S sanguinis

H. pylori ATCC 43504 was incubated in brain heart infusion (BHI) fluid medium with 5% Fetal Bovine Serum (FBS) that represented the H. pylori medium, in a microaerophilic chamber (6% O₂, 10% CO₂, and 84% N₂; Thermo Fisher Scientific, Inc., Waltham, MA, USA). S. mutans UA159 (ATCC 700610) and S. sanguinis (ATCC 10556) were maintained in BHI fluid medium in an anaerobic chamber (10% H₂, 5% CO₂, and 85% N₂; Thermo Fisher Scientific, Inc., Waltham, MA, USA) for planktonic growth. Both S. mutans and S. sanguinis were grown in BHI with 1% (w/v^-1) sucrose as a supplemental carbohydrate source, to allow biofilm formation. Biofilms were incubated at 37°C without agitation.

The strains used in the study came from the American Type Culture Collection (ATCC, Manassas, VA, USA). The cariogenic strains used were Streptococcus mutans (ATCC 25175), S. mitis (ATCC 49456), S. sanguinis (ATCC 10556), S. sobrinus (ATCC 33478), Lactobacillus paracasei (ATCC 11578), Enterococcus faecalis (ATCC 4082), and S. salivarius (ATCC 25975). For all assays performed the bacteria were incubated in Brain Heart Infusion agar (BHI), added with defibrinated sheep blood (5%) in a microaerophilia incubator for 24 h at 37 °C with 10% CO₂, except for E. faecalis and S. salivarius which were incubated aerobically at 37 °C for 24 h.

Authentic P. elliottii (batch number 21811-09) and P. tropicalis (batch number 20511-09) resin samples were obtained from the Brazilian Association of Resinators (ARESB), located in the city of Avaré – SP, Brazil. DHA was isolated from the P. elliottii resin and was obtained by the method previously described by Leandro et al. (2014) (link) (Supplementary Figures S1, S2).
The following bacteria from the American Type Culture Collection (ATCC) collection were used to conduct the antibacterial assays: S. mutans (ATCC 25175), S. mitis (ATCC 49456), S. sanguinis (ATCC 10556), S. sobrinus (ATCC 33478), S. salivarius (ATCC 25975), Lactobacillus casei (ATCC 11578), and Enterococcus faecalis (ATCC 4082). These microorganisms were maintained in a freezer at −80°C in 20% glycerol solution in the Laboratory of Applied Microbiology Research (LaPeMA) – University of Franca (Unifran).

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.

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.

Caries-related streptococci (Supplementary Table 1) which included S. mutans, S. gordonii and S. sanguinis, were obtained from American Type Culture Collection (ATCC, Manassas, VA), Guangdong Culture Collection Center (GS, Guangzhou, China), and Japan Collection of Microorganisms (JCM, Tokyo, Japan). In addition, clinical strains of S. mutans (Supplementary Table 1) were included in the assay and were collected, isolated and identified according to a previous study from West China Hospital of Stomatology, Chengdu, China (Lu et al., 2015 (link)). All the strains were grown in brain-heart infusion broth (BHI; Oxoid, Basingstoke, Hampshire, UK) anaerobically (85% N₂, 10% H₂ and 5% CO₂) at 37°C.