Osnat Feuerstein

Phototoxic Effect of Visible Light on Porphyromonas gingivalis and Fusobacterium nucleatum: An In Vitro Study¶,†

Abstract The antibacterial effect of visible light irradiation combined with photosensitizers has been reported. The objective of this was to test the effect of visible light irradiation without photosensitizers on the viability of oral microorganisms. Strains of Porphyromonas gingivalis, Fusobacterium nucleatum, Streptococcus mutans and Streptococcus faecalis in suspension or grown on agar were exposed to visible light at wavelengths of 400–500 nm. These wavelengths are used to photopolymerize composite resins widely used for dental restoration. Three photocuring light sources, quartz–tungsten–halogen lamp, light-emitting diode and plasma-arc, at power densities between 260 and 1300 mW/cm2 were used for up to 3 min. Bacterial samples were also exposed to a near-infrared diode laser (wavelength, 830 nm), using identical irradiation parameters for comparison. The results show that blue light sources exert a phototoxic effect on P. gingivalis and F. nucleatum. The minimal inhibitory dose for P. gingivalis and F. nucleatum was 16–62 J/cm2, a value significantly lower than that for S. mutans and S. faecalis (159–212 J/cm2). Near-infrared diode laser irradiation did not affect any of the bacteria tested. Our results suggest that visible light sources without exogenous photosensitizers have a phototoxic effect mainly on Gram-negative periodontal pathogens.

Mechanism of visible light phototoxicity on Porphyromonas gingivalis and Fusobacterium nucleatum.

Abstract Phototoxicity of visible light laser on the porphyrin-producing bacteria, Porphyromonas gingivalis, in the absence of photosensitizers and under aerobic conditions was shown in previous studies. Recently, we found that the noncoherent visible light sources at wavelengths of 400–500 nm, commonly used in restorative dentistry, induced a phototoxic effect on P. gingivalis, as well as on Fusobacterium nucleatum, and to a lesser extent on the Streptococci sp. To elucidate the mechanism of this phototoxic effect, P. gingivalis and F. nucleatum were exposed to light (1) under aerobic and anaerobic environments and (2) in the presence of scavengers of reactive oxygen species (ROS). Phototoxic effect was not observed when the bacteria were exposed to light under anaerobic conditions. Dimethyl thiourea, a hydroxyl radical scavenger, was effective in reducing phototoxicity (P ≤ 0.05). Other scavengers, such as catalase, superoxide dismutase and ascorbic acid, were less effective when applied separately. These results support the assumption that the phototoxic effect of blue light on the periopathogenic bacteria is oxygen dependent and that hydroxyl radicals play an important role in this process.

Genetic and Physiological Effects of Noncoherent Visible Light Combined with Hydrogen Peroxide on Streptococcus mutans in Biofilm

ABSTRACT Oral biofilms are associated with the most common infections of the oral cavity. Bacteria embedded in the biofilms are less sensitive to antibacterial agents than planktonic bacteria are. Recently, an antibacterial synergic effect of noncoherent blue light and hydrogen peroxide (H2O2) on planktonic Streptococcus mutans was demonstrated. In this study, we tested the effect of a combination of light and H2O2 on the vitality and gene expression of S. mutans embedded in biofilm. Biofilms of S. mutans were exposed to visible light (wavelengths, 400 to 500 nm) for 30 or 60 s (equivalent to 34 or 68 J/cm2) in the presence of 3 to 300 mM H2O2. The antibacterial effect was assessed by microbial counts of each treated sample compared with that of the control. The effect of light combined with H2O2 on the different layers of the biofilm was evaluated by confocal laser scanning microscopy. Gene expression was determined by real-time reverse transcription-PCR. Our results show that noncoherent light, in combination with H2O2, has a synergistic antibacterial effect through all of the layers of the biofilm. Furthermore, this treatment was more effective against bacteria in biofilm than against planktonic bacteria. The combined light and H2O2 treatment up-regulated the expression of several genes such as gtfB, brp, smu630, and comDE but did not affect relA and ftf. The ability of noncoherent visible light in combination with H2O2 to affect bacteria in deep layers of the biofilm suggests that this treatment may be applied in biofilm-related diseases as a minimally invasive antibacterial procedure.

