Highlights
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We propose a bioluminescence assay to quantify biofilm metabolism on resin composite.
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Positive correlations with viable colony counts validated the bioluminescence assay.
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Antibacterial dental materials could be tested with this non-disruptive, real-time assay.
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.
1
Introduction
The oral cavity is the habitat of a broad variety of microorganisms . Bacteria are the most common type of microorganism present in the oral milieu. Over 700 bacterial species have been detected in the oral microflora using various cultural and molecular methods . Streptococcus mutans has been implicated as the main causative agent of both primary and secondary caries . However, since S. mutans has been shown to account for only 1.6% of the total cariogenic biomass in active carious lesions , its role as the primary cause of tooth decay has been questioned . An uncontested attribute of active carious lesions is the presence of polymicrobial biofilms and particularly the acid-producing microorganisms within them. S. mutans has been investigated for many years as a model cariogenic organism for its ability to metabolize sugars to acid and because it forms biofilms by the deposition of water-insoluble glucans and other extracellular polymeric substances (EPS).
The viability of bacterial cells within biofilms is most commonly determined using the viable colony count method . The major advantage of this approach is the ability to quantify only the number of viable bacteria . This method’s main disadvantage is the requirement that bacteria be separated from the EPS by vortexing, sonication or matrix-dissolving enzymes. Significant errors could be introduced during that step because manipulation of the biofilms may impact the cells’ viability and does not guarantee complete or reproducible removal . In addition, microbial aggregation of bacteria like S. mutans that typically grow in dense microcolonies could lead to inaccurate counts of viable cells . Furthermore, resin composites release by-products and unpolymerized monomers that could influence biofilm growth . In our experience, biofilms are harder to remove from polymer-based materials as compared to other dental materials, thereby potentially limiting the accuracy of colony counts of biofilms on resin-based dental biomaterials.
These limitations have precipitated the development of simpler and less sensitive quantification methods, such as metabolic assays. Their mechanisms of action are based on either the quantification of extracellular metabolites (e.g., lactic acid) or on the cells’ capacity to reduce organic dyes (e.g., resazurin ). However, existing metabolic assays require the use of calibration curves derived from planktonic bacteria to quantify bacteria in biofilms, which introduces large errors in the assessment of cell viability due to the distinct metabolic rates between biofilms and their planktonic counterparts . In addition, metabolic assays typically do not permit evaluation of other parameters present in vivo, such as the effect of physiochemical characteristics of the substrate materials on which biofilms grow .
One of the major limitations of the resazurin (Alamar Blue) metabolic assay is the need for large numbers of cells (greater than 5- or 6-log CFU) to obtain results reasonably quickly (1–5 h) . In addition, Erb and Ehlers demonstrated that reduction of the fluorescent product (resorufin) into the nonfluorescent product (hydroresorufin) could lead to inaccurate results.
In light of the above limitations, a method is needed to quantify the viability of bacterial cells in biofilms in a non-disruptive, minimally invasive and real-time manner. One possible alternative is the utilization of bioluminescence assays based on the use of the North American firefly luciferase ( Photinus pyralis ; Fan and Wood ). These assays are considered highly efficient because nearly all of the ATP pool is converted into light (yield of 0.88 ). The equation below by Shama and Malik describes the basic mechanism.
Luciferin + O 2 + ATP → luciferase , M g 2 + Oxyluciferin + AMP + PPi + C O 2 + h v
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