Department of Stomatology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China.
The Academy of Medical Sciences, Zhengzhou University, Zhengzhou, China.
Appl Environ Microbiol. 2020 Aug 3;86(16). doi: 10.1128/AEM.01012-20.
Dental caries is a biofilm-mediated disease in which is the main pathogenic microorganism, and its incidence is closely related to sucrose. Rubusoside is a natural nonnutritive sweetener isolated from S. Lee. This study was designed to determine the effect of this sucrose substitute on the cariogenic properties and virulence gene expression of biofilms. was exposed to brain heart infusion (BHI) medium (as a control), 1% sucrose-supplemented medium, 1% rubusoside-supplemented medium, and 1% xylitol-supplemented medium. The growth curve of the biofilm was monitored by crystal violet staining, and the 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 amount of soluble proteins), numbers of CFU, and amounts of intra- and extracellular polysaccharides. Biofilm structural imaging was performed using a scanning electron microscope (SEM). Virulence gene expression (, , , , , , , , , and ) was determined by reverse transcription-quantitative PCR. Growth in rubusoside resulted in lower levels of acid production than observed during growth in sucrose, xylitol, and the control, while it also reduced the level of biofilm accumulation and bacterial viability and even reduced the level of production of extracellular polysaccharides. By SEM, the levels of biofilm formation and extracellular matrix during growth in rubusoside were lower than these levels during growth in sucrose and xylitol. From the perspective of virulence genes, growth in rubusoside and xylitol significantly inhibited the expression of virulence genes compared with their levels of expression after growth in sucrose. Among these genes, , , , , and downregulation was found with growth in rubusoside compared with their expression with growth in xylitol. Therefore, rubusoside appears to be less potentially cariogenic than sucrose and xylitol and may become an effective sucrose substitute for caries prevention. Further studies are needed to deepen these findings. Dental caries is a major public health challenge and places heavy biological, social, and financial burdens on individuals and health care systems. To palliate the deleterious effect of sucrose on the virulence factors of , massive commercial efforts have been oriented toward developing products that may act as sucrose substitutes. Rubusoside, a natural sucrose substitute, is a plant extract with a high level of sweetness. Although some studies have shown that rubusoside does not produce acids or inhibit the growth of , little attention has been paid to its effect on dental biofilm and the underlying mechanisms. Our study focuses on the effect of rubusoside on the formation and structure of biofilms and the expression of virulence genes. The results confirm that rubusoside can inhibit accumulation, bacterial viability, polysaccharide production by the biofilm, and related gene expression. These results provide further insight into the cariogenicity of biofilms and demonstrate a new perspective for studying the impact of sucrose substitutes on caries.
龋齿是一种生物膜介导的疾病，其中 是主要的致病微生物，其发病率与蔗糖密切相关。悬钩子苷是从 S. Lee 中分离出来的天然非营养性甜味剂。本研究旨在确定这种蔗糖替代品对 生物膜致龋特性和毒力基因表达的影响。将 暴露于脑心浸液（BHI）培养基（作为对照）、1%蔗糖补充培养基、1%悬钩子苷补充培养基和 1%木糖醇补充培养基中。通过结晶紫染色监测生物膜的生长曲线，并每隔 24 h 测量 pH 值。5 天后，从玻璃盖玻片上回收形成的生物膜以确定生物量（干重和总可溶性蛋白）、CFU 数量以及细胞内和细胞外多糖的量。使用扫描电子显微镜（SEM）进行生物膜结构成像。通过逆转录定量 PCR 确定毒力基因（ 、 、 、 、 、 、 、 和 ）的表达。在悬钩子苷中生长导致产酸水平低于在蔗糖、木糖醇和对照中观察到的水平，同时还降低了生物膜积累和细菌活力水平，甚至降低了细胞外多糖的产生水平。通过 SEM，在悬钩子苷中生长时形成生物膜和细胞外基质的水平低于在蔗糖和木糖醇中生长时的水平。从毒力基因的角度来看，与在蔗糖中生长相比，在悬钩子苷和木糖醇中生长显著抑制了毒力基因的表达。在这些基因中，与在木糖醇中生长相比，在悬钩子苷中生长时 、 、 、 、 和 下调。因此，悬钩子苷的致龋潜力似乎低于蔗糖和木糖醇，并且可能成为预防龋齿的有效蔗糖替代品。需要进一步的研究来深化这些发现。龋齿是一个主要的公共卫生挑战，给个人和医疗保健系统带来了沉重的生物、社会和经济负担。为了减轻蔗糖对 的毒力因子的有害影响，人们进行了大量的商业努力，开发可能作为蔗糖替代品的产品。悬钩子苷是一种天然的蔗糖替代品，是一种高甜度的植物提取物。尽管一些研究表明悬钩子苷不会产生酸或抑制 的生长，但很少关注它对牙菌斑生物膜的影响及其潜在机制。我们的研究重点是悬钩子苷对生物膜形成和结构以及毒力基因表达的影响。结果证实，悬钩子苷可以抑制生物膜的积累、细菌活力、多糖产生和相关基因表达。这些结果为生物膜的致龋性提供了进一步的见解，并为研究蔗糖替代品对龋齿的影响提供了新的视角。
