Bismuth plus metronidazole synergistically dampens Porphyromonas gingivalis persisters formation

Abstract

Objectives: P. gingivalis is the ‘keystone’ periodontopathogen. Our group has recently demonstrated that metronidazole-treated P. gingivalis persisters invade human gingival epithelial cells (HGECs) and perturb innate host response, and yet bismuth drugs markedly suppress P. gingivalis. This study explored the potential synergistic effects of bismuth drugs combined with commonly used antibiotics on the formation of P. gingivalis persisters. Methods: P. gingivalis cells (ATCC 33277) in planktonic and biofilm states were cultured to stationary phase and treated with colloidal bismuth subcitrate (CBS, 100 μM), metronidazole (100 μg/ml), amoxicillin (100 μg/ml) or different combinations of these medications. Drug concentrations of planktonic culture were measured after 6-h, 24-h, and 48-h treatments, and persister formation rate (PFR) was counted by colony-forming unit accordingly. P. gingivalis persisters in biofilms were detected by LIVE/DEAD staining after 72-h treatment. Cell viability and cytotoxicity of HGECs were tested after treatments with various concentrations of CBS for 24 h, 48 h, and 72 h. Results: P. gingivalis persisters in planktonic culture were identified (PFRs: 10-5 to 10-2) after 48-h treatments, and concurrently lethal dosages of CBS (> 20 μM), metronidazole (> 17 μg/ml) and amoxicillin (> 15 μg/ml) remained to be detectable. Notably, P. gingivalis persisters were remarkably eradicated (PFR:10-8), in both planktonic and biofilm states, by CBS combined with metronidazole, with reference to CBS or metronidazole alone. No such significant effects were observed in the combination of CBS and amoxicillin (PFR: 10-4 ) or metronidazole and amoxicillin (PFR: 10-4 ). Importantly, cell viability of HGECs was not affected by CBS, and very little cytotoxicity (< 5%) was detected even with a high concentration of CBS (400 μM). Conclusions: This study suggests that synergistic use of bismuth drugs and metronidazole dramatically tackles P. gingivalis persisters formation, and such novel approach may contribute to effectively controlling periodontitis and P. gingivalis-related systemic diseases.