Fernando Luis Esteban Florez1, Rochelle Denise Hiers2, Kristin Smart3, Jens Kreth4, Fengxia Qi5, Justin Merritt6, Sharukh Soli Khajotia7. 1. 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. 2. 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. 3. 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. 4. 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. 5. 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. 6. Restorative Dentistry, Oregon Health & Science University, MRB424, 3181 SW Sam Jackson Park Rd., Portland, OR 97239, United States. Electronic address: merrittj@ohsu.edu. 7. 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.
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, 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). 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.
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, 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). 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.
Authors: Christina Wilson; Rachel Lukowicz; Stefan Merchant; Helena Valquier-Flynn; Jeniffer Caballero; Jasmin Sandoval; Macduff Okuom; Christopher Huber; Tessa Durham Brooks; Erin Wilson; Barbara Clement; Christopher D Wentworth; Andrea E Holmes Journal: Res Rev J Eng Technol Date: 2017-10-24
Authors: Fernando Luis Esteban Florez; Rochelle Denise Hiers; Preston Larson; Matthew Johnson; Edgar O'Rear; Adam J Rondinone; Sharukh Soli Khajotia Journal: Mater Sci Eng C Mater Biol Appl Date: 2018-09-01 Impact factor: 7.328
Authors: Benjamin J Schneider; Rochelle D Hiers; G Frans Currier; Onur Kadioglu; Sarah E Johnston; Yan D Zhao; Fernando L Esteban Florez; Sharukh S Khajotia Journal: Am J Orthod Dentofacial Orthop Date: 2021-06-03 Impact factor: 2.711
Authors: Diane R Bienek; Anthony A Giuseppetti; Stanislav A Frukhtbeyn; Rochelle D Hiers; Fernando L Esteban Florez; Sharukh S Khajotia; Drago Skrtic Journal: J Funct Biomater Date: 2019-12-18