Vitamin k1

Five bacterial species were used for testing the sealers’ antimicrobial properties: the obligate anaerobes Fusobacterium nucleatum (ATCC 49256), Prevotella intermedia (ATCC 25611), Parvimonas micra (ATCC 33270), and Veillonella parvula (ATCC 17745) as well as the facultative anaerobe Streptococcus oralis (ATCC 35037). All strains were obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany) and grown on Schaedler agar plates supplemented with vitamin K₁ and 5% sheep blood (Becton Dickinson, Franklin Lakes, NJ, USA). Cultivation in liquid medium was performed in anaerobic brain–heart-infusion broth (BHI, Becton Dickinson) supplemented with hemin (5 µg/ml) and vitamin K₁ (1 µg/ml). The bacteria were incubated at 37 °C in an anaerobic atmosphere (5% H₂, 10% CO₂, 85% N₂).

Susceptibility to clindamycin, metronidazole, and vancomycin was evaluated using E-test strips (bioMérieux, Saint-Laurent, QC) on BBL Brucella agar with 5% horse blood, hemin, and vitamin K₁ (Becton, Dickinson and Co.) as previously described [37 (link)]. Briefly, bacteria were suspended in Brucella broth (Becton, Dickinson and Co.) prior to agar inoculation, and MICs were read after 48 hours of growth with Bacteroides fragilis ATCC 25285 and Staphylococcus aureus ATCC 29213 as controls. MIC interpretation for clindamycin and metronidazole was based on Clinical Laboratory Standards Institute (CLSI) breakpoints [38 ], while susceptibility to vancomycin was determined with European Committee on Antimicrobial Susceptibility Testing breakpoints [39 ] in the absence of established CLSI criteria.

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)].