Culturing and Metronidazole Treatment of P. gingivalis

P. gingivalis (ATCC 33277) was employed and cultured following our established protocol [19 (link)]. Frozen stocked bacteria were first grown on blood agar plates (44 g L⁻¹ BD Columbia agar base (Becton Dickinson GmbH, Heidelberg, Germany), 5% horse blood (Hemostat, Dixon, CA, USA), 5 mg L⁻¹ hemin (Sigma-Aldrich, St. Louis, MO, USA), 1 mg L⁻¹ vitamin K1 (Sigma-Aldrich, St. Louis, MO, USA)) in an anaerobic atmosphere at 37 °C (10% H₂, 5% CO₂ and 85% N₂). After one week, a single colony was picked and placed in liquid Trypticase soy broth (30 g L⁻¹ TSB; Becton Dickinson GmbH, Heidelberg, Germany) supplemented with 5 g L⁻¹ yeast extract, 5 mg L⁻¹ hemin and 1 mg L⁻¹ vitamin K1; it was then cultured under the same anaerobic conditions.
M-PgPs formation was performed following our previous study [19 (link)]. In brief, P. gingivalis was cultured in broth for 48 h and re-suspended in fresh broth to an OD600 of 0.1. Subsequently, the bacteria were incubated in the stationary phase (72 h) and further treated with metronidazole (MTZ, Sigma-Aldrich, St. Louis, MO, USA) at 100 mg L⁻¹ for 6 h. It was confirmed by our recent study that about 1% of the P. gingivalis cells remained viable after the 6 h MTZ treatment [51 (link)].