Effect of High Intensity Blue Light on Fusobacterium nucleatum Membrane Integrity.

Oral malodour is considered to be caused mainly by the production of volatile sulfide compounds (VSC) by anaerobic gram-negative oral bacteria. Previous studies showed that these bacteria were susceptible to blue light phototoxicity mediated by the production of reactive oxygen species (ROS). In the present study, we tested the effect of blue light on the integrity Fusobacterium nucleatum's membrane, cellular proteins and DNA. Bacterial samples were exposed to high intensity blue light for 0, 70, 140 and 280 s (i.e. fluences of 0, 96, 192 and 384 J cm-2 , respectively). Following light exposure, bacterial samples were examined for membrane damage using fluorescence microscopy, intra-cellular protein analysis using electrophoresis (SDS-PAGE) and DNA fragmentation using ultra-filtration. Results showed that the increasing exposure of bacterial samples to blue light caused increased membrane permeability concomitant with a reduction in intra-cellular proteins and DNA fragments content. These results suggest that membrane damage is the main effect of high intensity blue light exposure on malodour producing bacteria.

In Vitro Effect of Er:YAG Laser on Different Single and Mixed Microorganisms Being Associated with Endodontic Infections.

Objective: The purpose of this in vitro study was to evaluate the antimicrobial effect of activated irrigation with different modes of erbium-doped yttrium aluminum garnet (Er:YAG) laser application on microorganisms related to secondary endodontic infection. Background: Er:YAG laser has been recommended as an adjuvant tool for root canal disinfection during endodontic treatment. Materials and methods: Laser-activated irrigation (LAI) with 300 or 600 µm tips were tested with or without intermittent irrigation with 0.9% sodium chloride (NaCl) solution against different microorganisms (five single strains and dual species (Streptococcus gordonii combined with Actinomyces oris or Fusobacterium nucleatum) in root canals after 3 days of incubation. In a 21-day infection model, LAI was used together with intermittent rinsing with sodium hypochlorite (NaOCl) against the dual-species mixtures; here the incidence of microbial regrowth after up to 7 days was monitored. Results: In the 3-day root infection model, LAI protocols did not show any significant reduction of the microbial load when compared with manual irrigation with saline solution. In the 21-day infection, S. gordonii combined with A. oris were not detectable anymore after applying the LAI protocol with a 600 µm tip (30 mJ/10 pps) up to 7 days after treatment. Conclusions: Application of LAI with a 600 µm tip by using an Er:YAG laser might be advantageous in treatment of endodontic infections.

The Effects of Ultraviolet Light-Emitting Diodes with Different Wavelengths on Periodontopathic Bacteria In Vitro.

Objective: The aim of this study was to examine effects of recently developed ultraviolet light-emitting diodes (UV LEDs) wavelengths on in vitro growth and gene expression of cultural periodontopathic bacteria, and on viability of experimental gingival fibroblasts. Materials and methods: Porphyromonas gingivalis, Prevotella intermedia, Fusobacterium nucleatum, Aggregatibacter actinomycetemcomitans, and Streptococcus oralis were irradiated by UV LEDs (265, 285, 310, 365, and 448 nm) at 600 mJ/cm2 and grown anaerobically in vitro. The colony forming units were counted after 1 week. Cell morphology was observed using a scanning electron microscope (SEM). Quantitative real-time polymerase chain reaction was performed to investigate gene expression changes by 310 nm irradiation. Viability of the irradiated human gingival fibroblasts was evaluated using WST-8 assay. Results: Both 265 and 285 nm resulted in the complete death of bacteria and fibroblasts, whereas 310 nm caused partial killing and suppression of bacterial growth and much less damage to the fibroblasts in vitro. Both 365 and 448 nm resulted in no significant change. SEM showed that P. gingivalis cells gradually degraded from day 2 or 3 and were severely destructed on day 5 for 265, 285, and 310 nm. The 310 nm irradiation transiently suppressed the transcripts of SOS response- and cell division-relative genes. Conclusions: Both 265 and 285 nm may induce powerful bactericidal effects and severe fibroblast phototoxicity, and 310 nm may induce partial killing or growth suppression of bacterial cells with much less fibroblast phototoxicity. UV lights may have potential for bacterial suppression, with situations dependent on wavelength, in periodontal and peri-implant therapy.

