The effect of multiple-enzyme treatment on in situ oral biofilm formation in healthy participants

Abstract

Novel approaches for the prevention of biofilm-mediated oral diseases aim to control dental biofilms rather than eradicating bacteria in the mouth. One such approach is the use of enzymes that specifically target and degrade the dental biofilm matrix and thereby facilitate biofilm removal. Matrix-degrading enzymes have consistently shown promising results in vitro, but data on in situ-grown oral biofilms are limited. This study aimed to investigate the effect of combined treatment with mutanase, beta-glucanase and DNase on in situ biofilm formation and removal, microbial biofilm composition and biofilm pH. Biofilms from healthy participants were grown for 48 or 72 h on lower-jaw splints and enzyme or control-treated during (3x/day, 30 min) or after growth (30 min). Under the tested conditions, enzyme treatment had no significant effect on biofilm formation or removal compared to control, as assessed by optical coherence tomography and confocal microscopy. Likewise, enzymatic treatment did not induce significant changes in the microbial composition of the biofilms that were dominated by Streptococcus, Haemophilus, Neisseria, Veillonella and Fusobacterium species. The biofilm pH response to a sucrose challenge was assessed using confocal microscopy-based pH ratiometry, and the average biofilm pH was not significantly different between the intervention groups. Under the conditions employed in this study, the tested enzymes had no significant impact on in situ grown biofilms. The treatment regimen, the biofilm composition, or the analytical methods employed may explain the difference to previous results. Further studies are warranted to assess the therapeutic potential of multi-enzyme treatment for dental biofilm control.

Keywords: Confocal Microscopy; Dental plaque; Enzyme Therapy; Optical coherence tomography; Sequence Analysis, RNA.

Fig. 1

The effect of enzymatic treatment on biofilm removal. Biofilm thickness was measured by OCT. A) The biofilm thicknesses decreased after treatment with both enzyme and control solutions (∗p < 0.05), and there was no significant difference between the two groups (p = 0.73, gamma regression with log link function). Data from 95 biofilms (enzyme: n = 48; control: n = 47) from 11 subjects are shown (missing data: n = 12; outliers: n = 3). Bars and error bars show means and the upper and lower limits of 95 % confidence intervals, respectively. B) Representative 3D renderings of oral biofilms generated from images acquired by OCT before and after treatment.

Fig. 2

Effect of enzymatic treatment during biofilm formation. Enzyme or control treatment was performed three times per day for 30 min during in situ biofilm growth (48 h). Biofilm thickness was measured by OCT. No statistically significant difference in biofilm thickness was observed between groups (p = 0.15, gamma regression with log link function). Data from 129 biofilms (enzyme: n = 65; control: n = 64) from 11 participants (S01–S11) are shown (outliers: n = 3). Horizontal lines = means.

Fig. 3

The effect of enzymatic treatment on microbial biofilm composition. A) The heatmap shows the most abundant genera (mean abundance cutoff = 1 %) in each analyzed biofilm, sorted by subject (S01–S11), period (P1 and P2) and intervention (E = enzyme-treated biofilm; C = control-treated biofilm). Biofilms were dominated by typical genera for young dental biofilms, with no systematic differences between intervention groups. B) Principal component analysis showed no clustering according to intervention group.

Fig. 4

The effect of enzymatic treatment on biofilm pH. A) Average differences in pH between enzyme- and control-treated biofilms from the same individual (ΔpH) were 0.07 (T1) and 0.08 (T2) for sterile saline, and 0.04 (T1), 0.04 (T2) and 0.04 (T3) for cleared saliva. Each colored dot shows ΔpH for one participant. Horizontal lines = means; error bars = 95 % confidence intervals. Data from 43 biofilms (enzyme: n = 22; control: n = 21) from 11 participants (S01–S11) are shown (missing data: n = 1). B) Representative C-SNARF-4 images (left panels) of enzyme- and control-treated biofilms collected from one subject (S11) in period 1. The middle and right panels show the corresponding color-coded images of the extracellular pH at T1 (Control: pH 5.98; Enzyme: pH 5.90) and T2 (Control: pH 5.84; Enzyme: pH 5.83), respectively. T1 = 5 min after exposure to sucrose; T2 = 20 min after exposure to sucrose; T3 = 35 min after exposure to sucrose. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)