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Title: Development of in vitro microbiological systems for determining the antimcrobial potential of topical wound treatments
Author: Thorn, Robin Michael Statham
ISNI:       0000 0004 2694 5467
Awarding Body: University of the West of England, Bristol
Current Institution: University of the West of England, Bristol
Date of Award: 2009
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It is now widely accepted that most if not all open wounds will become contaminated with micro-organisms. This does not necessarily provide a barrier to wound healing, and it is only if the healing processes are disrupted by the presence and metabolic activities of these communities, that the wound will fail to heal. It was the aim of this thesis to develop in vitro microbiological models to simulate the growth of microbes within the wound environment, for testing and comparing the antimicrobial efficacy of novel or existing topical wound treatments. These models could then be applied towards the understanding of the behaviour of microbes when exposed to various biocidal or inhibitory agents, across a range of microbial physiological and environmental physicochemical conditions. An existing in vitro static diffusion model was modified to allow for 3 day repeat challenge capacity testing of topical wound treatments. This proved to be a powerful method able to differentiate the sustained antimicrobial potential of various test treatments. Association with surfaces is almost certainly the prevailing lifestyle in the wound bed, hence an in vitro perfusion biofilm model was developed. This enabled the growth of quasi-steady state P. aeruginosa (Jl = 0.13-1.16 h- l ) and S. aureus (Jl = 0.22-0.26 h- l ) biofilms, as determined by measuring the specific biofilm growth rate, facilitating topical antimicrobial therapies to be applied, and their effects continually monitored. This dynamic model produced antimicrobial kill kinetic profiles which could differentiate bactericidal from bacteriostatic effects, and could potentially predict the efficacy of a given therapy in vivo where a biofilm is present. A whole-cell bioluminescent P. aeruginosa reporter was integrated into both the static diffusion and perfusion biofilm models to monitor the effects of antimicrobial therapy in real time. There was found to be a strong correlation between bioluminescence readings and viable counting under the defined experimental conditions. This enabled the state of the systems to be continually monitored without disturbance, allowing more immediate and accurate calculations of antimicrobial kinetics. Microbial specific growth rate dramatically affects the structure and composition of microbial cells, therefore this was investigated using a chemostat, to ascertain whether it affected the sensitivity/resistance of cells to a range of bacteriostatic and bactericidal agents. The growth rate of target cells significantly affected their sensitivity/resistance to the test agents, although there were no general trends and the response was largely agent specific. Furthermore, biofilm cells grown within the flat bed perfusion model were growth rate matched to chemostat grown cells, and their antimicrobial sensitivity compared. The results largely showed that growth rate was the greatest predictor of antimicrobial sensitivity to the panel of test antimicrobial agents. The in vitro models developed, especially those that allow growth rate measurement and control, can further our understanding of the response of wound prevalent microorganisms to topical treatments, within an environment that models 'some' of the parameters that are believed to be present in at least some wounds. Therefore the antimicrobial kill kinetic data produced by these models could help direct research and development into novel antimicrobial therapies, as well as potentially helping to inform topical treatment selection in the clinical environment.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available