Fluorescence change of Fusobacterium nucleatum due to Porphyromonas gingivalis.

The aim of this study was to measure changes in the fluorescence of Fusobacterium nucleatum interacting with Porphyromonas gingivalis for excitation with blue light at 405-nm. P. gingivalis was mono- and co-cultivated in close proximity with F. nucleatum. The fluorescence of the bacterial colonies was photographed using a QLF-D (Quantitative Light-induced Fluorescence-Digital) Biluminator camera system with a 405 nm light source and a specific filter. The red, green and blue intensities of fluorescence images were analyzed using the image analysis software. A fluorescence spectrometer was used to detect porphyrin synthesized by each bacterium. F. nucleatum, which emitted green fluorescence in single cultures, showed intense red fluorescence when it was grown in close proximity with P. gingivalis. F. nucleatum co-cultivated with P. gingivalis showed the same pattern of fluorescence peaks as for protoporphyrin IX in the red part of the spectrum. We conclude that the green fluorescence of F. nucleatum can change to red fluorescence in the presence of adjacent co-cultured with P. gingivalis, indicating that the fluorescence character of each bacterium might depend on the presence of other bacteria.

Bactericidal effects of 310 nm ultraviolet light-emitting diode irradiation on oral bacteria.

BACKGROUND: Ultraviolet (UV) light is used for phototherapy in dermatology, and UVB light (around 310 nm) is effective for treatment of psoriasis and atopic dermatitis. In addition, it is known that UVC light (around 265 nm) has a bactericidal effect, but little is known about the bactericidal effect of UVB light. In this study, we examined the bactericidal effects of UVB-light emitting diode (LED) irradiation on oral bacteria to explore the possibility of using a 310 nm UVB-LED irradiation device for treatment of oral infectious diseases. METHODS: We prepared a UVB (310 nm) LED device for intraoral use to examine bactericidal effects on Streptococcus mutans, Streptococcus sauguinis, Porphyromonas gingivalis, and Fusobacterium nucleatum and also to examine the cytotoxicity to a human oral epithelial cell line (Ca9-22). We also examined the production of nitric oxide and hydrogen peroxide from Ca9-22 cells after irradiation with UVB-LED light. RESULTS: Irradiation with the 310 nm UVB-LED at 105 mJ/cm2 showed 30-50% bactericidal activity to oral bacteria, though 17.1 mJ/cm2 irradiation with the 265 nm UVC-LED completely killed the bacteria. Ca9-22 cells were strongly injured by irradiation with the 265 nm UVC-LED but were not harmed by irradiation with the 310 nm UVB-LED. Nitric oxide and hydrogen peroxide were produced by Ca9-22 cells with irradiation using the 310 nm UVB-LED. P. gingivalis was killed by applying small amounts of those reactive oxygen species (ROS) in culture, but other bacteria showed low sensitivity to the ROS. CONCLUSIONS: Narrowband UVB-LED irradiation exhibited a weak bactericidal effect on oral bacteria but showed low toxicity to gingival epithelial cells. Its irradiation also induces the production of ROS from oral epithelial cells and may enhance bactericidal activity to specific periodontopathic bacteria. It may be useful as a new adjunctive therapy for periodontitis.

Rose bengal uptake by E. faecalis and F. nucleatum and light-mediated antibacterial activity measured by flow cytometry.

