桑酮G对变异链球菌生长和生物被膜形成的影响

Effects of kuwanon G on the growth and biofilm formation of Streptococcus mutans

摘要:

【背景】变异链球菌(Streptococcus mutans)是导致龋齿的主要病原体，含氟产品虽然能治疗龋齿，但是会对人体造成危害。【目的】推动植物源抗龋齿药物的进一步开发利用，研究桑酮G对变异链球菌生长和生物被膜形成的影响。【方法】通过微量稀释法、平板计数法测定桑酮G对变异链球菌浮游菌的抗菌作用；通过pH计测量桑酮G对变异链球菌糖酵解产酸的影响；采用结晶紫染色测量生物被膜形成量；使用苯酚硫酸法测量水不溶性多糖的含量并通过激光扫描共聚焦显微镜观察胞外DNA量来评估桑酮G对生物被膜形成的影响；用逆转录实时荧光定量PCR测量生物被膜中毒力基因的mRNA水平。【结果】桑酮G对变异链球菌的最小抑菌浓度为8 μg/mL，最低杀菌浓度为64 μg/mL，高浓度桑酮G对变异链球菌呈现较强的杀菌效果；桑酮G能抑制生物被膜中gtfB、gtfC、gtfD、ldh、comD、vicK、atpF和gbpB的表达，降低水不溶性多糖的合成，抑制生物被膜的形成，并降低生物被膜中胞外DNA含量及生物被膜的形成量，同时还对变异链球菌的产酸耐酸能力产生负面影响。【结论】桑酮G能通过降低毒力基因的mRNA水平，使得生物被膜的黏附力降低，结构变得松散，对生物被膜形成起到抑制作用，期望能够为植物源天然产物的进一步应用提供一定的理论基础。

Abstract:

[Background] Streptococcus mutans stands as the primary pathogen responsible for dental caries. Despite the definite efficacy in treating dental caries, fluoride-containing products pose potential risks to human health. [Objective] We investigated the effects of kuwanon G on the growth and biofilm formation of S. mutans, aiming to facilitate the development of plant-derived anti-caries medications. [Methods] The microdilution and plate counting methods were employed to study the effects of kuwanon G on the viability and acid tolerance of planktonic S. mutans. Furthermore, the influence of kuwanon G on the acid production by glycolysis in S. mutans was gauged by a pH meter. Quantification of biofilm formation was executed via crystal violet staining, and the content of water-insoluble polysaccharides was determined by the phenol-sulfuric acid method. Additionally, laser scanning confocal microscopy was employed to observe the quantity of extracellular DNA, elucidating the effects of kuwanon G on biofilm development. Real-time fluorescence quantitative PCR was employed to assess the mRNA levels of virulence genes in the biofilm. [Results] Kuwanon G showed the minimum inhibitory concentration of 8 μg/mL and the minimum bactericidal concentration of 64 μg/mL against S. mutans. Notably, at higher concentrations, kuwanon G exhibited a potent bactericidal effect on S. mutans. Kuwanon G suppressed the expression of gtfB, gtfC, gtfD, ldh, comD, vicK, atpF, and gbpB in the biofilm milieu, thereby diminishing the synthesis of water-insoluble polysaccharides and impeding biofilm formation. Furthermore, it reduced the content of extracellular DNA in the biofilm matrix and curtailed the overall extent of biofilm formation. Moreover, kuwanon G adversely affected the acid production and acid tolerance of S. mutans. [Conclusion] Kuwanon G can down-regulate the mRNA levels of virulence genes to inhibit the adhesion and loosen the structure of the biofilm, thus exerting inhibitory effects on biofilm formation. This study offers a theoretical basis for further applications of plant-derived natural products.