Temperature changes in dental implants following exposure to hot substances in an ex vivo model.

OBJECTIVES The habitual consumption of extremely hot foods and beverages may affect implant treatment modality. Our objectives were to: (i) establish the maximum temperature produced intra-orally while consuming very hot substances and (ii) use these values in an ex vivo model to assess the temperature changes along the implant-bone interface. MATERIALS AND METHODS Temperatures were measured using thermocouples linked to a computer. The thermocouple electrodes were attached to the tooth-gum interface of the interproximal areas in 14 volunteers during consumption of extremely hot foods and beverages. The in vivo measured temperature values obtained were used in an ex vivo model of a bovine mandible block with an implant and with an assembled abutment. Temperatures were measured by thermocouple electrodes attached to five locations, three of them along the implant-bone interface. RESULTS During consumption of a hot beverage, a maximum temperature of up to 76.3 degrees C was recorded, and a calculated extreme intra-oral temperature of 61.4 degrees C was established. The ex vivo model showed a high correlation between the temperature measured at the abutment and that measured at the abutment-implant interface and inside the implant, reaching maximum temperatures close to 60 degrees C. At the mid-implant-bone and apical implant-bone interfaces, the maximum temperatures measured were 43.3 and 42 degrees C, respectively. CONCLUSIONS The maximum temperatures measured at the implant-bone interfaces reached the temperature threshold of transient changes in bone (42 degrees C). The results of this study support the notion that intra-oral temperatures, developed during the consumption of very hot substances, may be capable of damaging peri-implant tissues.

Influence of blue light on Streptococcus mutans re-organization in biofilm.

Our aim was to examine the viability and structure of new biofilm formed by Streptococcus mutans that was previously exposed to blue light. S. mutans bacteria were grown to form a mature biofilm, that was exposed to blue light (wavelengths, 400-500 nm) for 1-10 min (equivalent to 68-680 J/cm(2)). Biofilm was dispersed by sonication, and then the suspended bacteria were grown to re-organize as a new biofilm. Biofilm formation after 2, 4, and 6 h, was examined by viable counts and by confocal laser scanning microscopy using live/dead bacterial staining. A significant decrease in bacterial viability was found in the 6h biofilms formed by bacteria that had been previously exposed to blue light for 7 or 10 min. Confocal microscopy images showed a decrease in the live/dead bacterial ratio after 3-10 min of light exposures. Dead bacteria were mainly at the outer layers of the biofilm. Exposure of S. mutans in biofilm to blue light affected the re-formation of a new biofilm, showing an increase in the amount of dead bacteria. This phenomenon suggests that blue light has a delayed antibacterial effect, although it does not interfere with bacterial capability to reform an initial biofilm.

Light Therapy Complementary Antibacterial Treatment of Oral Biofilm

Conventional antibacterial treatment fails to eradicate biofilms associated with common infections of the oral cavity. Unlike chemical agents, which are less effective than anticipated, owing to diffusion limitations in biofilms, light is more effective on bacteria in biofilm than in suspension. Effectiveness depends also on the type and parameters of the light. We tested the phototoxic effects of non-coherent blue light (wavelengths, 400-500 nm) and CO2 laser (wavelength, 10.6 μm), which have different mechanisms of action on the oral bacterium Streptoccocus mutans, in biofilm and on tooth enamel. Exposure of S. mutans in biofilm to blue light had a delayed effect on bacterial viability throughout the biofilm and a sustained antibacterial effect on biofilm newly formed by previously irradiated bacteria. A synergistic antibacterial effect between blue light and H2O2 may enhance the phototoxic effect, which involves a photochemical mechanism mediated by reactive oxygen species (ROS) formation. The effect of CO2 laser irradiation on the viability of S. mutans in biofilm on enamel samples appeared to be higher in the deep layers, due to heating of the enamel surface by the absorbed energy. Biofilms do not interfere with the chemical changes resulting from irradiation, which may increase the enamel’s resistance to acid attack.

In vivo changes in dental implant temperatures during hot beverage intake: a pilot study.

