Chronic wounds are a rapidly growing clinical problem across the world, expensive to healthcare systems and severely injurious to the quality of life of those affected. Such wounds have been shown to contain bacteria within biofilms which can be difficult to identify and treat, and are implicated in the lack of healing. The aim of this study was to improve our understanding of the characterisation of these bacterial communities within chronic wounds. This was achieved through the collection of 18 unique chronic wound samples of various phenotypes and 9 acute wound samples. The two bacterial species, Staphylococcus .aureus and Pseudomonas. aeruginosa, most commonly isolated from chronic wounds and known to be prolific biofilm formers and formidable human pathogens were the focus of this study. Both PCR and culture demonstrated the presence of one or both species in all chronic wound samples and the presence of S. aureus in some acute samples. Novel monoclonal antibodies were developed to TNase (of S.aureus) and LPS (of P. aeruginosa) and their ability to detect the bacteria in vitro in a simulated chronic wound environment was evaluated. The action of these monoclonal antibodies were calibrated and evaluated through the use of fluorescent microscopy, and direct and competition ELISA. Following this biofilms of S.aureus and P. aeruginosa both single species and mixed were evaluated utilising Peptide Nucleic Acid Flourescent In Situ Hybridisation, and the Confocal Laser Scanning Microscope to demonstrate the architecture of the biofilms produced and the locations of the bacteria within the biofilm. Microbiologically using both culture and PCR, the widespread presence of S. aureus and P. aeruginosa throughout human chronic wound samples was demonstrated. The ELISA and fluorescent microscopy illustrated the feasibility of MABs as a rapid and accurate detection system for identifying bacteria within chronic wounds. The PNA FISH accurately identified individual species in mixed biofilms through multiplex staining. It also visually demonstrated the tissue invasion of S. aureus, adhesive properties of P. aeruginosa and the synergy of these virulence factors in mixed biofilms. This study has met its aims in that it has provided further evidence of techniques which could be successfully applied to rapidly identify bacteria within a chronic wound environment, and to characterise the structure and composition of bacterial biofilms formed within these wounds. It provides a basis for the development of future clinically relevant rapid bacterial detection systems, and for the academic study of bacteria within the biofilm phenotype.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:567440 |
| Date | January 2012 |
| Creators | Riddell, Andrew |
| Publisher | Cardiff University |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://orca.cf.ac.uk/41930/ |
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