Sterile phosphate buffered saline

Streaked and inoculated L. amylovorus SLZX20-1 onto a plate to activate the strain. In total, 1% of activated strains was anaerobically incubated in MRS broth at 37°C and tested OD600 nm value, pH, and counted viable bacteria numbers every 2 h. The L. amylovorus suspensions were diluted in a 10-fold gradient and streaked onto MRS medium, anaerobic incubated at 37°C to count viable bacteria numbers in plates, which colony numbers were between 30 and 300.
Tolerance to low pH and bile salts were assessed that 1% of activated strains was incubated with different pH (0, 2.5, 3.0, and 4.0) of sterile phosphate buffered saline (PBS) or with sterile PBS (Gibco, Brooklyn, NY, USA) containing different bile salts (Solarbio, Beijing, China) levels (0, 0.1, 0.2, and 0.3%). The mixture was incubated at 37°C for 0, 1, 2, 3, and 4 h to count viable bacteria numbers via the flat colony counting method. Taking the viable bacteria number of 0 h as the control, the survival rate of L. amylovorus SLZX20-1 in different pH conditions or different bile salt levels was calculated.

Streptococcus mutans (UA159) were obtained from ATCC (700610). Todd Hewitt Broth (THB) powder and agar were obtained from BD. Yeast extract, dextrose, and glycerol were obtained from Sigma. 96-well (Falcon tissue culture polystyrene) and 1-well (OmniTray) plates were obtained from Corning Life Sciences and Thermo Scientific, respectively. The automated liquid handler robot (Biomek FX), as well as regular (AP96 P250) and conductive (Span8 P250) pipette tips, were obtained from Beckman Coulter. XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-S-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide) was obtained from Fluka. Sterile phosphate buffered saline (PBS) was obtained from Gibco. Phenazine methosulfate (PMS) and cetylpyrininium bromide (CPB, 384.19 g/mol) were obtained from Acros Organics. Cetylpyrininium iodide (CPI, 431.79 g/mol) and chloride (CPC, 339.99 g/mol) were obtained from City Chemical, LLC and MP Biomedicals, Inc., respectively.

Reagents for cell culture were purchased from PAA laboratories GmbH (Cölbe, Germany) unless otherwise stated. Sterile phosphate‐buffered saline (PBS) was obtained from Life Technologies GmbH (Darmstadt, Germany). DMEM (high glucose) and DMEM/Ham's F12 (1:1) and fetal calf serum (FCS) were bought from Biochrom AG (Berlin, Germany). Reagents for agarose gels, primers, and chemicals for fluorometry experiments were purchased from Sigma‐Aldrich Chemie GmbH (Munich, Germany). 2,7‐biscarboxyethyl‐5(6)‐carboxyfluorescein‐acetoxymethylester (BCECF‐AM), Cytochalasin D, and Mitomycin C were obtained from Applichem GmbH (Darmstadt, Germany). RNA isolation and qRT‐PCR kits were purchased from Qiagen GmbH (Hilden, Germany) and Eurogentec GmbH (Cologne, Germany). WST‐1 for cell viability assays was bought from F. Hoffmann‐La Roche AG (Mannheim, Germany). 1,1′‐dioctadecyltetramethyl indotricarbocyanine iodide (DiR) was kindly provided by Professor Matthias Gaestel (Hannover Medical School, Germany). HOE642 (cariporide) was a kind gift by Sanofi‐Aventis GmbH (Frankfurt, Germany). 5‐(N,N‐dimethyl)‐amiloride hydrochloride (DMA), G418 and epidermal growth factor (EGF) were purchased from Sigma‐Aldrich Chemie GmbH (Munich, Germany). Rat NHE1‐expressing PS120 (PS120/rNHE1) fibroblasts were a kind gift of Prof. Mark Musch, University of Chicago.

Bacterial viability testing was performed following the method previously described by Foerster et al. [9 (link)]. Briefly, bacterial solution was incubated in a 96-well plate. At each time point, 100 µL aliquots, which amounts to the complete volume of the wells to be tested, were transferred to another plate. Subsequently, cultures were serially diluted in seven subsequent 1:10 dilutions (20 µL culture in 180µL sterile phosphate-buffered saline (PBS) (Gibco, Lougborough, UK). Five µL droplets of every dilution were spotted on pre-dried CHOC-GC agar plates and incubated at 37 °C in a 5 % CO₂-enriched atmosphere. The density of viable bacteria, measured in colony forming units per ml (CFU/mL), was calculated from the first dilution that resulted in a countable range of 3–30 colonies.