Antibacterial photodynamic therapy (aPDT) using rose bengal (RB) and blue-light kills bacteria through the production of reactive oxygen derivates. However, the interaction mechanism of RB with bacterial cells remains unclear. This study investigated the uptake efficiency and the antibacterial activity of blue light-activated RB against Enterococcus faecalis and Fusobacterium nucleatum. Spectrophotometry and epifluorescence microscopy were used to evaluate binding of RB to bacteria. The antibacterial activity of RB after various irradiation times was assessed by flow cytometry in combination with cell sorting. Uptake of RB increased in a concentration dependent manner in both strains although E. faecalis displayed higher uptake values. RB appeared to bind specific sites located at the cellular poles of E. faecalis and at regular intervals along F. nucleatum. Blue-light irradiation of samples incubated with RB significantly reduced bacterial viability. After incubation with 10µM RB and 240s irradiation, only 0.01% (±0.01%) of E. faecalis cells and 0.03% (±0.03%) of F. nucleatum survived after treatment. This study indicated that RB can bind to E. faecalis and F. nucleatum in a sufficient amount to elicit effective aPDT. Epifluorescence microscopy showed a yet-unreported property of RB binding to bacterial membranes. Flow cytometry allowed the detection of bacteria with damaged membranes that were unable to form colonies on agars after cell sorting.

Potential of shock waves to remove calculus and biofilm.

Effective calculus and biofilm removal is essential to treat periodontitis. Sonic and ultrasonic technologies are used in several scaler applications. This was the first feasibility study to assess the potential of a shock wave device to remove calculus and biofilms and to kill bacteria. Ten extracted teeth with visible subgingival calculus were treated with either shock waves for 1 min at an energy output of 0.4 mJ/mm(2) at 3 Hz or a magnetostrictive ultrasonic scaler at medium power setting for 1 min, which served as a control. Calculus was determined before and after treatment planimetrically using a custom-made software using a grey scale threshold. In a second experiment, multispecies biofilms were formed on saliva-preconditioned bovine enamel discs during 64.5 h. They were subsequently treated with shock waves or the ultrasonic scaler (N = 6/group) using identical settings. Biofilm detachment and bactericidal effects were then assessed. Limited efficiency of the shock wave therapy in terms of calculus removal was observed: only 5% of the calculus was removed as compared to 100% when ultrasound was used (P ≤ 0.0001). However, shock waves were able to significantly reduce adherent bacteria by three orders of magnitude (P ≤ 0.0001). The extent of biofilm removal by the ultrasonic device was statistically similar. Only limited bactericidal effects were observed using both methods. Within the limitations of this preliminary study, the shock wave device was not able to reliably remove calculus but had the potential to remove biofilms by three log steps. To increase the efficacy, technical improvements are still required. This novel noninvasive intervention, however, merits further investigation.

Energy dose parameters affect antimicrobial photodynamic therapy-mediated eradication of periopathogenic biofilm and planktonic cultures.

OBJECTIVE: This study evaluated the in vitro efficacy of a commercially available aPDT system in eradication of the periopathogens Porphyromonas gingivalis, Fusobacterium nucleatum, and Aggregatibacter actinomycetemcomitans in both planktonic and biofilm cultures. BACKGROUND DATA: Antimicrobial photodynamic therapy (aPDT) is an effective antibacterial approach in vitro; however, few data are available regarding effective light-energy parameters. MATERIALS AND METHODS: Planktonic and biofilm cultures of periopathogens were exposed to a methylene blue-based formulation and irradiated with a 670-nm nonthermal diode laser. Energy doses were varied from 2.3 to 9.4 J/cm(2) through adjustments in illumination time and a constant power density. Controls consisted of no treatment, light only, and photosensitizer only. Temperature changes were recorded in experimental samples before and after illumination. RESULTS: aPDT with an energy dose of 9.4 J/cm(2) was effective in eradicating P. gingivalis, F. nucleatum, and A. actinomycetemcomitans in biofilm and planktonic form. Reductions from control in planktonic cultures at this energy dose were 6.8 +/- 0.7, 5.2 +/- 0.6, and 1.9 +/- 0.6 log(10), respectively, whereas biofilm reductions were 4.5 +/- 1.2, 3.4 +/- 1.1, and 4.9 +/- 1.4 log(10). Decreasing the treatment time produced an energy dose-dependent killing effect in both models. Changes in sample temperature did not exceed 3 degrees C under these exposure parameters. CONCLUSION: This study demonstrated that three important periopathogens are susceptible to aPDT-mediated killing, regardless of whether they are present in planktonic or biofilm form. Furthermore, a clear energy dose-dependence exists with this treatment that should to be taken into account when determining optimal treatment times in clinical application.

