Optimization of a real-time high-throughput assay for assessment of Streptococcus mutans metabolism and screening of antibacterial dental adhesives

Journal Article · 14 January 2020 · Dental Materials

DOI: https://doi.org/10.1016/j.dental.2019.12.007 · OSTI ID:1815606

Esteban Florez, Fernando Luis  ^[1]; Hiers, Rochelle Denise  ^[2]; Zhao, Yan  ^[2]; Merritt, Justin  ^[3]; Rondinone, Adam Justin ^[4]; Khajotia, Sharukh Soli  ^[2]

---

1. Univ. of Oklahoma Health Sciences Center, Oklahoma City, OK (United States); OSTI
2. Univ. of Oklahoma Health Sciences Center, Oklahoma City, OK (United States)
3. Oregon Health & Science Univ., Portland, OR (United States)
4. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Center for Nanophase Materials Sciences (CNMS)

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.

• Research Organization: Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States). Center for Nanophase Materials Science (CNMS)
• Sponsoring Organization: National Institute of Dental and Craniofacial Research (NIDCR); National Institute of General Medical Sciences (NIGMS); National Institutes of Health (NIH); USDOE Office of Science (SC), Basic Energy Sciences (BES)
• Grant/Contract Number: AC05-00OR22725
• OSTI ID: 1815606
• Journal Information: Dental Materials, Journal Name: Dental Materials Journal Issue: 3 Vol. 36; ISSN 0109-5641
• Publisher: ElsevierCopyright Statement
• Country of Publication: United States
• Language: English