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.
| Original language | English (US) |
|---|---|
| Journal | Dental Materials |
| DOIs | |
| State | Accepted/In press - Jan 1 2020 |
Fingerprint
Keywords
- Antibacterial
- Bioluminescence
- Dental adhesive resins
- High-throughput
- Streptococcus mutans
ASJC Scopus subject areas
- Materials Science(all)
- Dentistry(all)
- Mechanics of Materials
Cite this
Optimization of a real-time high-throughput assay for assessment of Streptococcus mutans metabolism and screening of antibacterial dental adhesives. / Esteban Florez, Fernando Luis; Hiers, Rochelle Denise; Zhao, Yan; Merritt, Justin; Rondinone, Adam Justin; Khajotia, Sharukh Soli.
In: Dental Materials, 01.01.2020.Research output: Contribution to journal › Article
}
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
PY - 2020/1/1
Y1 - 2020/1/1
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
UR - http://www.scopus.com/inward/record.url?scp=85077929010&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85077929010&partnerID=8YFLogxK
U2 - 10.1016/j.dental.2019.12.007
DO - 10.1016/j.dental.2019.12.007
M3 - Article
AN - SCOPUS:85077929010
JO - Dental Materials
JF - Dental Materials
SN - 0109-5641
ER -