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Title: Proteomics as a tool for the characterisation of nosocomial pathogens
Author: Rajakaruna, L. K.
Awarding Body: Nottingham Trent University
Current Institution: Nottingham Trent University
Date of Award: 2010
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The aim of the present investigation was to develop and assess the potential of various proteomic approaches for the characterisation of two major nosocomial pathogens, S. aureus and C. difficile and to further investigate the intraspecies diversity of C. difficile using a genotypic approach. The surface-associated proteins of S. aureus and intracellular stable ribosomal proteins of C. difficile were analysed by MALDI-TOF-MS and the resulting spectra interrogated using two databases viz. MMU (Waters®) and SARAMIS™ (AnagnosTec) respectively. A total of 134 clinical S. aureus isolates were tested using the MMU database and the MicrobeLynx™ software. All were successfully identified with minor contamination errors that corroborated with 16S rRNA sequencing. By contrast, C. difficile isolates were only partially identified (to the genus level) using the MMU database and protocol. Changes in the matrix solution and use of the new database (SARAMIS™) resulted in the correct identification of all C. difficile isolates and, detailed ultra-structural studies indicated that intracellular proteins were the new diagnostic biomarkers. The cytosolic/membrane-bound proteins of S. aureus and C. difficile were investigated using SELDI-TOF-MS and, potential biomarkers for MRSA and MSSA studied using Artificial Neural Networks. Seven key ions were detected for predicting MRSA and MSSA correctly. 1D- gel electrophoresis was also carried out on both taxa. To detect novel loci to differentiate the different ribotypes (027 and 001) and other ribotypes of C. difficile a molecular method, ‘VNTR’, was undertaken using 92 isolates belonging to three ribotype groups. Ten novel loci were detected and could be used to differentiate between isolates of different ribotypes belonging to 027 and 001. This is now being implemented as an epidemiological tool because of its high reproducibility and throughput for genotyping of the invasive ribotype 027 and 001 of C. difficile.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available