherry, broadleaf maple, and on Scouler willow; Melanconis sp. on bitter cherry, western dogwood, broadleaf maple and on Scouler willow. It was shown that all of these parasites extended their host ranges, to varying extent when the bark moisture level was reduced to levels within the range of 69 to 77 per cent, or in some instances to the range of 41 to 67 per cent of saturation. Cuttings with as low bark moisture levels as 41 per cent appeared to be viable as indicated by the production of roots and (or) shoots. / Science, Faculty of / Botany, Department of / Graduate

Mycobacterium tuberculosis, the causative agent for the disease Tuberculosis, is a serious public health problem that is responsible for 1.6 million deaths each year. The WHO’s recent report on Tuberculosis estimates that a third of the world’s population is latently infected with the bacteria, and, of those, 10% will progress to active disease. M. tuberculosis is a successful pathogen mainly due to its ability to adapt and survive in changing environments. It can survive a dormant state with limited metabolic activity during latent infection, while also being able to escape the macrophage and disseminate into active disease. Efforts to eradicate the disease must be based on understanding the biology of this organism, and the mechanisms it uses to infect, colonize, and evade the immune system. Understanding the behaviour of pathogenic mycobacteria in the macrophage is also important to the discovery of new drug targets. In this thesis, we employed state of the art mass spectrometry techniques, which allowed us to unpack the biology of this bacterium in different growth environments and expand our understanding of the mechanisms it employs to adapt and survive. We investigated protein regulation by the process of phosphorylation, through sensory kinases, which add a phosphate group to a protein of interest, thereby regulating its function. First, we interrogated the phosphoproteomic landscape between M. bovis BCG and M. smegmatis to explain how differential protein regulation results in the differences between slow and fast growth of mycobacteria. Second, we focused on Protein Kinase G (PknG), which plays an important role in bacterial survival by blocking phagosome/lysosome fusion. We identified the in vivo physiological substrates of this kinase in actively growing M.bovis BCG culture. Our results revealed that this kinase is a regulator of protein synthesis. We then examined the mechanisms of survival in murine RAW 246.7 macrophages mediated by PknG, using M. bovis BCG reference strain and PknG knock-out mutant. Our results indicated strong evidence that pathogenic mycobacteria disrupt the macrophagic cytoskeleton, through phosphorylation of proteins that are involved in cytoskeleton rearrangement. These results explain the strategies that pathogenic mycobacteria employ mediated by PknG to block phagosome-lysosome fusion and evade the host immune system and survive for prolonged periods in the macrophages. The findings of this thesis contribute to our understanding of the physiology of pathogenic mycobacteria and their interaction with the host.

Members of the Staphylococcus genus are found as a part of normal microflora in humans and can commonly be found on the skin or in the nasal cavity. However, these microorganisms can cause serious and life-threatening opportunistic infections when there is a break in the physical barrier of skin. These infections have become difficult to treat as resistant strains emerge, particularly Methicillin Resistant Staphylococcus aureus (MRSA). MRSA has become a commonly acquired nosocomial infection which is difficult to treat with conventional antibiotics of the blactam class. Even Vancomycin, a last resort antibiotic, has been ineffective on some infections. Furthermore, S. aureus readily forms biofilms on implanted medical devices which establishes a hardy and difficult to treat infection. These biofilms serve as a point of infection to the bloodstream. Research involving polymicrobial interactions and the inhibitory effects of bacterial-bacterial interactions could be a starting point for the discovery of a new therapeutic treatment for infections. It has been shown in our lab that Alcaligenes faecalishas inhibitory effects on Staphylococcus aureusplanktonic growth. Therefore, in this study, we wanted to examine 1) The mechanism by which A. faecalisinhibitsS. aureus growth and 2) how A. faecalisimpacts the various phases of S. aureusbiofilm growth. It was found that A. faecalislikely inhibits S. aureususing a physical mechanism that requires close contact, rather than using a secreted molecule. However, a Type VI secretion system could also produce similar results. Further research involving the formation of mutants to find the gene allowing A. faecalisto inhibit S. aureuswas started, but no viable mutants were created during the course of this research. A. faecaliswas found to inhibit the formation of S. aureus biofilm growth, but when added to a mature S. aureusbiofilm, the slow growth rate of A. faecaliscould not overtake the quickly replicating S. aureus. Further research in the polymicrobial interactions between S. aureus and A. faecaliscould lead to a finding of a new therapeutic target for antibiotics or the use of A. faecalisin infections.

This document only includes an excerpt of the corresponding thesis or dissertation. To request a digital scan of the full text, please contact the Ruth Lilly Medical Library's Interlibrary Loan Department (rlmlill@iu.edu).

No description available.

No description available.

No description available.

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 185-203).