Thesis (M.S.)--University of Wisconsin--Madison. / Typescript. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 60-66).

The purpose of this study was to determine the effect of varying concentrations of geosmin on the growth of Bacillus cereus.

This thesis concentrates on the Bacillus cereus group of organisms and interactions that they may encounter in their natural environment. Inorganic polyphosphate has been identified as an important factor of stress and survival in B. cereus. One of the aims of this project was to create knock out mutants of certain enzymes involved in polyphosphate metabolism in B. anthracis, the etiological agent of anthrax. Unfortunately, even though B. anthracis is very closely related to B. cereus and despite the application of published methods it was not possible to create these B. anthracis knockout mutants. In order to address the importance of inorganic polyphosphate in B. anthracis, a real time RT‐PCR assay was developed to monitor the mRNA levels of these enzymes when the bacterium is faced with harsh nutrient environments Real time RT‐PCR analysis showed that mRNA levels of the metabolizing enzymes were upregulated in low nutrient conditions but that the profiles of gene expression were varied when grown in a chemically defined media. In addition to abiotic stresses such as low nutrients, B. anthracis is also likely to face biotic stress such as predation by amoeba in the soil. Investigations were performed into the outcome of the interaction of B. cereus group bacteria with a model amoeba, Acanthamoeba polyphaga. Amoebae are bacterial predators but can also be utilised as hosts by bacterial symbionts and pathogens, such as Legionella pneumophila. It was theorised that amoebae may provide a host environment similar to that of the professional macrophages, which B. anthracis encounters in mammalian infection. These investigations confirmed that the B. cereus group bacteria demonstrate a range of interactions with amoeba cells, from surface attachment through to intracellular persistence. These studies went on to show that B. cereus, B. thuringiensis and B. anthracis can all be engulfed by amoebae when challenged in their vegetative form and that spores were able to survive, and apparently germinate. Finally these studies have identified a new developmental stage of the B. cereus group bacteria. When grown in static conditions, especially in the presence of amoeba, the bacterial cells cease to septate and large (often motile) continuous hyphae like filaments form. These filaments can be seen to “weave” together to form large “rope” like macrofibre structures which can even become visible by eye. Previously this macrofibre growth has also been seen in B. subtilis, suggesting it may be common to the whole genus. In the light of these findings we speculate that this group of pathogens have evolved complex behaviours to interact with soil amoeba in order to facilitate survival in harsh environmental conditions.

The Bacillus cereus sensu lato group is composed of six closely-related bacterial species, exhibiting a broad range of virulence activities. B. anthracis causes anthrax to human and mammals and B. thuringiensis is an entomopathogen against Lepidopteran, Coleopteran and Dipteran larvae. B. cereus s.s. and B. weihenstephanensis are opportunistic bacteria, causing gastro-enteritis and more acute diseases in immuno-compromised people. B. mycoides and B. pseudomycoides are generally considered as non-pathogenic. In addition to the specific virulence factors, several generic toxins have been described in the B. cereus s.l. members (phospholipases, enterotoxins, proteases and hemolysins). Among these virulence factors, cereolysin O was first characterised in 1967 by Cowell and Bernheimer, who suggested it to be a member of the cholesterol-dependent cytolysins. This toxin family cause the lysis of cholesterol-containing membranes and their activity, inhibited by oxidising agents, is restored by reducing agents. Moreover, small amount of free cholesterol irreversibly inactivate the toxins. The first part of this thesis gives a description of the hemolytic activities of the different species of the B. cereus s.l. members, with regard to the international ISO norm for the characterisation of B. cereus s.s. in food. One hundred strains were analysed for their hemolytic and lecithinase activities on different blood-containing media, and the results showed that the most sensitive was the sheep erythrocytes-containing medium. The hemolytic activity of B. cereus s.l. is due to at least five hemolysins whose precise roles in pathogenesis are largely unknown. The continuation of this thesis was focused on one of these hemolysins, cereolysin O. PCR, RFLP and Southern blotting analysis pointed out that the clo gene was present and highly conserved in all the strains of the six tested species. The results also indicated that two copies of the clo gene are present in at least 20% of the strains. The last part of this work was to evaluate the hemolytic activity of Clo by knock-out and cloning experiments. The results revealed the importance of cereolysin O in the lysis of human blood as it is not the case on sheep blood.

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Tübingen, Univ., Diss., 2009.

Thesis (M.S.)--University of Wisconsin--Madison, 1969. / eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (M.S.)--University of Wisconsin--Madison, 1971. / eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.