Peptide mediated inhibition of Porphyromonas gingivalis dual and three species biofilms.

The colonization of Porphyromonas gingivalis is the key phase in transformation of bacterial biofilm from a commensal plaque to pathogenic form observed in periodontitis. Objectives: The goals of this study are to establish optimum levels of Porphyromonas gingivalis, Streptococcus gordonii and Fusobacterium nucleatum to reproducibly generate dual and three species biofilms and examine inhibition of biofilm formation by BAR peptide and BAR analogs C1-24 R1182 I1185 BAR, R1182 I1185 BAR and C1-24 BAR. Methods: S. gordonii was grown in BHIY media; P. gingivalis and F. nucleatum were grown in TSBY and BHI media respectively supplemented with hemin and menadione. Labeled S. gordonii was adhered to a cover glass in wells of a culture plate for 18-24 hours and then labeled P. gingivalis or P. gingivalis and F. nucleatum were added. After incubating for 18-24 hours, the cover glass is fixed, mounted onto a slide glass and visualized by confocal laser scanning microscopy. Biofilm images were assembled and analyzed using Volocity software. Results: For establishing the optimum inocula for Streptococcus gordonii, Porphyromonas gingivalis and Fusobacterium nucleatum- biofilms were formed using cultures of optical densities 0.05, 0.1, 0.2, 0.5, 0.8, 1.0 and 2.0. Optimal conditions were found to use S. gordonii at O.D 0.8, P. gingivalis at O.D 0.1(for 2 species), 0.2(for 3species) and Fusobacterium nucleatum at O.D 2.0. After the conditions were established, P. gingivalis was incubated with BAR peptide or BAR peptide analogs (0 -50µg/ml) prior to addition to S. gordonii to assess inhibition of biofilm formation. BAR peptide and peptide analogs significantly inhibited the biofilms. Inhibition of dual species biofilms by R1182 I1185BAR is statistically greater (P < 0.001) compared to parent BAR. Conclusion: Ideal conditions for biofilms were established. BAR peptide and peptide analogs significantly inhibited adherence of P. gingivalis in both dual and three species complex biofilms.