Combination chemotherapy using Autoinducer-analog and Antibiotics for Porphyromonas gingivalis Biofilm

Objectives: Quorum sensing is a bacterial cell-to-cell communication system using the signaling molecules called autoinducer (AI), and some AI analogs inhibit the biofilm formation. Porphyromonas gingivalis locates in subgingival and extraradicular biofilm, and is associated with severe marginal periodontitis and refractory periapical periodontitis. We reported that 3 out of 17 AI analogs examied decreased the numbers of biofilm-forming P. gingivalis cells in vitro. P. gingivalis biofilm became thinner in the presence of analogs, it is assumed that antibiotics are more effective for analog-treated biofilms. The purpose of this study was to investigate the effects of combination chemotherapy using AI analog and antibiotics on P. gingivalis biofilm. Methods: The 3 antibiotics (sefloxime, ofloxacin and minocycline) were prepared. The analog was C₁₀H₂₀N₂O₂. P. gingivalis 381 biofilms were anaerobically performed on the hydroxyapatite and celluloid disks by the flow-cell system. Samples were divided into 4 groups, which were control group, antibiotics-treated group, analog-treated group and combination-treated group. The biofilm forming cells were assessed using adenosine triphosphate (ATP) bioluminescence assay, scanning electron microscopy (SEM) and confocal laser microscopy (CLSM). Results: In the combination-treated group, ATP value was significantly decreased compared with those of antibiotics- and analog-treated groups. In CLSM observation, the proportion of dead cells were highest in combination-treated groups. In SEM observation, part of the extracellular matrix structures had disappeared in minocycline- and sefuroxime-treated groups. In ofloxacin-treated group, the outline of each cell was not well defined because biofilm-forming cells were covered with extracellular matrix structure. Conclusion: Combination chemotherapy using AI analog and antibiotics were effective to inhibit P. gingivalis biofilm, and may be successful for control the oral biofilm. This study was supported by a Grant-in-Aid for Scientific Research (#22791833 and #21390508) from the Japan Society for the Promotion of Science.