Sucrose promotes caries progression by disrupting the microecological balance in oral biofilms: an in vitro study

Abstract

Sucrose has long been regarded as the most cariogenic carbohydrate. However, why sucrose causes severer dental caries than other sugars is largely unknown. Considering that caries is a polymicrobial infection resulting from dysbiosis of oral biofilms, we hypothesized that sucrose can introduce a microbiota imbalance favoring caries to a greater degree than other sugars. To test this hypothesis, an in vitro saliva-derived multispecies biofilm model was established, and by comparing caries lesions on enamel blocks cocultured with biofilms treated with sucrose, glucose and lactose, we confirmed that this model can reproduce the in vivo finding that sucrose has the strongest cariogenic potential. In parallel, compared to a control treatment, sucrose treatment led to significant changes within the microbial structure and assembly of oral microflora, while no significant difference was detected between the lactose/glucose treatment group and the control. Specifically, sucrose supplementation disrupted the homeostasis between acid-producing and alkali-producing bacteria. Consistent with microbial dysbiosis, we observed the most significant disequilibrium between acid and alkali metabolism in sucrose-treated biofilms. Taken together, our data indicate that the cariogenicity of sugars is closely related to their ability to regulate the oral microecology. These findings advance our understanding of caries etiology from an ecological perspective.

Figure 1

Biofilm preparation and treatment regimen. The saliva inoculum was inoculated in 1.5 ml of SHI medium and cultured without disturbance during the first 24 h to form an initial biofilm community on the enamel block surface. Starting at 72 h, the biofilms were transferred to SHI medium containing different dietary sugars (i.e., 2% sucrose, 2% lactose and 2% glucose) to simulate cariogenic challenges until the endpoint (144 h). The culture medium was changed every 24 h until the end of the experimental period. The control group was saliva derived biofilm cultured with SHI medium only.

Figure 2

Sucrose-treated biofilms exhibit the most significant demineralization effect on enamel blocks. (a) Representative transverse microradiography images of enamel blocks from sucrose, lactose and glucose treated groups at 144 h. Quantification of lesion depth (b) and mineral loss (c) of enamel blocks at the endpoint. All results are presented as mean ± standard deviation (n = 5; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant).

Figure 3

Sucrose influences the microbial structure and assembly of the oral microecology. (a) The microbial richness as revealed by OTU number. (b) Venn diagram showing shared OTUs among biofilms treated without/with sucrose, lactose and glucose. (c–e) The PCoA and dissimilarity analysis (including Adonis and ANOSIM) of polymicrobial oral biofilm among groups. Each dot stands for a biofilm sample. (f) Network inferences of microbial relationships in biofilms without/with sucrose, lactose or glucose treatment. Each node represents an OTU, and each edge represents a significant pairwise association. Red lines indicate synergistic relationships while green lines represent antagonistic relationships (n ≥ 4; ns, not significant; OTU, operational taxonomic unit; PCoA, principal coordinates analysis).

Figure 4

Sucrose significantly decreases the level of bacteria contributing to acid reduction. (a) Taxa distribution at the genus level. Only genera showed significantly different distribution were included. (b,c) Different distributed Veillonella species among samples. Each column stands for one sample. (d) qPCR quantification of V. parvula in saliva derived biofilm without or with sucrose/lactose/glucose treatment. Results were presented as mean ± standard deviation (n ≥ 4; *p < 0.05; **p < 0.01; ****p < 0.0001; ns, not significant; V. p = V. parvula).

Figure 5

Sugar disrupts the equilibrium between acid- and alkali-producing bacteria. qPCR quantification of S. mutans (a), S. sanguinis (b), S. gordonii (c), and the ratios of S. mutans/S. sanguinis (d) and S. mutans/S. gordonii (e) in biofilm treated without or with sucrose/lactose/glucose. Results were presented as mean ± standard deviation. (n = 5; *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant; S. m = S. mutans; S. s = S. sanguinis; S. g = S. gordonii).

Figure 6

Sugar exposure increases the cariogenicity of saliva-derived biofilms. (a) Dynamic pH values of spent media for 6 days and (b) lactic acid production in the end of 6^th day. (c) Dynamic NH₃ production of spent media for 6 days. All results are presented as mean ± standard deviation, each group was compared with the control group, saliva derived biofilm cultured with SHI medium, at the corresponding time point (n = 5; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant).