Gingival infection with periodontal pathogens often persists after treatment, and mucosa are thought to be a reservoir for recolonization. Invaded mucosal cells may provide a protected environment for these fastidious anaerobes. Preliminary studies used fluorescent in situ hybridization (FISH) with universal and specific rRNA probes and confocal microscopy (LSCM) to detect Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, and unidentified bacteria inside buccal cells from 23 of 24 subjects. This suggests that intracellular mucosal bacteria exist in a multi-species community which may maintain itself by modulating or overcoming host cell defenses, with exfoliated cells providing a protected route for bacterial transmission. Those postulates will be tested by these specific aims: 1.) Determine the composition of the mucosal intracellular community by probing for additional periodontal pathogens and other mucosal species. 16S rRNA temperature gradient electrophoresis will be used to design probes for uncultured species. Multicolor FISH will determine whether more than one species can occupy a cell. 2.) Determine whether intracellular bacteria modulate or overcome cell defenses in vivo by using multicolor FISH/LCSM to compare invaded and uninvaded cells for expression of cytokine and antimicrobial peptide mRNA. 3.) Determine whether exfoliated invaded cells may be a vector for transmission by using a tissue culture model. Washed mucosal cells will be suspended in autologous or heterologous clarified saliva with or without antibiotics, and then incubated with green fluorescent protein (GFP)-labeled KB cells. Transmission will be evaluated by multicolor FISH/LSCM. 4.) Determine whether the intracellular mucosal community can establish and maintain itself in the absence of the gingival crevice by using FISH/LSCM to look for such communities in predentate infants, and to verify whether they occur in edentulous adults. 5.) Determine whether mucosal invasion protects pathogens from elimination in periodontal patients who require aggressive treatment, by using FISH/LSCM and multiplex PCR to compare mucosal and gingival colonization before and after scaling and root planing, topical chlorhexidine, and systemic antibiotics.
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