Award details

Design sysnthesis and analyisis of novel derivatives of industrially important chromogens for improved bacterial detection

Reference	BB/H015825/1
Principal Investigator / Supervisor	Professor Helen Osborn
Co-Investigators / Co-Supervisors	Dr Richard Bovill
Institution	University of Reading
Department	Pharmacy
Funding type	Skills
Value (£)	75,281
Status	Completed
Type	Training Grants
Start date	01/10/2010
End date	30/09/2014
Duration	48 months

Abstract

unavailable

Summary

In the medical and food sectors it is vitally important to quickly and accurately detect and identify pathogenic or food spoilage bacteria in samples. Molecular methods are showing great promise in this area but there is still a need to obtain the organism as a pure viable entity on a culture medium for further identification or typing and to obtain details of antibiotic susceptibility that may be used by the clinician for effective treatment of infection [1]. Many selective culture media formulations have changed little since their introduction 50 years or more ago with the exception of the introduction of chromogenic substrates to agar media some 15 years ago. The latter are generally in the form of a sugar joined to a chromogen to form a glycoside that is colourless. Hydrolysis of the glycoside by an appropriate enzyme releases the chromogen and a coloured halo appears around the bacterial colony. Glycosidase enzymes are not widespread and some may be restricted to only a few species that grow on a particular selective medium. This allows a presumptive identification of the organism saving the analyst a great deal of time and effort [2]. Different sugars may be added to the same chromogen to alter specificity since only the sugar affects enzyme activity [3]. In this multidisciplinary project the student will be involved in the design, synthesis and analysis of novel derivatives of industrially useful chromogens for bacterial detection. They will use computational methods to predict the visual absorption maxima and hence the colour of novel derivatives of known chromogens, for example by using software programmes such as WinPPP and/or PISYTEM. Chromogens of interest within the project include the reactive dyes such as Black 5 and Orange G. A particular aim of the project will be to develop novel chromogens with colours that complement the existing range of available chromogenic substrates. This will allow a greater multiplicity of chromogenic substrates to be used in media to separate organisms in mixtures that are difficult to separate on the presence or absence of one or two particular enzymes. A further aim will be to extend the range of sugars linked to chromogens to determine if glycosides not currently commercially available are useful for the differention of bacterial species. Glycosides will be synthesised using organic chemistry techniques and the student will be trained to perform both large and small scale reactions, and also to handle air sensitive materials. The development of some new methodology will be required. Materials will be purified using a range of techniques such as chromotography and recrystallisation. Glycoside products will then be characterised using state of the art analytical chemistry techniques, such as NMR and UV-Vis spectroscopy, and mass spectrometry. Biological analysis will then be performed to determine the ability of specific glycosidase enzymes to release the chromogen from the substrates, to afford coloured precipitates. Studies using whole bacteria present within food and clinical samples will then be performed to determine whether the substrates do indeed allow identification of particular bacteria, based on specific glycosidase enzymes that they contain. Due to the stringent specificity of glycosidase enzymes, it is essential that individual isomers (anomers) of the targets are prepared for the biological results to be meaningful. Relevant synthetic expertise in both carbohydrate chemistry and prodrug therapy within Professor Osborn's group will ensure that pure individual isomers will indeed be prepared. This synthetic expertise will be complemented by that within Dr Bovill's group, which specialises in the analysis and inhibition of growth of food borne pathogens. [1] Orenga, S. et al., J. Microbiol. Methods, 2009, in press [2] Kiernan, J.A. Biotech. Histochem., 2007, 82, 73 [3] Butterworth, L.A. et al., J. Appl. Microbiol., 2004, 96, 170

Committee	Not funded via Committee
Research Topics	X – not assigned to a current Research Topic
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	Training Grant - Industrial Case

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