Optimization of an ultra-bright real-time high-throughput renilla luciferase assay for antibacterial assessment of Streptococcus mutans biofilms.

OBJECTIVE: The present work demonstrates the optimization of a renilla-based real-time, ultra-bright, non-disruptive, high-throughput bioluminescence assay (HTS) to assess the metabolism of intact Streptococcus mutans biofilms and its utility in screening the antibacterial efficacy of experimental nanofilled dental adhesive resins containing varying concentrations of nitrogen-doped titanium dioxide nanoparticles (N_TiO2). METHODS: Optimization of the assay was achieved by screening real-time bioluminescence changes in intact Streptococcus mutans biofilms imposed by the various experimental biofilm growth parameters investigated (bacterial strain, growth media, sucrose concentration, dilution factor, and inoculum volume). The optimized assay was then used to characterize the antibacterial efficacy of experimental nanofilled dental adhesive resins. The assay's ability to discriminate between bacteriostatic and bactericidal approaches was also investigated. RESULTS: Relative Light Units (RLU) values from the HTS optimization were analyzed by multivariate ANOVA (α = 0.05) and coefficients of variation. An optimized HTS bioluminescence assay was developed displaying RLUs values (brightness) that are much more intense when comparing to other previously reported bioluminescence assays, thereby decreasing the error associated with bioluminescence assays and displaying better utility while investigating the functionalities of antimicrobial nanofilled experimental dental adhesive resins with proven long-term properties. SIGNIFICANCE: The present study is anticipated to positively impact subsequent research on dental materials and oral microbiology because it serves as a valuable screening tool in metabolic-based assays with increased sensitivity and robustness. The assay reported is anticipated to be further optimized to be used as a co-reporter for other Luc based assays.

Characterization of Experimental Nanoparticulated Dental Adhesive Resins with Long-Term Antibacterial Properties.

Experimental adhesives with functional nitrogen-doped titanium dioxide nanoparticles (N_TiO2) have been shown to display improved properties. However, these materials have not been characterized regarding their degree of conversion (DC), biaxial flexure strength (BFS), surface roughness (SR), elastic modulus (EM), and long-term antibacterial functionalities. Experimental adhesives were synthesized by dispersing N_TiO2 (10%, 20%, or 30%, v/v%) into OptiBond Solo Plus (OPTB, Kerr Corp., USA). Unpolymerized adhesives (volume = 50 µL/drop, n = 3/group) were individually placed onto a heated (37 °C) attenuated total reflectance (ATR) monolithic diamond crystal (Golden Gate, Specac). The spectra of composites were obtained with a Fourier-transform infrared (FTIR) spectrometer (Nicolet IS50; 500-4500 cm-1; resolution = 4 cm-1, 10 internal scans/spectrum) before and after polymerization. Disk-shaped specimens (diameter = 6.0 mm, thickness = 0.5 mm) for BFS (n = 12/group), SR and EM (n = 3/group), and for antibacterial testing (n = 18/group/time-point) were fabricated and photopolymerized (1 min each; 385-515 nm, 1000 mW/cm2; VALO). DC values (%) were calculated from pre- and post-polymerization spectra using the two-frequency method and tangent-baseline technique. BFS was assessed using a universal testing machine (Instron 68TM-5, crosshead speed = 1.27 mm/min, 25 °C). SR and EM were investigated using an atomic force microscope (Multimode 8) with aluminum-coated silicon probes (8 nm pyramidal tip, spring constant 40 N/m, Bruker). Antibacterial testing was performed by growing Streptococcus mutans biofilms (UA159-ldh, 37 °C, microaerophilic) on the surfaces of specimens for 24 h and then measuring the relative luminescence units (RLU) with a Biotek Synergy HT multi-well plate reader. Results demonstrate that experimental materials containing 10%, 20%, and 30% of N_TiO2 displayed higher levels of DC, had better mechanical properties, and were able to exert strong and durable antibacterial properties without visible light irradiation and after extended periods of simulated shelf-life and aging in PBS. The reported experimental materials are expected to increase the service lives of polymer-based bonded restorations by decreasing the incidence of secondary caries.

Antibacterial efficacy of non-thermal atmospheric plasma against Streptococcus mutans biofilm grown on the surfaces of restorative resin composites.

The aim of this study was to evaluate the antimicrobial efficacy of non-thermal atmospheric plasma (NTAP) against Streptococcus mutans biofilms. Resin discs were fabricated, wet-polished, UV sterilized, and immersed in water for monomer extraction (37 °C, 24 h). Biofilms of bioluminescent S. mutans strain JM10 was grown on resin discs in anaerobic conditions for (37 °C, 24 h). Discs were divided into seven groups: control (CON), 2% chlorhexidine (CHX), only argon gas 150 s (ARG) and four NTAP treatments (30 s, 90 s, 120 s, 150 s). NTAP was applied using a plasma jet device. After treatment, biofilms were analyzed through the counting of viable colonies (CFU), bioluminescence assay (BL), scanning electron microscopy (SEM), and polymerase chain reaction (PCR). All NTAP-treated biofilm yielded a significant CFU reduction when compared to ARG and CON. BL values showed that NTAP treatment for 90 s, 120 s or 150 s resulted in statistically significantly lower metabolic activity when compared to the other groups. CHX displayed the lowest means of CFU and BL. SEM showed significant morphological changes in NTAP-treated biofilm. PCR indicated damage to the DNA structure after NTAP treatment. NTAP treatment was effective in lowering the viability and metabolism of S. mutans in a time-dependent manner, suggesting its use as an intraoral surface-decontamination strategy.

