Background: Periodontitis is a complex infectious disease and P.gingivalis and F.nucleatum are considered important pathogens in disease pathogenesis. The two bacteria also linked to each other via coaggregation and metabolic synergism. Previous studies have shown a destructive synergistic effect of mixed infection with the two bacteria. However, the host response to the mixed infection in the molecular level was never investigated. Objectives: (1) To investigate the local inflammatory response to mixed infection of P.gingivalis and F.nucleatum (Fn/Pg). (2) To investigate the effect of immunization on the host response to the mixed infection. Methods: The subcutaneous chamber model in mice was used. Different concentrations of Fn/Pg were injected into the chambers, and compared to infection with single bacterium. In addition, the effect of immunization on the mixed infected animals was evaluated using animals immunized with F.nucleatum or P.gingivalis. The mice were challenged with mixed infection of Fn/Pg. The content of the chambers were collected at baseline, 2 hours and 24 hours post-challenge for cell and cytokine analysis. Results: Infection with Fn/Pg significantly increased all the intra-chamber cytokines levels. Cytokines levels induced by 10⁷ and 10⁸ CFU bacteria were significantly higher than 10⁵ and 10⁶ CFU bacteria. In addition, animals infected with 10⁷ and 10⁸ Fn/Pg, but not with 10⁵ and 10⁶ bacteria, showed clinical signs of sickness. Using the sub-clinical doses of Fn/Pg, mixed infection induced higher levels of TNFα, IL1β and IL10 (but not IFNγ) levels compared to single bacterium infection (F.nucleatum or P.gingivalis). Immunized animals against either bacterium showed lower TNFα and IFNγ compared to non-immunized animals infected with low concentration of Fn/Pg. Conclusions: Mixed infection with Fn/Pg induced stronger inflammatory response than single bacterium infection, and the combination of the two bacteria were found to be synergistic. Immunization with either bacterium was found to suppress the inflammatory response.

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