Within the five experimental chapters of this doctoral thesis (i) the eubacterial diversity of the microbiota of chronic wounds and healthy skin was investigated, (ii) biofilm formation and associated coaggregation interactions of wound and skin-associated bacterial isolates was examined, (iii) formulation of media which reproduced some aspects of the nutritional conditions of wounds and healthy skin were developed, (iv) novel wound biofilm models were developed and validated and (v) microbial population interactions associated with healthy skin and chronic wounds were investigated using a novel model system. (i) The microbial diversity of chronic wounds and contralateral skin swabs was investigated using culture, denaturing gradient gel electrophoresis (DGGE) and microscopy. Intrapersonal analysis identified that non-infected wounds had a proportionally higher incidence of bacteria which were identified on contralateral healthy skin according to DGGE analysis when compared to infected wounds indicating that taxonomically distinct consortia are associated with infection. Microcolonies and putative biofilms structures were identified in both culture-defined infected and non-infected wounds indicating that the presence of biofilms may not be linked to infection. (ii) By assessing pair-wise combinations of skin and wound-associated bacteria, the role of coaggregation in the formation of wound polymicrobial communities was assessed using a quantitative spectrophotometric assay. Aggregation interactions were weak or not detectable, apart from those associated with Corynebacterium xerosis. This bacterium produced a high autoaggregation score (c. 50%). The limited coaggregation interactions suggest that coaggregation may be comparatively unimportant in the development of wound biofilms. (iii) In order to facilitate the development of biofilm models specific to chronic wounds, the formulation of representative growth media is important in order to reproduce the in situ nutrient environment. Therefore complex, artificial sweat and serum media broadly reflective of the nutrient availability in wounds and healthy skin were developed and validated based upon their ability to support realistic phenotypes (assessed by proteomics) and the growth of a range bacterial isolates. Developed media maintained the sessile growth the test bacteria and produced broadly similar proteomic profiles to foetal calf serum. (iv) Two novel model systems were developed to study cross-sectional population interactions and to investigate longitudinal population development of wound consortia and biofilm formation. A fine celled foam (FCF) multi-well wound model and a multiple membrane FCF model maintained dynamic steady state of axenic and mixed populations of bacteria associated with chronic wounds and supported the development of biofilms. (v) The FCF multi-well wound model was used to investigate population interactions in environments broadly reflective of healthy skin and wounds. When grown in artificial sweat prior colonisation with Staphylococcus saprophyticus resulted in a significant reductions in methicillin resistant Staphylococcus aureus (99%) and P. aeruginosa (75%) whilst prior colonisation by C. xerosis resulted in a significant reduction in P. aeruginosa (91%) only. However no significant reductions in pathogenic bacteria were noted in artificial serum indicating colonisation resistance could be simulated in the model and the outcome of immigration was markedly influenced by the species of established bacterium and nutrient availability.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:764196 |
| Date | January 2011 |
| Creators | Oates, Angela |
| Contributors | Mcbain, Andrew |
| Publisher | University of Manchester |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://www.research.manchester.ac.uk/portal/en/theses/the-microbial-ecology-of-chronic-wounds(54e6dce0-cffa-4f03-9701-7bc1ea907af9).html |
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