Sorption, solubility and cytotoxicity of novel antibacterial nanofilled dental adhesive resins.

Dental adhesives hydrolyze in the mouth. This study investigated the water sorption (SOR), solubility (SOL) and cytotoxicity (CYTO) of experimental adhesives containing nitrogen-doped titanium dioxide nanoparticles (N_TiO2). Specimens (n = 15/group [SOR, SOL]; n = 10/group [CYTO]) of unaltered Clearfil SE Protect (CSP), OptiBond Solo Plus (OSP), Adper Scotchbond (ASB) and experimental adhesives (OSP + 25% or 30% of N_TiO2) were fabricated, desiccated (37 °C) and tested for SOR and SOL according to ISO Specification 4049 (2009). CYTO specimens were UV-sterilized (8 J/cm2) and monomer extracted in growth medium (1, 3 or 7 days). Human pulp cells were isolated and seeded (0.5 × 104) for MTT assay. SOR and SOL data was analyzed using GLM and SNK (α = 0.05) and CYTO data was analyzed with Kruskal-Wallis and SNK tests (α = 0.05). SOR and SOL values ranged from 25.80 µg/mm3 (30% N_TiO2) to 28.01 µg/mm3 (OSP) and 23.88 µg/mm3 (30% N_TiO2) to 25.39 µg/mm3 (25% N_TiO2). CYTO results indicated that pulp cells exposed to experimental materials displayed comparable viabilities (p > 0.05) to those of OSP. Experimental materials displayed comparable SOR, SOL and CYTO values (p > 0.05) when compared to unaltered materials. N_TiO2 incorporation have not adversely impacted SOR, SOL and CYTO properties of unaltered adhesives.

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

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.

Antibacterial dental adhesive resins containing nitrogen-doped titanium dioxide nanoparticles.

The development of dental adhesive resins with long-lasting antibacterial properties is a possible solution to overcome the problem of secondary caries in modern adhesive dentistry. OBJECTIVES: (i) Synthesis and characterization of nitrogen-doped titanium dioxide nanoparticles (N_TiO2), (ii) topographical, compositional and wettability characterization of thin-films (unaltered and experimental) and, (iii) antibacterial efficacy of N_TiO2-containing dental adhesives against Streptococcus mutans biofilms. MATERIALS AND METHODS: Nanoparticles were synthesized and characterized using different techniques. Specimens (diameter = 12 mm, thickness ≅ 15 µm) of OptiBond Solo Plus (Kerr Corp., USA) and experimental adhesives [50, 67 and 80% (v/v)] were fabricated, photopolymerized (1000 mW/cm2, 1 min) and UV-sterilized (254 nm, 800,000 µJ/cm2) for microscopy, spectroscopy, wettability and antibacterial testing. Wettability was assessed with a contact angle goniometer by dispensing water droplets (2 µL) onto four random locations of each specimen (16 drops/group). Drop profiles were recorded (1 min, 25 frames/s, 37 °C) and contact angles were calculated at time = 0 s (θINITIAL) and time = 59 s (θFINAL). Antibacterial testing was performed by growing S. mutans (UA159-ldh, JM10) biofilms for either 3 or 24 h (anaerobic conditions, 37 °C) with or without continuous light irradiation (410 ±â€¯10 nm, 3 h = 38.75 J/cm2, 24 h = 310.07 J/cm2) against the surfaces of sterile specimens. RESULTS: N_TiO2 was successfully prepared using solvothermal methods. Doped-nanoparticles displayed higher light absorption levels when compared to undoped titania. Experimental adhesives demonstrated superior antibacterial efficacy in dark conditions. CONCLUSIONS: The findings presented herein suggest that N_TiO2 is a feasible antibacterial agent against cariogenic biofilms.

Bioluminescence Analysis of Antibacterial Photodynamic Therapy Using Methylene Blue Mediated by Low-Intensity Level Laser Against Cariogenic Biofilms.

OBJECTIVE: A non-destructive and real-time bioluminescence (BL) assay was used to determine the utility of antimicrobial photodynamic therapy (aPDT) treatments mediated by methylene blue (MB) and laser irradiation (LI) against intact biofilms that are capable of producing caries (cariogenic). The efficacy of antibacterial photodynamic treatments has been currently determined by using either viable colony counts (VCC) or metabolic assays (Alamar Blue) that were demonstrated to have critical limitations when used on microcolony-forming bacteria such as Streptococcus mutans. MATERIALS AND METHODS: Resin composite specimens were fabricated, wet-polished, ultraviolet-sterilized, and stored in water (72 h). S. mutans (strain JM10) biofilms were grown [24 h; 0.65 × THY with 0.1% (w/v) sucrose] on the surfaces of sterile specimens. Antibacterial treatments were performed by using MB (0.0005% and 0.001%) with or without LI (660 ± 10 nm, 6 J/cm2). Specimens treated with chlorhexidine gluconate served as the negative control group. The efficacy of aPDT treatments was determined in terms of BL for intact biofilms and VCC for sonicated bacteria. RESULTS: BL results were corrected by using the Greenhouse-Geisser method and were analyzed with repeated-measures analysis of variance and post hoc Bonferroni test (α = 0.05). VCC results were analyzed by using Kruskal-Wallis and Dunn's multiple-comparisons post hoc tests (α = 0.05). Our findings demonstrated that experimental treatments significantly decreased the viability of S. mutans biofilms (p < 0.05). Moderate reductions in cellular viability were observed on biofilms subjected to aPDT treatments. CONCLUSIONS: This study demonstrated that aPDT has promising potential to be used as an additional method to control oral cariogenic biofilms.

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

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.