Real-time assessment of Streptococcus mutans biofilm metabolism on resin composite

Fernando Luis Esteban Florez, Rochelle Denise Hiers, Kristin Smart, Jens Kreth, Fengxia Qi, Justin Merritt, Sharukh Soli Khajotia

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

Objective The release of unpolymerized monomers and by-products of resin composites influences biofilm growth and confounds the measurement of metabolic activity. Current assays to measure biofilm viability have critical limitations and are typically not performed on relevant substrates. The objective of the present study was to determine the utility of firefly luciferase assay for quantification of the viability of intact biofilms on a resin composite substrate, and correlate the results with a standard method (viable colony counts). Methods Disk-shaped specimens of a dental resin composite were fabricated, wet-polished, UV-sterilized, and stored in water. Biofilms of Streptococcus mutans (strain UA159 modified by insertion of constitutively expressed firefly luc gene) were grown (1:500 dilution; anaerobic conditions, 24 h, 37 °C) in two media concentrations (0.35x and 0.65x THY medium supplemented with 0.1% sucrose; n = 15/group). An additional group of specimens with biofilms grown in 0.65x + sucrose media was treated with chlorhexidine gluconate solution to serve as the control group. Bioluminescence measurements of non-disrupted biofilms were obtained after addition of D-Luciferin substrate. The adherent biofilms were removed by sonication, and bioluminescence of sonicated bacteria was then measured. Viable colony counts were performed after plating sonicated bacteria on THY agar plates supplemented with spectinomycin. Bioluminescence values and cell counts were correlated using Spearman correlation tests (α = 0.05). Results Strong positive correlations between viable colony counts and bioluminescence values, both before- and after-sonication, validated the utility of this assay. Significance A novel non-disruptive, real-time bioluminescence assay is presented for quantification of intact S. mutans biofilms grown on a resin composite, and potentially on antibacterial materials and other types of dental biomaterials.

Original languageEnglish (US)
Pages (from-to)1263-1269
Number of pages7
JournalDental Materials
Volume32
Issue number10
DOIs
StatePublished - Oct 1 2016

Fingerprint

Streptococcus mutans
Composite Resins
Biofilms
Metabolism
Bioluminescence
Resins
Composite materials
Assays
Sonication
Sugar (sucrose)
Sucrose
Bacteria
Substrates
Fireflies
Spectinomycin
Luminescent Measurements
Firefly Luciferases
Dental Materials
Biocompatible Materials
Plating

Keywords

  • Biofilms
  • Bioluminescent assays
  • Colony count
  • Composite resins
  • Dental materials
  • Microbial
  • Streptococcus mutans

ASJC Scopus subject areas

  • Materials Science(all)
  • Dentistry(all)
  • Mechanics of Materials

Cite this

Real-time assessment of Streptococcus mutans biofilm metabolism on resin composite. / Esteban Florez, Fernando Luis; Hiers, Rochelle Denise; Smart, Kristin; Kreth, Jens; Qi, Fengxia; Merritt, Justin; Khajotia, Sharukh Soli.

In: Dental Materials, Vol. 32, No. 10, 01.10.2016, p. 1263-1269.

Research output: Contribution to journalArticle

Esteban Florez, Fernando Luis ; Hiers, Rochelle Denise ; Smart, Kristin ; Kreth, Jens ; Qi, Fengxia ; Merritt, Justin ; Khajotia, Sharukh Soli. / Real-time assessment of Streptococcus mutans biofilm metabolism on resin composite. In: Dental Materials. 2016 ; Vol. 32, No. 10. pp. 1263-1269.
@article{721fbe31cae3474c9dd3c997a1cf02ad,
title = "Real-time assessment of Streptococcus mutans biofilm metabolism on resin composite",
abstract = "Objective The release of unpolymerized monomers and by-products of resin composites influences biofilm growth and confounds the measurement of metabolic activity. Current assays to measure biofilm viability have critical limitations and are typically not performed on relevant substrates. The objective of the present study was to determine the utility of firefly luciferase assay for quantification of the viability of intact biofilms on a resin composite substrate, and correlate the results with a standard method (viable colony counts). Methods Disk-shaped specimens of a dental resin composite were fabricated, wet-polished, UV-sterilized, and stored in water. Biofilms of Streptococcus mutans (strain UA159 modified by insertion of constitutively expressed firefly luc gene) were grown (1:500 dilution; anaerobic conditions, 24 h, 37 °C) in two media concentrations (0.35x and 0.65x THY medium supplemented with 0.1{\%} sucrose; n = 15/group). An additional group of specimens with biofilms grown in 0.65x + sucrose media was treated with chlorhexidine gluconate solution to serve as the control group. Bioluminescence measurements of non-disrupted biofilms were obtained after addition of D-Luciferin substrate. The adherent biofilms were removed by sonication, and bioluminescence of sonicated bacteria was then measured. Viable colony counts were performed after plating sonicated bacteria on THY agar plates supplemented with spectinomycin. Bioluminescence values and cell counts were correlated using Spearman correlation tests (α = 0.05). Results Strong positive correlations between viable colony counts and bioluminescence values, both before- and after-sonication, validated the utility of this assay. Significance A novel non-disruptive, real-time bioluminescence assay is presented for quantification of intact S. mutans biofilms grown on a resin composite, and potentially on antibacterial materials and other types of dental biomaterials.",
keywords = "Biofilms, Bioluminescent assays, Colony count, Composite resins, Dental materials, Microbial, Streptococcus mutans",
author = "{Esteban Florez}, {Fernando Luis} and Hiers, {Rochelle Denise} and Kristin Smart and Jens Kreth and Fengxia Qi and Justin Merritt and Khajotia, {Sharukh Soli}",
year = "2016",
month = "10",
day = "1",
doi = "10.1016/j.dental.2016.07.010",
language = "English (US)",
volume = "32",
pages = "1263--1269",
journal = "Dental Materials",
issn = "0109-5641",
publisher = "Elsevier Science",
number = "10",

