Menadione

Porphyromonas gingivalis JCM12257 (ATCC33277), JCM8525, and JCM19600 were purchased from RIKEN BioResource Research Center (Ibaraki, Japan) [14 (link)]. All three strains were grown at 37 °C under anaerobic conditions (AnaeroPack™-Anaero, Tokyo, Japan) in a Gifu anaerobic medium broth (Nissui, Tokyo, Japan) supplemented with 5 µg/mL hemin (Sigma-Aldrich, St. Louis, MO, USA) and 1 µg/mL menadione (Fujifilm Wako Pure Chemical, Osaka, Japan) (GAM medium). Ampicillin sodium (ABPC) (Fujifilm) was only used as a positive control at a final concentration of 50 µg/mL.

These homogenized saliva samples were serially diluted by 10-fold with sterile phosphate buffer saline (PBS) from 10
^-3 to 10
^-7. Aliquots (100 µl) of each diluted sample were inoculated into Bacto
^TM Brain Heart Infusion (BHI, Difco Laboratories, Detroit, MI, USA) supplemented with 5% (volume/volume) defibrinated sheep blood (BHI agar), hemin (10 μg/mL, Wako, Osaka, Japan), menadione (5 µg/ml, Wako), and the selective medium
Veillonella agar
^{23 (link)}. After inoculation, all media were incubated under anaerobic conditions with 10% H
₂, 85% N
₂, and 5% CO
₂ at 37°C.
Veillonella agar was incubated for 5 days and BHI agar was incubated for 7 days. The bacterial colonies grown on BHI and
Veillonella agar were counted as the total number of bacteria and typical
Veillonella colonies in the saliva sample, respectively. Bacterial cells of typical
Veillonella colonies were confirmed as gram-negative cocci with light microscopy after gram staining. Standard strains consisted of
V. atypica ATCC 17744
^T,
V. denticariosi JCM 15641
^T,
V. dispar ATCC 17748
^T,
V. parvula ATCC 10790
^T,
V. rogosa JCM 15642
^T, and
V. tobetsuensis ATCCBAA-2400
^T.