Effects of DJK-5 and chlorhexidine on exopolysaccharide volume and pH in oral biofilms

Abstract

Background: Exopolysaccharides (EPS) are essential constituents of the extracellular matrix within oral biofilms and are significantly influenced by the local microenvironment. This study aimed to investigate the impact of two distinct antimicrobial agents, DJK-5 and chlorhexidine (CHX), on the EPS volume and pH levels in oral biofilms.

Methods: Oral biofilms obtained from two donors were cultured on hydroxyapatite discs for durations of 3 days, 1 week, 2 weeks, 3 weeks, and 4 weeks. Subsequently, these biofilms were subjected to treatment with 10 µg/mL DJK-5 or 2% CHX for 3 min. The impact of these antimicrobial treatments on factors such as the proportion of dead bacterial, in situ pH, and EPS volume within the biofilms was assessed using corresponding fluorescent probes. The examination was carried out utilizing confocal laser scanning microscopy, and the resulting images were analyzed with a focus on the upper and lower layers of the biofilm, respectively.

Results: DJK-5 exhibited a more potent bactericidal effect compared to CHX across the 3-day to 4-week duration of the biofilm (P < 0.05). The biofilms were acidic, with the upper layer being less acidic than the lower layer (P < 0.05). Both antimicrobial agents increased the pH, but DJK-5 had a greater effect than CHX (P < 0.05). The volume of EPS was significantly lower in DJK-5 treated biofilms compared to that of CHX, regardless of age or layer (P < 0.05).

Conclusion: DJK-5 exhibited superior effectiveness in reducing viable bacteria and EPS volume, as well as in raising extracellular pH, as compared to chlorhexidine.

Keywords: Chlorhexidine; Confocal laser scanning microscope; DJK-5; Exopolysaccharides; Oral biofilms; pH.

Fig. 1

Amino acid structure of DJK-5

Fig. 2

The proportion of dead bacterial within biofilms of different ages from both donors, treated with different antimicrobial agents. (A, B, C: donor 1; D, E, F: donor 2; A and D: whole biofilm; B and E: upper layer of the biofilm; C and F: lower layer of the biofilm)

Fig. 3

Representative CLSM images of live and dead bacteria within 3-day and 3-week biofilms that underwent treatment with different antimicrobial agents. (A, C, and E: 3-day; B, D and F: 3-week; Green: live bacteria; Red: dead bacteria)

Fig. 4

The pH levels of biofilms of different ages from the two donors, treated with different antimicrobial agents. (A, B, C: donor 1; D, E, F: donor 2; A and D: whole biofilm; B and E: upper layer of the biofilm; C and F: lower layer of the biofilm)

Fig. 5

The biovolume of EPS and live bacteria in biofilms of different ages from both donors, subject to different antimicrobial treatments. (A, D, G and J: whole biofilm; B, E, H and K: upper layer; C, F, I and L: lower layer). Different lowercase letters represent significant differences within the group (P < 0.05)

Fig. 6

The ratio of live bacteria to EPS within biofilms of different ages from both donors, treated with different antimicrobial agents. (A and D: whole biofilm; B and E: upper layer of biofilm; C and F: lower layer of biofilm)

Fig. 7

Representative CLSM images of live bacteria and EPS within 3-day and 3-week biofilms treated with different antimicrobial agents. (A, C and E: 3-day; B, D and F: 3-week; Green: live bacteria; Red: EPS)