Endodontic photoactivated disinfection using a conventional light source: an in vitro and ex vivo study.

OBJECTIVE: The antimicrobial effect of photoactivated disinfection (PAD) using toluidine blue and an LED lamp was tested on endodontic pathogens in planktonic suspension and after inoculation into extracted teeth. Irradiation time was limited to 30 seconds. STUDY DESIGN: The effect of PAD on planktonic suspensions of Escherichia coli, Candida albicans, Enterococcus faecalis, Fusobacterium nucleatum, and Streptococcus intermedius was analyzed using Poisson regression. Moreover, cultures of S. intermedius were inoculated into prepared root canals of extracted molars. The effect of PAD performed immediately after inoculation or after overnight bacterial incubation was determined by a 2-sample t test. RESULTS: Photoactivated disinfection yielded significant reductions (P < .001) in the viable counts of all organisms in planktonic suspension. The PAD treatment of S. intermedius in root canals yielded a mean log10 reduction of 2.60 (P < .001) immediately after inoculation and of 1.38 (P < .001) after overnight incubation. CONCLUSION: Photoactivated disinfection using a conventional light source strongly reduces the number of viable endodontic pathogens in planktonic suspension and in root canals.

Photodynamic therapy in periodontal therapy: microbiological observations from a private practice.

In recent years, the combination of laser light and photosensitizer known as photodynamic therapy (PDT) has been used in periodontal therapy. However, there are not enough clinical studies to fully evaluate the effects of PDT on the periodontal tissues. This microbiological study examined the effects of PDT on the periodontal bacteria in combination with scaling and root planing (SRP) in the same group of patients by randomly selecting PDT or SRP for use in different quadrants of the mouth. For the present study, PDT was compared with a diode laser (980 nm) and an Nd:YA G laser (1,064 nm). Microbiological samples were examined and evaluated over a period of three months. Significant bacterial reduction has been observed in all cases. The diode laser with SRP presented long-term positive results, while PDT showed a significant bacteria reduction during the entire observation period.

The bactericidal effect of a Genius Nd:YAG laser.

PURPOSE: To evaluate the 'in vitro' bactericidal effect of the Nd:YAG laser (Genius, MØlsgaard Dental, Copenhagen, Denmark) on six periodontal pathogens. METHODS: Suspensions of six different periodontal pathogens (Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythia, Fusobacterium nucleatum and Parvimonas micra) were prepared in small Eppendorff tubes, and exposed to a Nd:YAG laser for five different periods of time. Laser settings used: Power 6 Watt (on a scale of 1-12 W), Frequency 50 Hz, Pulse duration 250 mus. After exposure to the laser, aliquots of the suspensions were spread on blood agar plates for bacterial counting. RESULTS: After 5 s of laser exposure, there was a decrease in total colony forming units for all six selected microorganisms. After 15, 30 and 45 s, no viable bacterial cells could be retrieved. CONCLUSION: In this 'in vitro' model, 15 s of Nd:YAG laser use was found to be effective for total killing of the six tested periodontal pathogens.

Effects of low-energy shock waves on oral bacteria.

