Vitamin k

Streptococcus oralis (ATCC 9811; American Type Culture Collection, Manassas, VA, USA) was cultured overnight on Tryptone Soy Agar (TSA) plates at 37 °C. A few colonies were inoculated into Tryptone Soy Broth (TSB) (Oxoid Limited, Hamsphire, UK) supplemented with 10% yeast extract (Carl Roth GmbH + Co. KG, Karlsruhe, Germany) and 50 mM glucose (Carl Roth GmbH + Co. KG, Karlsruhe, Germany) at 37 °C under constant shaking. Aggregatibacter actinomycetemcomitans (DSM 11123; German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) or Porphyromonas gingivalis (DSM 20709; German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) were cultured for 48 h on fastidious anaerobe agar (FAA) plates (LabM, Heywood, UK), supplemented with 5% sheep blood at 37 °C under anaerobic conditions (80% N₂, 10% H₂, 10% CO₂). A few colonies of A. actinomycetemcomitans or P. gingivalis were inoculated overnight into brain heart infusion medium (BHI; Oxoid, Wesl, Germany) supplemented with 10 μg/mL vitamin K (Roth, Karlsruhe, Germany) under anaerobic conditions [36 (link)]. Treponema denticola (DSM 14222; German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) was cultured in new oral spirochete (NOS) medium at 37 °C for 72 h under static anaerobic conditions [37 (link)].

Streptococcus oralis (ATCC 9811, American Type Culture Collection, Manassas, VA, USA), Aggregatibacter actinomycetemcomitans (DSM 11123, German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany), and Porphyromonas gingivalis (DSM 20709) were cultured on tryptone soy agar (TSA) or fastidious anaerobe agar (FAA) plates supplemented with 5% sheep blood at 37 °C under anaerobic conditions (80% N₂, 10% H₂, 10% CO₂). Few colonies of S. oralis were inoculated into tryptone soy broth (TSB) (Oxoid Limited, Hamsphire, United Kingdom) supplemented with 10% yeast extract (Carl Roth GmbH + Co. KG, Karlsruhe, Germany) and 50 mM glucose (Carl Roth GmbH + Co. KG, Karlsruhe, Germany) at 37 °C under constant shaking. A few colonies of A. actinomycetemcomitans or P. gingivalis were inoculated overnight into brain heart infusion medium (BHI; Oxoid, Wesl, Germany) supplemented with 10 μg/mL vitamin K (Roth, Karlsruhe, Germany) under anaerobic conditions [21 (link)]. Treponema denticola (DSM 14222) cultures were prepared in new oral spirochete (NOS) medium at 37 °C for 72 h under static anaerobic conditions [22 (link)].

Aggregatibacter actinomycetemcomitans (DSM 11123, German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) were cultured on fastidious anaerobe agar (FAA) plates (LabM, Heywood, UK), supplemented with 5% sheep blood under anaerobic conditions (80% N₂, 10% H₂, 10% CO₂) for 48 h at 37°C. A few colonies of A. actinomycetemcomitans from the FAA plate were inoculated overnight into a brain heart infusion medium (BHI; Oxoid, Wesl, Germany) supplemented with 10 μg/mL vitamin K (Roth, Karlsruhe, Germany) under anaerobic conditions (Kommerein et al., 2017 (link)). The overnight cultures were adjusted to OD₆₀₀ = 0.1 in BHI and used for in vitro cell culture assays and infections in the mouse model.