L 1 hemin

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)].

The bacterial strains Streptococcus oralis CECT 907T, Veillonella parvula NCTC 11810, Actinomyces naeslundii ATCC 19039, Fusobacterium nucleatum DMSZ 20482, Aggregatibacter actinomycetemcomitans DSMZ 8324, and Porphyromonas gingivalis ATCC 33277 were selected for bacterial growth in blood agar plates (Blood Agar Oxoid N° 2, Oxoid, Basingstoke, UK), supplemented with 5% (v/v) sterile horse blood (Oxoid), 5.0 mg L-1 hemin (Sigma, St. Louis, MO, USA), and 1.0 mg L-1 menadione (Merck, Darmstadt, Germany), under anaerobic conditions (10% H₂, 10% CO₂, and balance N₂) at 37 °C for 24–72 h.