Effect of Rubusoside, a Natural Sucrose Substitute, on Streptococcus mutans Biofilm Cariogenic Potential and Virulence Gene Expression In Vitro

Abstract

Dental caries is a biofilm-mediated disease with Streptococcus mutans as the main pathogenic microorganism, and its incidence is closely related to sucrose. Rubusoside is a natural non-nutritive sweetener isolated from Rubus suavissimu S.Lee. This study was designed to determine the effect of the sucrose substitute on cariogenic properties and virulence gene expression of S. mutans biofilms. S. mutans was exposed to: BHI medium (as a control), BHI+1% sucrose, BHI+1% rubusoside, and BHI +1% xylitol. The growth curve of biofilm was monitored with crystal violet staining and pH was measured every 24 h. After 5 days, the biofilms formed on the glass coverslips were recovered to determine the biomass (dry weight and total soluble proteins), colony-forming units, and intra- and extracellular polysaccharides. Biofilm structural imaging was observed using a scanning electron microscope (SEM). The virulence gene expression (gtfB, gtfC, gtfD, ftf, spaP, gbpB, ldh, atpF, vicR, and comD) was determined by rt-qPCR. Compared to growth in sucrose, xylitol and control, growth in rubusoside resulted in a lower acid production, while also reducing the biofilm accumulation, bacterial viability, and even reducing the production of extracellular polysaccharides. And less biofilm formation and extracellular matrix was observed through SEM in rubusoside than sucrose and xylitol. From the perspective of virulence genes, rubusoside and xylitol significantly inhibited expression of virulence genes compared with sucrose, among which, down-regulation of the gtfB, gtfC, gbpB, ldh, and comD was found in rubusoside than xylitol. Therefore, rubusoside appears to be less potential cariogenicity than sucrose and xylitol, and may become an effective sucrose substitute for caries prevention. Further studies need to deepen these findings. Importance Dental caries, as a major public health challenge, places a heavy biological, social and financial burden on individuals and health care systems. To palliate the deleterious effect of sucrose on the virulence factors of S. mutans, massive commercial products have oriented towards developing sucrose substitutes. Rubusoside, a natural sucrose substitute, is a plant extract with high sweetness. Although some studies have shown that rubusoside do not produce acids and inhibit the growth of S. mutans, little attention has been paid to dental biofilm and underlying mechanisms. Our study focuses on the effect of rubusoside on formation and structure of biofilm, and expression of virulence genes. Results confirms that rubusoside can inhibit accumulation, bacterial viability, polysaccharide production of biofilm, and inhibit related gene expression. These results provide further insight into the cariogenicity of S. mutans biofilm, and demonstrate a new perspective for studying the impact of sucrose substitutes on caries.

Inhibitory effects of Stevioside on Streptococcus mutans and Candida albicans dual-species biofilm

“…Additionally, since stevioside is about 300 times sweeter than sucrose on an equal weight basis, we evaluated another experiment group in which we used a stevioside concentration with the same sweetness as sucrose, providing a reference for economic application and popularization. Many studies confirm our results regarding sucrose's strong cariogenic ability from the perspective of bacterial and fungal planktonic accumulation, biofilm formation, EPS, and acid production (Klein et al, 2009;Forssten et al, 2010;Cai et al, 2018;Cavazana et al, 2018;Guan et al, 2020). From the planktonic growth curve and crystal violet assay, we conclude that stevioside restrains the accumulation of suspension cells and significantly inhibits the growth of hybrid biofilm compared with sucrose.…”

“…Overnight cultures of S. mutans and C. albicans were standardized to approximately 10 4 CFU/mL and deposited into a 24-well microplate plate containing various growth media. The culture medium was renewed every 24 h ( Guan et al, 2020 ). The culture supernatants were centrifuged at 5000 × g for 15 min, and the precipitation of bacterial/fungal cells was removed.…”

Lactobacillus cell envelope-coated nanoparticles for antibiotic delivery against cariogenic biofilm and dental caries

“…The plates were incubated for an additional 24 h. The biofilm was rinsed with physiological saline to remove free TCS and NPs, and the medium was replaced with fresh medium every 24 h. After incubation for 8 days, the biofilms were scraped off the glass coverslips with a sterile cell scraper, suspended in PBS and divided into aliquots for analysis. The biofilm biomass assay was conducted as proposed by Chunru [ 60 ].…”

Streptococcus mutans suppresses filamentous growth of Candida albicans through secreting mutanocyclin, an unacylated tetramic acid

“…Cells of single colonies were then collected, dilutedand used for morphological analysis. S. mutans strains were cultured in brain heart infusion medium (BHI; Oxoid, Basingstoke, United Kingdom) plus .5% sucrose [ 28 ], referred to herein as BHI-sucrose medium [ 29 ]. Sucrose was purchased from Beijing Chemical Works (Beijing, China).…”