Lactobacillus plantarum Lipoteichoic Acids Possess Strain-Specific Regulatory Effects on the Biofilm Formation of Dental Pathogenic Bacteria

Abstract

Bacterial biofilm residing in the oral cavity is closely related to the initiation and persistence of various dental diseases. Previously, we reported the anti-biofilm activity of Lactobacillus plantarum lipoteichoic acid (Lp.LTA) on a representative dental cariogenic pathogen, Streptococcus mutans. Since LTA structure varies in a bacterial strain-specific manner, LTAs from various L. plantarum strains may have differential anti-biofilm activity due to their distinct molecular structures. In the present study, we isolated Lp.LTAs from four different strains of L. plantarum (LRCC 5193, 5194, 5195, and 5310) and compared their anti-biofilm effects on the dental pathogens, including S. mutans, Enterococcus faecalis, and Streptococcus gordonii. All Lp.LTAs similarly inhibited E. faecalis biofilm formation in a dose-dependent manner. However, their effects on S. gordonii and S. mutans biofilm formation were different: LRCC 5310 Lp.LTA most effectively suppressed the biofilm formation of all strains of dental pathogens, while Lp.LTAs from LRCC 5193 and 5194 hardly inhibited or even enhanced the biofilm formation. Furthermore, LRCC 5310 Lp.LTA dramatically reduced the biofilm formation of the dental pathogens on the human dentin slice infection model. Collectively, these results suggest that Lp.LTAs have strain-specific regulatory effects on biofilm formation of dental pathogens and LRCC 5310 Lp.LTA can be used as an effective anti-biofilm agent for the prevention of dental infectious diseases.

Keywords: Lactobacillus plantarum; biofilm; dental pathogens; human dentin slice; lipoteichoic acid.

Figure 1

Elution profiles and molecular characteristics of LTAs purified from four different strains of L. plantarum. (A–D) LTAs were purified from four different strains of L. plantarum by sequential application of butanol extraction, hydrophobic interaction chromatography, and ion-exchange chromatography. Inorganic phosphate assays were then performed to measure the quantity of LTAs in each fraction after hydrophobic interaction chromatography (Octyl-Sepharose, left) and ion-exchange chromatography (DEAE-Sepharose, right) as described in Materials and Methods. (E) Purified LTAs were subjected to Western blot analysis using antibody specific for LTA.

Figure 2

Lp.LTAs dose-dependently inhibit E. faecalis biofilm formation. Four strains of E. faecalis, including (A,E) a laboratory (ATCC 29212) and (B–D,F–H) three clinically isolated strains (KCOM 1083, KCOM 1161, and KCOM 5291), at 1×10⁷CFU/ml were grown on 96-well plates in the presence or absence of the indicated concentrations of (A–D) various Lp.LTA or (E–H) LRCC 5310 Lp.LTA at 37°C for 24h. After the incubation, biofilm formations were determined by crystal violet staining as described in Materials and Methods. Biofilm formation is presented as percentage change±standard deviation of triplicates against the non-treatment group set as 100%. (E–H) After collecting the biofilm, it was serially diluted and incubated on the THY agar plate at 37°C for 48h, and CFU formation was examined. Asterisk (*) indicates statistical significance at p<0.05 between the non-treatment and each Lp.LTA treatment groups.

Figure 3

Lp.LTAs have different anti-biofilm activity on S. gordonii. Total of four strains of S. gordonii, including (A,E) a laboratory (CH1) and (B–D,F–H) three clinically isolated strains (KCOM 1967, KCOM 2106, and KCOM 2867), were grown on 96-well plates in the presence or absence of the indicated concentrations of (A–D) various Lp.LTA or (E–H) LRCC 5310 Lp.LTA at 37°C for 24h. After the incubation, biofilm formations were determined by crystal violet staining assay as described in Materials and Methods. Biofilm formation is presented as percentage change±standard deviation of triplicates against the non-treatment group set as 100%. (E–H) After collecting the biofilm, it was serially diluted and incubated on the THY agar plate at 37°C for 48h, and CFU formation was examined. Asterisk (*) indicates statistical significance at p<0.05 between the non-treatment and each Lp.LTA treatment groups.

Figure 4

Lp.LTAs differently suppress S. mutans biofilm formation. Four different strains of S. mutans, including (A,E) a laboratory (ATCC 25175) and (B–D,F–H) three clinically isolated strains (KCOM 1054, KCOM 1116, and KCOM 1223), were grown on 96-well plates in the presence or absence of the indicated concentrations of (A–D) various Lp.LTA or (E–H) LRCC 5310 Lp.LTA at 37°C for 24h. After the incubation, biofilm formations were determined by crystal violet staining as described in Materials and Methods. Biofilm formation is presented as percentage change±standard deviation of triplicates against the non-treatment group set as 100%. (E–H) After collecting the biofilm, it was serially diluted and incubated on the THY agar plate at 37°C for 48h, and CFU formation was examined. Asterisk (*) indicates statistical significance at p<0.05 between the non-treatment and each Lp.LTA treatment groups.

Figure 5

LRCC 5310 Lp.LTA inhibits biomass and thickness distribution of biofilm formed by clinically isolated E. faecalis, S. gordonii, and S. mutans. Clinically isolated strains of (A) E. faecalis (KCOM 1083), (B) S. gordonii (KCOM 1967), and (C) S. mutans (KCOM 1223) at 1×10⁷CFU/ml were grown on glass-bottom confocal dishes at 37°C for 24h in the presence or absence of 30μg/ml of LRCC 5310 Lp.LTA. After the incubation, biofilms were stained with the LIVE/DEAD BacLight Bacterial Viability Kit containing SYTO9 and propidium iodide and then visualized by confocal laser microscopy (green SYTO9; red, propidium iodide). The biomass and thickness distribution of biofilms were quantified using COMSTAT2 software. Asterisk (*) indicates statistical significance at p<0.05 between the non-treatment and LRCC 5310 Lp.LTA treatment groups.

Figure 6

LRCC 5310 Lp.LTA suppresses biofilm formation of clinically isolated dental pathogens on human dentin slices. Clinically isolated strains of (A) E. faecalis (KCOM 1083), (B) S. gordonii (KCOM 1967), and (C) S. mutans (KCOM 1223) at 1×10⁷CFU/ml was grown on human dentin slices in the presence or absence of 30μg/ml of LRCC 5310 Lp.LTA at 37°C for 24h. Images were obtained by scanning electron microscopy with magnification at ×5,000 and ×30,000. Scale bars given in each image indicate 2 or 10μm.