S oralis

Standard strains were used for evaluating species specificity of the PCR amplifications and as controls in the bile solubility test. These strains were S. oralis ATCC 35037, S. parasanguinis ATCC 15912, S. gordonii ATCC 33399, S. mitis ATCC 49456, S. constellatus ATCC 27823, S. mutans ATCC 25175, S. sanguinis ATCC 10556, S. intermedius ATCC 27335, S. sobrinus ATCC 33478, S. anginosus ATCC 33397, S. vestibularis ATCC 49124, S. salivarius ATCC 25975, S. cristatus ATCC 51100, S. infantis ATCC 700779, S. pseudopneumoniae ATCC BAA-960, and S. pneumoniae ATCC 10813.

Four Gram-positive strains were used in this research, S. mutans (ATCC 25175), S. aureus (ATCC 25923), S. oralis (ATCC 6249), and S. salivarius (ATCC 13419), obtained from Microbiologics, St. Cloud, MN, USA, and cultivated on Mueller-Hinton Agar with 5% Sheep Blood (HiMedia Laboratories Pvt. Ltd., Mumbai, India) for 48 h at 37 °C, with 5% CO2. After this period, bacteria colonies were picked up from the new culture and suspended in 2 mL of sterile distilled water, then serial diluted (fivefold) from 1:10 to 1:10⁵.

The bacterial species used were S. sanguinis ATCC 10,556 (ATCC, American Type Culture Collection, Manassas, VA, USA), S. gordonii ATCC 10,558, and S. oralis ATCC 35,037. The bacteria were cultured on plates containing brain–heart infusion (BHI; Sigma-Aldrich, St. Louis, MO, USA) and 1.5% agar (Wako, Tokyo, Japan)^{36 (link)}. Pre-culture was performed in an anaerobic chamber (N₂: 80%, H₂: 10%, and CO₂: 10%) at 37 °C for 1 day　(Anaerobox ANX-5; Hirasawa, Tokyo, Japan). A single colony on the plate was cultured in BHI broth for another 24 h. After liquid culture for another 4 h, a fresh culture was used for the adherence experiment. The optical density of each bacterial suspension was adjusted with BHI broth to 0.2 at 660 nm using a spectrophotometer (Ultrospec 2100 pro; Amersham Biosciences, Piscataway, NJ, USA); it corresponds to 6.0 × 10⁷ CFU/ml. For subsequent experiments, the bacteria were cultured on disks in 24-well plates.