It has been suggested that fungal-bacterial mixed-species biofilms may be more virulent as a result of cross-kingdom interaction mediated by fungal cell-wall's mannans. We aim to evaluate the role of beta-mannanase on mixed-species biofilm development through the disruption of mannan-mediated bacterial-fungal interaction. Specifically, this study aims to determine the effects of beta-mannanase on bacterial-fungal binding forces and on 3D structure and on mechanical stability of biofilms. A well-characterized and validated biofilm model based on the co-cultivation of C. albicans and S. mutans will be used to better standardize experimental conditions for mannanase activity (Study 1). Then, mannanase will be tested against S. schenckii and Staphylococci using the above mentioned model and previously standardized experimental conditions (Study 2). C. albicans and S. schenckii will be exposed to beta-mannanase and after 60 minutes cell viability will be assessed by Time-Lapse Confocal Fluorescence Imaging, cell morphology will be assessed by Scanning Electron Microscopy and mannanprotein will be quantified on fungal cell-wall after exposure to beta-mannanase. Additionally, the effect of beta-mannanase on binding forces between C. albicans and S. mutans and between S. schenckii and Staphylococcus aureus or Staphylococcus epidermidis will be assessed by Single-Molecule Atomic Force Microscopy. Moreover, S. mutans/C. albicans single-species or mixed-species biofilms and S. schenckii/ S. aureus or S. epidermidis single-species or mixed-species biofilms will be grown for up to 72 hours and exposed to beta-mannanase twice a day. Biofilms will be assessed via Confocal Laser Scanning Microscopy to determine their 3D structure as well as number of microcolonies and microcolonies size. Yet, the effect of beta-mannanase on cohesiveness and mechanical stability of biofilms will be assessed by a Shear-induced Biofilm Mechanical Strength Tester under constant shear stress. It is expected that virulence and development of fungal-bacterial mixed-species biofilms be controlled by the disruption of mannan-mediated interactions.
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