}

TY - JOUR

T1 - Real-time assessment of Streptococcus mutans biofilm metabolism on resin composite

AU - Esteban Florez, Fernando Luis

AU - Hiers, Rochelle Denise

AU - Smart, Kristin

AU - Kreth, Jens

AU - Qi, Fengxia

AU - Merritt, Justin

AU - Khajotia, Sharukh Soli

PY - 2016/10/1

Y1 - 2016/10/1

N2 - Objective The release of unpolymerized monomers and by-products of resin composites influences biofilm growth and confounds the measurement of metabolic activity. Current assays to measure biofilm viability have critical limitations and are typically not performed on relevant substrates. The objective of the present study was to determine the utility of firefly luciferase assay for quantification of the viability of intact biofilms on a resin composite substrate, and correlate the results with a standard method (viable colony counts). Methods Disk-shaped specimens of a dental resin composite were fabricated, wet-polished, UV-sterilized, and stored in water. Biofilms of Streptococcus mutans (strain UA159 modified by insertion of constitutively expressed firefly luc gene) were grown (1:500 dilution; anaerobic conditions, 24 h, 37 °C) in two media concentrations (0.35x and 0.65x THY medium supplemented with 0.1% sucrose; n = 15/group). An additional group of specimens with biofilms grown in 0.65x + sucrose media was treated with chlorhexidine gluconate solution to serve as the control group. Bioluminescence measurements of non-disrupted biofilms were obtained after addition of D-Luciferin substrate. The adherent biofilms were removed by sonication, and bioluminescence of sonicated bacteria was then measured. Viable colony counts were performed after plating sonicated bacteria on THY agar plates supplemented with spectinomycin. Bioluminescence values and cell counts were correlated using Spearman correlation tests (α = 0.05). Results Strong positive correlations between viable colony counts and bioluminescence values, both before- and after-sonication, validated the utility of this assay. Significance A novel non-disruptive, real-time bioluminescence assay is presented for quantification of intact S. mutans biofilms grown on a resin composite, and potentially on antibacterial materials and other types of dental biomaterials.

AB - Objective The release of unpolymerized monomers and by-products of resin composites influences biofilm growth and confounds the measurement of metabolic activity. Current assays to measure biofilm viability have critical limitations and are typically not performed on relevant substrates. The objective of the present study was to determine the utility of firefly luciferase assay for quantification of the viability of intact biofilms on a resin composite substrate, and correlate the results with a standard method (viable colony counts). Methods Disk-shaped specimens of a dental resin composite were fabricated, wet-polished, UV-sterilized, and stored in water. Biofilms of Streptococcus mutans (strain UA159 modified by insertion of constitutively expressed firefly luc gene) were grown (1:500 dilution; anaerobic conditions, 24 h, 37 °C) in two media concentrations (0.35x and 0.65x THY medium supplemented with 0.1% sucrose; n = 15/group). An additional group of specimens with biofilms grown in 0.65x + sucrose media was treated with chlorhexidine gluconate solution to serve as the control group. Bioluminescence measurements of non-disrupted biofilms were obtained after addition of D-Luciferin substrate. The adherent biofilms were removed by sonication, and bioluminescence of sonicated bacteria was then measured. Viable colony counts were performed after plating sonicated bacteria on THY agar plates supplemented with spectinomycin. Bioluminescence values and cell counts were correlated using Spearman correlation tests (α = 0.05). Results Strong positive correlations between viable colony counts and bioluminescence values, both before- and after-sonication, validated the utility of this assay. Significance A novel non-disruptive, real-time bioluminescence assay is presented for quantification of intact S. mutans biofilms grown on a resin composite, and potentially on antibacterial materials and other types of dental biomaterials.

KW - Biofilms

KW - Bioluminescent assays

KW - Colony count

KW - Composite resins

KW - Dental materials

KW - Microbial

KW - Streptococcus mutans

UR - http://www.scopus.com/inward/record.url?scp=84991093572&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84991093572&partnerID=8YFLogxK

U2 - 10.1016/j.dental.2016.07.010

DO - 10.1016/j.dental.2016.07.010

M3 - Article

C2 - 27515531

AN - SCOPUS:84991093572

VL - 32

SP - 1263

EP - 1269

JO - Dental Materials

JF - Dental Materials

SN - 0109-5641

IS - 10

ER -