Streptococcus mutans atcc 25175

The human salivary gland (HSG) cell line was provided by the Shanghai Engineering Research Center of Tooth Restoration and Regeneration (Tongji University, Shanghai, China). HSG cells were incubated with Dulbecco's modified Eagle's medium (Hyclone) containing penicillin, streptomycin (Hyclone), and 10% fetal bovine serum (Gibco, Grand Island, NY, USA) at 37°C in a 5% CO₂ incubator. The culture solution was replaced every day, and HSG cells were passaged every 2 days. HSG cells were adjusted to ~5 × 10⁵ cells per 6 cm culture plate for bacterial co-incubation.
Streptococcus mutans (ATCC 25175) was purchased from ATCC (ATCC, Manassas, VA, USA) and grown to mid-log phase [optical density (OD) = 0.30, according to the growth curve shown in Supplementary Figure 1] in brain heart infusion (BHI) broth (Hopebio, Qingdao, China) at 37°C. The colony-forming units (CFU)/mL of Streptococcus mutans at 0.30 OD was estimated (data not shown). Bacteria were then heat-killed in BHI broth at 60°C for 30 min and added to the HSG cells at a ratio of 100 bacteria to 1 cell and co-incubated for 24 h, these were determined as optimal co-incubation conditions by preliminary experiments (data not shown).

Strains of Lacticaseibacillus, Streptococcus and Enterococcus were isolated and purified as follows: Dental supragingival plaque samples were collected from 50 Algerian patients (aged between 17 to 72 years old) consulting different dental offices in Bejaia city. Written informed consent was obtained from each participant. The samples were tenfold serially diluted in a tryptone salt (TS) solution (0.9% [w/v] NaCl supplemented with 0.1% [w/v] tryptone; Sigma) and inoculated onto de Man, Rogosa and Sharpe (MRS) agar (Sigma; pH 5.4) and Mitis-Salivarius bacitracin agar (Sigma; pH 7). The plates were incubated at 37°C/48 h. After incubation, the colonies were sub-cultured on new plates and their purity was checked by Gram staining and catalase activity testing. Gram-positive and catalase-negative bacilli were stored in MRS broth (Sigma; pH 5.4) supplemented with 20% [v/v] glycerol (Sigma), whereas the Gram-positive and catalase-negative cocci regrouped in chains were stored in Brain Heart Infusion (BHI) broth (Sigma; pH 7) supplemented with 20% [v/v] glycerol.
Three laboratory reference strains of oral pathogens were included in this study: Streptococcus mutans ATCC 25175, Candida albicans ATCC 28366 and Fusobacterium nucleatum ATCC 25586. These strains were obtained from the American Type Culture Collection (ATCC; Manassas, VA, United States).

Brain-Heat Infusion (BHI) agar was purchased from Becton Dickinson and Company (Franklin Lakes, NJ). Modified GAM broth and modified GAM agar were purchased from Nissui Pharmaceutical Co., LTD (Tokyo, Japan). Porphyromonas gingivalis ATCC 33277, Fusobacterium nucleatum ATCC 25586, Streptococcus sanguinis ATCC 10556, and Streptococcus mutans ATCC 25175 were purchased from the American Type Culture Collection (Manassas, VA). P. gingivalis and F. nucleatum were grown on GAM broth and GAM agar, and S. sanguinis and S. mutans were grown on BHI broth and BHI agar in an anaerobic chamber with Anaeropack Kenki (Mitsubishi Gas Chemical Company, Tokyo, Japan) at 37 °C.

Brain heart infusion (BHI) broth was purchased from Difco Laboratories (Detroit, MI, USA). Glucose and dimethyl sulfoxide (DMSO) were obtained from Sigma-Aldrich Co. (St. Louis, MO, USA). Streptococcus mutans ATCC 25175 was purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA).

Streptococcus mutans ATCC 25175 obtained from American Type Culture Collection was used in the study. The strain was grown on brain heart infusion broth (Mast Group, Bootle, UK) with 2% sucrose (BHIS) until, unless mentioned otherwise. For longer storage and preservation, the strain was stored in 20% glycerol at −80 ℃.