OBJECTIVE The present work shows the optimization of a high-throughput bioluminescence assay to assess the metabolism of intact Streptococcus mutans biofilms and its utility as a screening method for nanofilled antibacterial dental materials. METHODS The assay was optimized by monitoring changes in bioluminescence mediated by variation of the experimental parameters investigated (growth media and sucrose concentration, inoculum:D-Luciferin ratio, dilution factor, inoculum volume, luminescence wavelength, replicate and luciferase metabolic activity). Confocal microscopy was then used to demonstrate the impact of biofilm growth conditions on the 3-D distribution of extracellular polymeric substance (EPS) within Streptococcus mutans biofilms and its implications as confounding factors in high-throughput studies (HTS). RESULTS Relative Luminescence Unit (RLU) values from the HTS optimization were analyzed by multivariate ANOVA (α = 0.05) and coefficients of variation, whereas data from 3-D structural parameters and RLU values of biofilms grown on experimental antibacterial dental adhesive resins were analyzed using General Linear Models and Student-Newman-Keuls post hoc tests (α = 0.05). Confocal microscopy demonstrated that biofilm growth conditions significantly influenced the quantity and distribution of EPS within the 3-D structures of the biofilms. An optimized HTS bioluminescence assay was developed and its applicability as a screening method in dentistry was demonstrated using nanofilled experimental antibacterial dental adhesive resins. SIGNIFICANCE The present study is anticipated to positively impact the direction of future biofilm research in dentistry, because it offers fundamental information for the design of metabolic-based assays, increases the current levels of standardization and reproducibility while offering a tool to decrease intra-study variability.

中文翻译：

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用于评估变形链球菌代谢和筛选抗菌牙科粘合剂的实时高通量分析的优化。

目的本工作显示了一种高通量生物发光测定法的优化，以评估完整的变形链球菌生物膜的代谢及其作为纳米填充抗菌牙科材料的筛选方法的实用性。方法通过监测所研究的实验参数（生长培养基和蔗糖浓度，接种物：D-荧光素比率，稀释因子，接种物体积，发光波长，复制和荧光素酶代谢活性）的变化介导的生物发光变化来优化测定方法。然后使用共聚焦显微镜来证明生物膜生长条件对变形链球菌生物膜内细胞外聚合物（EPS）的3-D分布的影响及其在高通量研究（HTS）中作为混杂因素的影响。结果通过多变量方差分析（α= 0.05）和变异系数分析了HTS优化过程中的相对发光单位（RLU）值，而使用抗菌牙科粘合树脂在生物膜上生长的3D结构参数和生物膜的RLU值数据进行了分析。通用线性模型和Student-Newman-Keuls事后检验（α= 0.05）。共聚焦显微镜表明，生物膜的生长条件显着影响生物膜3-D结构内EPS的数量和分布。开发了一种优化的HTS生物发光测定法，并使用纳米填充的实验性抗菌牙科粘合剂树脂证明了其在牙科筛查方法中的适用性。