Saudi Cultural Missions Theses & Dissertations
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Item Restricted In vitro and in vivo models to study the effect of biofilms and antimicrobial wound dressings on wound healing(The University of Manchester, 2024-06-25) Baqader, Sajwa; Thomason, Helen; Humphrey, Gavin; McBain, AndrewBackground: Biofilms have been strongly implicated in delayed wound healing. Reduced oxygen and low microbial growth rates within wound biofilms are a recognized driver of tolerance towards antimicrobial agents, and poor treatment outcomes. There is however uncertainty about the contribution of biofilm to delayed healing and the mechanisms involved. This emphasises the need to examine the differences in the wound healing process between the planktonic and biofilm-infected wounds through preclinical models. Improved knowledge in this area may lead to the development of improved treatment strategies. Antimicrobial chemicals, especially silver-based ones, are frequently employed in wound dressings, but little is known about their efficacy and there are few reliably predictive preclinical wound models. This doctoral thesis focuses on visualising the variations in metabolic activity in bacterial cells during biofilm development and studying the differences in the wound healing process between planktonic and biofilm-infected wounds. This thesis also evaluates the antibacterial efficacy of innovative wound dressings that incorporate silver oxynitrate, which produces highly reactive Ag2+ and Ag3+ ions, using in vitro and in vivo models. Methods: Initially, a 96 well plate-based assay was used to determine the bactericidal activity of dressings containing silver oxynitrate and untreated controls against planktonic S. aureus in both log- and stationary phases. A colony biofilm model was then used to assess the efficacy of dressings containing silver oxynitrate in eradicating biofilms over 1, 3, and 7 days. Next, an in vivo excisional wound model either uninfected or infected with 104 S. aureus in planktonic form, or as preformed biofilms was established, and used to assess effects of dressings containing silver oxynitrate compared to non-antimicrobial controls and uninfected wound controls. Bromodeoxyuridine (BrdU), a nucleotide analogue that incorporates into nascent DNA in actively dividing cells was used to visualise proliferating. Biofilms were exposed to BrdU for 4 h before harvesting at 1, 3 and 7 days. Wound healing was characterised by quantification of wound width, area, and re-epithelialisation from histological sections. Inflammation was determined based on neutrophil/macrophage marker immunohistochemistry. Wound biofilms were characterised by anti- BrdU immunofluorescence, fluorescence in-situ hybridisation specific prob, viability mapping, tissue Gram stain, quantitative bacterial culture, and scanning electron microscopy. Results: Strong BrdU staining was observed corresponding to active growth of 1 day S. aureus biofilm and slow metabolic rates occurred following 3 days old biofilms. No BrdU signal was detected in the 7 days old biofilms of S. aureus. Over 7 days, wound width and areas increased significantly in wounds infected with preformed biofilm and in planktonic infected wounds compared to uninfected wounds. A significant reduction in the reepithelialisation percentage was observed in wounds infected with preformed biofilms and infected planktonic wounds. In contrast, uninfected wounds exhibited a small wound width, area, and comparatively high reepithelialisation percentage. The number of neutrophils and macrophages was significantly higher in wounds infected with preformed biofilm than wounds that infected with planktonic bacteria. S. aureus abundance on surface of both planktonic and biofilm infected wounds was markedly increased after seven days. Dressings containing silver oxynitrate significantly reduced planktonic bacterial cell counts more during log phase than stationary phase. Furthermore, this dressing effectively decreased number of bacterial cells in colony biofilm following a 7-day exposure. The dressings containing silver oxynitrate also significantly reduced S. aureus viable counts, wound width, area, inflammation, and increased reepithelialisation more in planktonic infected wounds than biofilm infected wounds over 7 days. SEM imaging revealed that infected wounds treated with silver oxynitrate-incorporating dressings contained fewer bacteria and biofilm extracellular polymeric substance was disrupted. Conclusion: The in vitro colony biofilm method of labelling metabolically active S. aureus with BrdU over 7 days demonstrated the bacterial cells dormancy. The murine wound model provides evidence of the impact of S. aureus in planktonic and biofilm form in delaying healing process. The use of dressings containing silver oxynitrate demonstrated a greater impact on the treatment of infected murine wounds in both states, with greater efficacy against planktonic infected wounds. The in vitro and in vivo models mentioned above can be used to evaluate effectiveness of other wound treatments obtain better wound management and patient outcomes.19 0