Furst, Vanessa Wyndham; (1993) Bacterial taxonomy - Analytical biochemical methods with special reference to Mycobacteria. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Bacterial taxonomy is particularly important in medicine, where the correct identification of an organism enables the optimal treatment to be prescribed. Currently medical microbiology uses a compilation of sequential morphological, staining, biochemical and serological tests to identify an organism. Many of the routine identification tests do not enable sub-speciation of an organism. Sub-speciation can be important in routine circumstances, particularly for infection control; studying the epidemiology of an organism by sub-speciation is vital. Thus there is a need for new microbiological tools and methods to advance microbiological knowledge and this is the basis on which this study was undertaken. A universal feature of all cells is the uptake and utilisation of certain substances for the synthesis of proteins, the essential tools of cell metabolism. The bacteria were incubated with [35S] methionine and the radiolabelled samples were separated by SDS PAGE. The resulting "bar code" type pattern was then examined by autoradiography and more importantly using a Radioanalytic imaging system that enabled computer analysis of the data. The data were extracted as histograms and normalisation strategies assessed. The data were then grouped and analysed by dendrogram or used in a database for identification. The methods were standardized to allow comparisons of different species and the use of other sources of [35S] such as inorganic sulphate and thio ATP were investigated. All organisms gave labelled patterns with the methionine (except for Mycobacterium leprae) and most did so with the sulphate and ATP and the data were used to investigate speciation and sub-speciation. Mycobacteria were of particular interest because they are slow growing and there is a need for rapid identification and sub-speciation techniques. Mycobacteria have very thick cell walls with a very high lipid content, this made standardisation of cell break down for analysing the cell content difficult, so the secreted proteins were predominantly analysed. Mycobacterium leprae presented particular problems and some studies of the metabolism of this organism were carried out in order to try and apply the labelling methods to this organism. It was not possible to reproducibly label M. leprae proteins but some useful metabolic studies were made. Mycobacteria are unique bacteria in producing iron binding compounds such as mycobactins which are known to be species specific. In this study the methods for extracting, labelling with [55Fe] and separating mycobactins by TLC were improved to increase detection sensitivity and their potential for rapid identification of mycobacteria from clinical specimens was assessed.

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