Biofilm formation on titanium (Ti) implant surfaces can trigger inflammation in the adjacent mucosal tissues. Mixed fungal-bacterial biofilms, consisting of Candida albicans and oral streptococci of the mitis group, have been shown to trigger an exaggerated inflammatory response. However, the growth of mixed fungal-bacterial biofilms on Ti and its effect on mucosal inflammation has not been studied. Moreover, a reciprocal effect of mucosal inflammatory products on such biofilm growth has been suggested but never been tested. Additionally, the role of extracellular matrix and glucosyltransferases (GTF) in the interaction of Candida and members of the mitis group remains unknown. To test these hypotheses, (1) single species (Streptococcus oralis, Streptococcus mitis, Streptococcus sanguinis, Streptococcus gordonii or C. albicans alone) and dual species (C. albicans with each streptococcal species together) biofilms will be formed on Ti disks. Furthermore, the microorganisms will be inoculated on the disks and will be suspended above an organotypic oral mucosal constructed to test whether the mucosal environment promotes the interaction. (2) To test the effect of single and dual species biofilms on the mucosal inflammatory response, the mucosal will be exposed to pre-formed mature Ti surface biofilms. Tissue media will be collected at different time points and analyzed simultaneously for multiple proinflammatory cytokines using Luminex technology. Tissue supernatants with increasing concentrations (serially dilution) to supplement biofilm growth media and mix of recombinant cytokines will be used to test the effect of cytokines on biofilm growth. (3) To study the role of biofilm matrix and streptococcal GTF in mixed biofilm development, biofilms will be grown under two different conditions, 0% or 2% sucrose, since sucrose serves as a substrate for synthesis of extracellular polymers. Additionally, to assess the role of GTF to promote intercellular fungal-bacterial binding, mixed Candida-S. oralis or -S. gordonii biofilms with gtfR-negative mutant in S. oralis strain 34 and a gtfG-negative mutant in S. gordonii strain CH1 will be used.
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