We have recently demonstrated that extracorporeal shock-wave therapy (ESWT) is effective in promoting the healing of dermal wounds and in regenerating alveolar bone lost through periodontal disease. The objective of the present study was to determine any antibacterial effect of ESWT on oral bacteria. Monoculture suspensions of 6 bacterial species were treated with 100 to 500 pulses of ESWT at energy flux densities (EFD) of 0.12 mJ/mm(2), 0.22 mJ/mm(2), and 0.3 mJ/mm(2). Following treatment, aliquots were plated for viability determination and compared with untreated controls. ESWT showed a significant microbicidal effect for Streptococcus mutans and an unencapsulated strain of Porphyromonas gingivalis following as few as 100 pulses at 0.3 mJ/mm(2) (p 0.05). These findings suggest that low-energy ESWT may be bactericidal for selected oral bacteria.

Effect of photo-activated disinfection on endodontic pathogens ex vivo.

AIM: To test the hypothesis that photo-activated disinfection (PAD) has a bactericidal effect on pathogens inoculated in root canals, with emphasis on biofilm formation/destruction. METHODOLOGY: Root canals of extracted teeth (n = 38) were prepared (size 30, 0.10 taper), autoclaved, divided into three groups and two negative controls inoculated (Streptococcus anginosus, Enterococcus faecalis or Fusobacterium nucleatum) and treated (PAD, laser, dye or positive control) according to a cross-sectional design. Resultant colony-forming unit counts were associated with observations of cell structural changes using environmental scanning electron microscopy (ESEM) on inoculated dentinal surfaces (n = 22, two controls) before (1, 2 and 6 days of incubation) and after treatment with PAD. RESULTS: Treatment of root canals with PAD (15 J) caused a significant reduction of the bacterial load, resulting in a 93.8% kill of S. anginosus (P < 0.0001), a 88.4% kill of E. faecalis (P < 0.05) and a 98.5% kill of F. nucleatum (P < 0.0001), but no sterilization. Laser alone had no significant effect on the load nor did the dye without laser. The ESEM experiment showed that individual cells or monolayers were easily eliminated with PAD. But when biofilms were present (2 and 6 days for E. faecalis, 6 days for S. anginosus), bacterial eradication was substantially reduced in deep layers. CONCLUSIONS: Photo-activated disinfection is not an alternative but a possible supplement to the existing protocols for root canal disinfection as the interaction between light (diode laser) and associated dye (TBO) provides a broad-spectrum effect. Some endodontic pathogens that grow as single-species biofilms, however, are difficult to eradicate.

Sonoporation is an efficient tool for intracellular fluorescent dextran delivery and one-step double-crossover mutant construction in Fusobacterium nucleatum.

Studies of microorganisms are often hindered by a lack of effective genetic tools. One such example is Fusobacterium nucleatum, a gram-negative anaerobe associated with various human infections, including those causing periodontal disease and preterm birth. The first double-crossover allelic-exchange mutant in F. nucleatum was recently constructed using sonoporation, a novel ultrasound-mediated intracellular delivery method, demonstrating potential for bacterial gene transfection. To better unveil its mechanism, the current study examines the factors affecting the outcome of sonoporation. Delivery of Texas Red-conjugated dextran into F. nucleatum by sonoporation was at least twice as efficient as that by electroporation, and sonoporation was nonbactericidal, unlike electroporation. The delivery efficiency was affected by the acoustic pressure amplitude, the duty cycle, and the quantity of microbubbles used to initiate cavitation but not by the pulse repetition frequency of ultrasound application. To examine the involvement of homologous recombination in sonoporation-mediated mutant construction, the highly conserved recA gene, which carried most of the consensus residues, including the P loop, was identified in F. nucleatum, and a double-crossover recA mutant of F. nucleatum 12230, US1610, was constructed by sonoporation. The mutant exhibited increased sensitivity to UV exposure compared with that of the wild type, indicating that the RecA function in F. nucleatum was conserved. Interestingly, US1610 was also sensitive to ultrasound treatment, suggesting the likely involvement of RecA in postsonoporation repair and survival. Since sonoporation has consistently generated one-step double-crossover mutants in F. nucleatum by use of intact suicide plasmids, this technology may be developed into an efficient tool for streamlining mutant construction in bacteria.

