Human cathelicidin LL-37 enhance the antibiofilm effect of EGCG on Streptococcus mutans

Abstract

Background: Streptococcus mutans forms biofilms as a resistance mechanism against antimicrobial agents in the human oral cavity. We recently showed that human cathelicidin LL-37 exhibits inhibitory effects on biofilm formation of S. mutans through interaction with lipoteichoic acid (LTA), but without antibacterial or biofilm dispersal abilities. (-)-Epigallocatechin gallate (EGCG) is the most abundant constituent of tea catechins that has the greatest anti-infective potential to inhibit the growth of various microorganisms and biofilm formation. Therefore, in this study, we evaluated whether LL-37 interacts with EGCG to enhance the antibiofilm effect of EGCG on S. mutans biofilm formation.

Methods: Clinical S. mutans strains (n = 10) isolated from children's saliva were tested in a biofilm formation assay. The antibiofilm effect of EGCG with and without LL-37 was analyzed by the minimum biofilm eradication concentration assay and confirmed using field emission-scanning electron microscopy. In addition, the interaction among EGCG, LL-37, and LTA of S. mutans was determined using quartz crystal microbalance analysis.

Results: EGCG killed 100 % of planktonic S. mutans within 5 h, inhibited biofilm formation within 24 h, and reduced bacteria cells in preformed biofilms within 3 h at a concentration of 0.2 mg/mL. However, EGCG did not appear to interact with LTA. LL-37 effectively enhanced the bactericidal activity of EGCG against biofilm formation and preformed biofilms as determined by quantitative crystal violet staining and field emission-scanning electron microscopy. In addition, quartz crystal microbalance analysis revealed that LL-37 interacted with EGCG and promoted binding between EGCG and LTA of S. mutans.

Conclusions: We show that LL-37 enhances the antibiofilm effect of EGCG on S. mutans. This finding provides new knowledge for dental treatment by using LL-37 as a potential antibiofilm compound.

Keywords: Biofilm; EGCG; LL-37; Streptococcus mutans.

Fig. 1

Short-term killing assays for EGCG against S. mutans (n = 10). Standardized overnight cultures of S. mutans strains (3000 CFU/mL) were seeded in a 96-well microtiter plate with varying concentrations of EGCG and incubated for 3 and 5 h at 37 °C. Data are presented as the mean ± SEM from three independent experiments. *, significant difference compared with untreated control (p < 0.05)

Fig. 2

Biofilm inhibition assay. Standardized overnight cultures of S. mutans strains (n = 10) were grown on MBEC pegs incubated with 0.2 mg/mL EGCG in the presence or absence of 80 μg/mL LL-37 for 12, 24, or 36 h at 37 °C. Adherent cells were then fixed with ethanol, stained with crystal violet, eluted with ethanol, and the absorbance was measured. Data are presented as the mean ± SD from three independent experiments. *, significant difference compared with untreated control (p < 0.05)

Fig. 3

Biofilm susceptibility assays. Standardized overnight cultures of S. mutans strains grown on MBEC pegs and incubated overnight at 37 °C to form mature biofilms. The pegs were immersed in wells containing 0.2 mg/mL EGCG in the presence or absence of 80 μg/mL LL-37 for 3 and 5 h to allow the biofilm to disperse from the pegs to the wells below, and the absorbance was measured. Data are presented as the mean ± SD from three independent experiments. *, significant difference compared with untreated control (p < 0.05)

Fig. 4

Changes in number of planktonic cells and preformed biofilms following incubation of EGCG with/without LL-37. Representative images of bacterial cells observed by field emission-scanning electron microscope (working distance: 10 mm, field width: 5 μm) are shown. Streptococcus mutans suspensions incubated in the presence (a) or absence (b) of 0.2 mg/mL EGCG for 24 h. Streptococcus mutans biofilms on MBEC pegs incubated with 0.2 mg/mL EGCG (c), 0.2 mg/mL EGCG, and 80 μg/mL LL-37 (d), or BHI medium (e) for 24 h. The white arrow indicates the “ring” phenomena around damaged cells

Fig. 5

Interaction among EGCG, LL-37, and LTA of S. mutans. Graphs show the change in delta frequency after injecting LTA and/or LL-37 on the sensor of quartz crystal microbalance. Five microliters of LTA (1 mg/mL) and/or LL-37 (8 mg/mL) were injected. The final concentrations of LTA and LL-37 were 10 and 80 μg/mL, respectively. LTA without initially binding EGCG was injected on the sensor as a control. Data presented are representative of three independent experiments with similar results