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Chronic wound infections represent a worldwide problem, generating high morbidity and medical expense. Failure to control infections such as MRSA in the reparative process of a wound can cause disruption of normal anatomical structure and function, resulting in a chronic wound. Existing approaches to identifying infection largely involve surveying a range of physical parameters, and a limited use of non-invasive technologies. Evaluation is time consuming, and often results in inconsistencies in patient care. This project researches three possible alternative methodologies/technologies for the monitoring of wounds, by measuring components of wound fluid. Two of the three technologies are designed to be used by physicians and patients, similarly to commercially available home blood glucose test kits, and are based on the measurement of three biomarkers: glucose, ethanol and H2O2 in PBS, and in serum as surrogate wound fluid. The first is a voltammetric technique known as dual pulse staircase voltametry (DPSV), which produces peaks characteristic of particular analytes at an electrode. The second is an amperometric biosensor array, based on screen printed three electrode assembies of carbon, rhodinised carbon (glucose biosensor only) and Ag/AgCl reference. The glucose biosensor uses glucose oxidase enzyme as the biorecognition agent, the H2O2 biosensor is a mediated system using horseradish peroxidase enzyme and dimethylferrocene mediator, and the ethanol biosensor is a bienzyme mediated system utilising alcohol oxidase enzyme horseradish peroxidase enzyme and coupled dimethylferrocene mediator. Wounds are known to produce characteristic odours, therefore the third technology studied is a single sensor odour analyser with advanced data analysis to detect five commonly occuring wound bacteria, S.aureus, K.pneumoniae, S.pyogenes, E.coli and P.aeruginosa in growth media and surrogate wound fluid. This technology would be used as a 'near patient' monitoring system and is based on machine olfaction similar to that of a commercial electronic nose, but uses a single metal oxide sensor in combination with principle components analysis. DPSV scans of the individual analytes demonstrated distinctive peaks, exhibiting nonlinear relationships with concentration. A great deal of useful information was generated using this technique, however, limitations were discovered regarding repeatability and inter-analyte interference in mixtures. Limits of detection in surrogate wound fluid with the glucose biosensor, hydrogen peroxide biosensor, and ethanol biosensor were as follows: 169.5 µM glucose, 8.43 µM hydrogen peroxide, and 7.94 µM ethanol respectively (all at 99.7% confidence). Direct detection of ethanol from metabolically active S.aureus in surrogate wound fluid yielded a limit of detection of 1.23 x 108 CFU/ml at 99.7% confidence, and 19 µM in terms of ethanol specific response. The single sensor odour analyser demonstrated the ability to detect and discriminate between the three biomarkers, between five bacteria individually, and partial discrimination of paired bacteria (in broth and surrogate wound fluid). It was also found that S.aureus could be detected down to a cell density of 5x106CFU/ml in surrogate wound fluid, lower than that found for the biosensor concept.

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Biorilms are structured communities of bacterial cells enclosed in a self-produced polymeric matrix that are adherent to an inert or living surface. Their growth on the surface of catheter lines, artificial joints and other surgical implants is of major medical concern. As many laboratory strains have become accustomed to standardised conditions, they are not necessarily representative of clinical strains. This study investigated the effect of horse blood, human blood and human blood components on biofilm. formation by clinical and laboratory isolates of Escherichia coli and Campylobacter jejuni using a 96-well microtitre plate method, crystal violet staining and confocal laser scanning microscopy. The results for the E. coli laboratory isolatesi ndicated a decreasein biofilm fonnation in Luria-Bertani (LB) broth supplemented with lysed horse blood, whole and lysed human blood, human plasma, human serum and whole and lysed human red blood cells. Conversely, the results for the E. coli clinical isolates showed an increase in biofilm formation in LB broth supplemented with either lysed horse blood or lysed human blood. Supplementation with human serum decreased biofilm formation by these clinical strains, whilst the addition of human plasma appeared to be inhibitory to biofilm formation. Of the clinical E. coli, only the urine derived strains showed a significant increase in biofilm formation in LB broth with added whole human red blood cells or whole human blood and produced haemolysin. The results for the C. jejuni isolates used in this study indicated a large increase in biofilm formation when grown in Bolton Broth (BB) supplemented with lysed horse blood, lysed human blood or lysed human red blood cells. This increased biofilm formation was not observed for cultures grown in BB supplemented with human serum, human plasma, whole human blood, iron or whole human red blood cells.