title: Real-time assessment of Streptococcus mutans biofilm metabolism on resin composite.
abstract: 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).
  
  
  
  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, 24h, 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).
  
  
  
  Strong positive correlations between viable colony counts and bioluminescence values, both before- and after-sonication, validated the utility of this assay.
  
  
  
  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.
authors: Dental Materials, College of Dentistry, University of Oklahoma Health Sciences Center, 1201 N. Stonewall Avenue, Oklahoma City, OK 73117, United States. Electronic address: fernando-esteban-florez@ouhsc.edu.
authors: Fernando Luis
authors: FL
authors: Esteban Florez
authors: Dental Materials, College of Dentistry, University of Oklahoma Health Sciences Center, 1201 N. Stonewall Avenue, Oklahoma City, OK 73117, United States. Electronic address: Shelley-Hiers@ouhsc.edu.
authors: Rochelle Denise
authors: RD
authors: Hiers
authors: Dental Materials, College of Dentistry, University of Oklahoma Health Sciences Center, 1201 N. Stonewall Avenue, Oklahoma City, OK 73117, United States. Electronic address: kristin.smart@gmail.com.
authors: Kristin
authors: K
authors: Smart
authors: Microbiology and Immunology, College of Medicine, University of Oklahoma Health Sciences Center, 940 Stanton L. Young Blvd., BMSB 1053, Oklahoma City, OK 73104, United States; Restorative Dentistry, Oregon Health & Science University, MRB424, 3181 SW Sam Jackson Park Rd., Portland, OR 97239, United States. Electronic address: kreth@ohsu.edu.
authors: Jens
authors: J
authors: Kreth
authors: Microbiology and Immunology, College of Medicine, University of Oklahoma Health Sciences Center, 940 Stanton L. Young Blvd., BMSB 1053, Oklahoma City, OK 73104, United States. Electronic address: Felicia-Qi@ouhsc.edu.
authors: Fengxia
authors: F
authors: Qi
authors: Restorative Dentistry, Oregon Health & Science University, MRB424, 3181 SW Sam Jackson Park Rd., Portland, OR 97239, United States. Electronic address: merrittj@ohsu.edu.
authors: Justin
authors: J
authors: Merritt
authors: Dental Materials, College of Dentistry, University of Oklahoma Health Sciences Center, 1201 N. Stonewall Avenue, Oklahoma City, OK 73117, United States. Electronic address: sharukh-khajotia@ouhsc.edu.
authors: Sharukh Soli
authors: SS
authors: Khajotia
mesh_terms: D000900:Anti-Bacterial Agents; D018441:Biofilms; D001681:Biological Assay; D003188:Composite Resins; D003764:Dental Materials; D008163:Luminescent Measurements; D013295:Streptococcus mutans
chemical_list: D000900:Anti-Bacterial Agents; D003188:Composite Resins; D003764:Dental Materials
keywords: Biofilms; Bioluminescent assays; Colony count; Composite resins; Dental materials; Microbial; Streptococcus mutans