This study employed the S. mutans ATCC 25175 (KN88) strain. Bacteria were incubated in brain-heart infusion broth (BHI; Becton, Dickinson, Franklin Lakes, NJ) at 37°C under aerobic conditions with 5% CO₂ for 18 h. Suspension turbidity was measured by a spectrophotometer (Smart Plus 2700; Youngwoo inst., Seoul, Korea). A standard curve of culture turbidity and bacterial cell numbers was established and utilized for cell dilutions. The bacteria were diluted to 10⁷ colony-forming units (CFU)/mL with phosphate-buffered saline (PBS) to determine MIC and MBC and diluted to 10⁸ CFU/mL with PBS to determine MBIC and MBEC.

A solution of 10000 μg/L (10% w/v) erythrosine (Sigma-Aldrich, St Louis, MO) was prepared in PBS. This solution was filtered-sterilized and stored at 4°C in the dark. The solution was then diluted in BHI broth at a concentration range of 0.02 - 10000 μg/L.

To investigate the antimicrobial activity of erythrosine, the MIC was measured by the microdilution method. The susceptibility test was conducted according to the criteria recommended by the Clinical and Laboratory Standards Institute (CLSI, 2016). The bacterial suspension was serially diluted in 96-well plates (SPL Life Sciences, Pocheon, Korea) using BHI medium. After incubation at 37°C for 18 h in a 5% CO₂ incubator, the minimum concentration at which bacterial growth was inhibited was determined as the MIC. The culture broths of wells with higher MICs were plated on blood agar plates (Hangang, Gunpo, Korea), and the concentration at which 99.9% of bacteria were killed was determined as the MBC. If the same results were obtained in 2 repeated experiments, the values were set as MIC and MBC values. If experiment results were different, the tests were repeated to determine the MIC and MBC.

To form S. mutans biofilms on the bottom of 96-well plates (Flat-Bottom Plate; SPL Life Sciences), 180 μL BHI medium and 20 μL bacterial suspension (10⁸ CFU/mL) were added to the plates and cultured in a 5% CO₂ incubator at 37°C for 18 h. After incubation, each microplate was washed 3 times with BHI medium to remove any non-adherent bacterial cells. Erythrosine was serially diluted in BHI medium in a new microplate and then the diluted solution was transferred to the microplates with biofilms, which were then cultured at 37°C for 18 h. After incubation, supernatant turbidity was examined and the minimum concentration at which bacterial growth was inhibited was determined as the MBIC. After MBIC determination, the erythrosine-containing microbial culture was washed 3 times with BHI medium. Then, fresh BHI culture medium without erythrosine was added to the microplates and incubation was carried out for 18 h at 37°C. Supernatant turbidity of this biofilm culture was observed visually to determine the minimum concentration at which the bacteria no longer grew, which was designated the MBEC. All experiments were repeated twice, and if the results were inconsistent, the experiment was repeated until consistent MBIC and MBEC values were obtained.

Erythrosine MIC and MBC against S. mutans in the planktonic state are shown in Table 1. The determined MIC and MBC values after repeated experiments were both 19.5 μg/L.

Erythrosine MBIC and MBEC against S. mutans biofilms are shown in Table 1. The determined MBIC and MBEC values after repeated experiments were 313 μg/L and 2500 μg/L, respectively.