Sensitivity of oral bacteria to 254 nm ultraviolet light.

AIM: To explore the sensitivity of bacteria commonly found in root canals to 254 nm ultraviolet (UV) light, either as individual cells or as participants of a bacterial multilayer. METHODOLOGY: The sensitivity of oral bacteria, as individual cells, to UV light was tested by subjecting plates streaked with bacteria to 254 nm UV, at a fluence of 1-20 mJ cm(-2). An experimental model was designed to produce a bacterial multilayer and to study absorption of UV light by bacteria in an outer layer and its effect on the elimination of bacteria in the inner layer. RESULTS: Direct exposure to relatively low doses of UV light (2-7 mJ cm(-2)) effectively eliminated all bacterial strains tested. Furthermore, an Enterococcus faecalis strain, partially resistant to a 24 h exposure to calcium hydroxide, was effectively eliminated within several seconds of exposure to UV light (P < 0.001). UV was absorbed by a multilayer of bacteria. When 4 bacterial cells microm(-2) were present in the light path, the UV light dose had to be increased by a factor of x10 to achieve 100% elimination of the bacteria in an inner layer. CONCLUSIONS: The application of UV light to eliminate endodontic pathogens may be possible. Nevertheless, its absorbance by outer layers of bacteria should be considered and the UV light dose adapted accordingly.

An antibacterial surface on dental implants, based on the photocatalytic bactericidal effect.

BACKGROUND: It is well known that the moderately roughened surfaces of dental implants enhance direct bone-implant contact. However, rough implant surfaces, as compared to smooth surfaces, are thought to pose a higher risk of bacterial infection when exposed to the oral cavity. PURPOSE: This study was focused on evaluating the photocatalytic bactericidal effects of anatase titanium dioxide (TiO(2)) on gram-negative anaerobic bacteria known to be associated with periimplantitis. MATERIALS AND METHODS: A film of photocatalytic anatase TiO(2) was added onto the surface of commercially pure titanium disks by plasma source ion implantation (PSII) followed by annealing. The photocatalytic properties of the film were confirmed by the degradation of methylene blue. Actinobacillus actinomycetemcomitans and Fusobacterium nucleatum cells were incubated anaerobically and seeded on the disk. The disks were then exposed to ultraviolet A (UVA) illumination from black light in an anaerobic environment. After illumination, the number of viable cells was counted in terms of colony-forming units. RESULTS: The anatase TiO(2) film added by the PSII method and annealing exhibited a strong photocatalytic reaction under UVA illumination. The viability of both types of bacteria on the photocatalytic TiO(2) film was suppressed to less than 1% under UVA illumination within 120 minutes. CONCLUSION: The bactericidal effect of the TiO(2) photocatalyst is of great use for sterilizing the contaminated surface of dental implants.

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

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

Phototoxic effect of visible light on Porphyromonas gingivalis and Fusobacterium nucleatum: an in vitro study.

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.

Sensitization of periodontopathogenic bacteria to killing by light from a low-power laser.

Cultures of Porphyromonas gingivalis, Fusobacterium nucleatum and Actinobacillus actinomycetemcomitans were treated with a range of photosensitizers and then exposed to light from a 7.3 mW helium/neon laser for up to 80 s. Toluidine blue O (25 micrograms/ml) and methylene blue (25 micrograms/ml) were effective lethal photosensitizers of all 3 target organisms, enabling substantial light dose-related reductions in viable counts. Dihaematoporphyrin ester and aluminium disulphonated phthalocyanine were lethal photosensitizers only of P. gingivalis. In the absence of a photosensitizer, exposure to laser light had no significant effect on the viability of the cultures. If such low doses of light (22 J/cm2) are effective at killing bacteria in vivo, the technique may be useful as a means of eliminating periodontopathogenic bacteria from diseased sites.