Purpose:The aim of this study was to measure the increase in temperature in dental implants during the intake of hot beverages in vivo. Materials and Methods:Eight successfully osseointegrated implants in 7 subjects were examined. Each subject was asked to drink the same volume of hot beverage. While drinking, temperature changes were recorded via 3 embedded thermocouples placed (i) in the implant’s internal space, (ii) at the implant-abutment interface, and (iii) at the abutment. All thermocouples were linked to a computer and analyzed with appropriate software. Results:The maximum temperatures were 47.3°C at the abutment, 45.6°C at the implant’s internal space, and 44.6°C at the implant-abutment interface. A linear correlation was found between the temperatures measured (i) at the implant abutment and in the implant’s internal space, and (ii) at the abutment and at the abutment-implant interface. Conclusions:Further clinical studies are required to determine whether the habitual consumption of hot food and beverages may be considered a risk factor in the success of implant-supported prostheses.

Effects of CO2 laser irradiation on tooth enamel coated with biofilm

CO2 laser irradiation of tooth enamel can inhibit demineralization of tooth enamel, by changing enamel composition and resistance to acid attack. The aim of this work was to examine these effects of CO2 laser irradiation on enamel covered by biofilm.

Effect of Sublethal CO2 Laser Irradiation on Gene Expression of Streptococcus mutans Immobilized in a Biofilm

Streptococcus mutans colonizing on tooth surfaces is one of the major causative agents of human dental caries. Despite numerous studies conducted on lasers and oral tissue interactions, little is known about the effect of laser energy on S. mutans gene expression in a biofilm form. The aim of this study was to investigate the effect of sublethal energies of CO2 laser on biofilm and gene expression of the oral bacteria S. mutans immobilized in biofilm. S. mutans biofilm was irradiated with CO2 laser. Vitality and construction of the biofilm were observed by confocal laser scanning microscopy and scanning electron microscopy. The effect of laser irradiation on gene expression was evaluated by DNA microarray. CO2 laser irradiation had a dose effect on the viability of S. mutans immobilized in biofilm. A nonsignificant lethal effect was observed at 31 J/cm2 while at higher energy of 70 and 144 J/cm2 an antibacterial effect was recorded. The mode of antibacterial action seems to be from the inner layers toward the outer layer of the biofilm, indicating the influence of the surface on the killing effect. At 31 J/cm2, microarray analysis indicated a moderate effect on S. mutans gene expression due to CO2 laser irradiation, mainly down-regulating genes related to bacterial stress response. In conclusion, laser irradiation at sublethal energy had an effect on gene expression of S. mutans.

Bacillus subtilis Biofilm Development – A Computerized Study of Morphology and Kinetics

Biofilm is commonly defined as accumulation of microbes, embedded in a self-secreted extra-cellular matrix, on solid surfaces or liquid interfaces. In this study, we analyze several aspects of Bacillus subtilis biofilm formation using tools from the field of image processing. Specifically, we characterize the growth kinetics and morphological features of B. subtilis colony type biofilm formation and compare these in colonies grown on two different types of solid media. Additionally, we propose a model for assessing B. subtilis biofilm complexity across different growth conditions. GFP-labeled B. subtilis cells were cultured on agar surfaces over a 4-day period during which microscopic images of developing colonies were taken at equal time intervals. The images were used to perform a computerized analysis of few aspects of biofilm development, based on features that characterize the different phenotypes of B. subtilis colonies. Specifically, the analysis focused on the segmented structure of the colonies, consisting of two different regions of sub-populations that comprise the biofilm – a central “core” region and an “expanding” region surrounding it. Our results demonstrate that complex biofilm of B. subtillis grown on biofilm-promoting medium [standard lysogeny broth (LB) supplemented with manganese and glycerol] is characterized by rapidly developing three-dimensional complex structure observed at its core compared to biofilm grown on standard LB. As the biofilm develops, the core size remains largely unchanged during development and colony expansion is mostly attributed to the expansion in area of outer cell sub-populations. Moreover, when comparing the bacterial growth on biofilm-promoting agar to that of colonies grown on LB, we found a significant decrease in the GFP production of colonies that formed a more complex biofilm. This suggests that complex biofilm formation has a diminishing effect on cell populations at the biofilm core, likely due to a combination of reduced metabolic rate and increased levels of cell death within this region.