TY - JOUR
T1 - Optimization of a real-time high-throughput assay for assessment of Streptococcus mutans metabolism and screening of antibacterial dental adhesives
AU - Esteban Florez, Fernando Luis
AU - Hiers, Rochelle Denise
AU - Zhao, Yan
AU - Merritt, Justin
AU - Rondinone, Adam Justin
AU - Khajotia, Sharukh Soli
N1 - Funding Information:
The research results discussed in this publication were made possible in part by funding through the award project number HR16-131, from the Oklahoma Center for the Advancement of Science and Technology. The present work was also partially funded by the Oklahoma Shared Clinical and Translational Resources grant number NIGMS U54GM104938. The synthesis and characterization of metaloxide nanoparticles was conducted at the Center for Nanophase Materials Sciences (CNMS2013-331 and CNMS2015-331), which is a DOE Office of Science user facility. JM is funded by N.I.HNIDCR grants DE018893 and DE022083.
Funding Information:
The research results discussed in this publication were made possible in part by funding through the award project number HR16-131 , from the Oklahoma Center for the Advancement of Science and Technology . The present work was also partially funded by the Oklahoma Shared Clinical and Translational Resources grant number NIGMS U54GM104938 . The synthesis and characterization of metaloxide nanoparticles was conducted at the Center for Nanophase Materials Sciences (CNMS2013-331 and CNMS2015-331), which is a DOE Office of Science user facility. JM is funded by N.I.H NIDCR grants DE018893 and DE022083 .
Publisher Copyright:
© 2019 The Academy of Dental Materials
PY - 2020/3
Y1 - 2020/3
N2 - 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.
AB - 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.
KW - Antibacterial
KW - Bioluminescence
KW - Dental adhesive resins
KW - High-throughput
KW - Streptococcus mutans
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U2 - 10.1016/j.dental.2019.12.007
DO - 10.1016/j.dental.2019.12.007
M3 - Article
C2 - 31952798
AN - SCOPUS:85077929010
VL - 36
SP - 353
EP - 365
JO - Dental Materials
JF - Dental Materials
SN - 0109-5641
IS - 3
ER -