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

Fernando Luis Esteban Florez; Rochelle Denise Hiers; Yan Zhao; Justin Merritt; Adam Justin Rondinone; Sharukh Soli Khajotia

doi:10.1016/j.dental.2019.12.007

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

Dent Mater. 2020 Mar;36(3):353-365. doi: 10.1016/j.dental.2019.12.007. Epub 2020 Jan 15.

Authors

Fernando Luis Esteban Florez¹, Rochelle Denise Hiers², Yan Zhao³, Justin Merritt⁴, Adam Justin Rondinone⁵, Sharukh Soli Khajotia⁶

Affiliations

¹ The University of Oklahoma Health Sciences Center, Department of Restorative Sciences, Division of Dental Biomaterials, College of Dentistry, 1201 N. Stonewall Avenue, Oklahoma City, OK, 73117, USA. Electronic address: fernando-esteban-florez@ouhsc.edu.
² The University of Oklahoma Health Sciences Center, Department of Restorative Sciences, Division of Dental Biomaterials, College of Dentistry, 1201 N. Stonewall Avenue, Oklahoma City, OK, 73117, USA. Electronic address: shelley-hiers@ouhsc.edu.
³ The University of Oklahoma Health Sciences Center, Department of Biostatistics and Epidemiology, College of Public Health, 801 NE 13th Street, Oklahoma City, OK, 73126, USA. Electronic address: daniel-zhao@ouhsc.edu.
⁴ Oregon Health & Science University, Department of Restorative Dentistry, School of Dentistry, MRB424, 3181 SW Sam Jackson Park Rd., Portland, OR, 97239, USA. Electronic address: merrittj@ohsu.edu.
⁵ Oak Ridge National Laboratory, Center for Nanophase Materials Sciences, Oak Ridge, TN, 37831, USA. Electronic address: rondinoneaj@ornl.gov.
⁶ The University of Oklahoma Health Sciences Center, Department of Restorative Sciences, Division of Dental Biomaterials, College of Dentistry, 1201 N. Stonewall Avenue, Oklahoma City, OK, 73117, USA. Electronic address: sharukh-khajotia@ouhsc.edu.

Abstract

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.

Keywords: Antibacterial; Bioluminescence; Dental adhesive resins; High-throughput; Streptococcus mutans.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

Anti-Bacterial Agents
Biofilms
Dental Cements*
Extracellular Polymeric Substance Matrix
Humans
Reproducibility of Results
Streptococcus mutans*

Substances

Anti-Bacterial Agents
Dental Cements

Abstract

Publication types

MeSH terms

Substances

Grants and funding