Photodynamic inactivation of Streptococcus mutans and Streptococcus sanguinis biofilms in vitro

The purpose of this study was to evaluate specific effects of photodynamic inactivation (PDI) using erythrosine (ER) and Rose Bengal (RB) photosensitizers and a blue light-emitting diode (LED) on the viability of Streptococcus mutans and Streptococcus sanguinis biofilms. Biofilms were grown in acrylic disks immersed in broth to production of biofilms, inoculated with microbial suspension (106 cells/mL) and incubated for 48 h. After the formation of biofilms, the effects of the photosensitizers ER and RB at a concentration of 5 μM for 5 min and blue LED (455 ± 20 nm) for 180 s, photosensitizers alone and conjugated were evaluated. Next, the disks were placed in tubes with sterile physiological solution (0.9 % sodium chloride) and sonicated for to disperse the biofilms. Tenfold serial dilutions were carried and aliquots seeded in brain heart infusion agar which were then incubated for 48 h. Then the numbers colony-forming units per milliliter (CFU/mL; log 10) were counted and analyzed statistically (ANOVA, Tukey test, P ≤ 0.05). Significant decreases in the viability of all microorganisms were observed for biofilms exposed to PDI mediated by both photosensitizers. The reductions with RB and ER were, 0.62 and 0.52 log10 CFU mL -1 for S. mutans biofilms (p = 0.001), and 0.95 and 0.88 log 10 CFU mL-1 for S. sanguinis biofilms (p = 0.001), respectively. The results showed that biofilms formed in vitro by S. mutans and S. sanguinis, were sensitive to PDI using a blue LED associated with photosensitizers ER or RB, indicating its use in the control of caries and periodontal diseases. © 2012 Springer-Verlag London Ltd.

Photodynamic antimicrobial therapy of curcumin in biofilms and carious dentine

Photodynamic therapy (PDT) is a technique that involves the activation of photosensitizers by light in the presence of oxygen, resulting in the production of reactive radicals that are capable of inducing cell death. The present study evaluated the susceptibility of Streptococcus mutans and Lactobacillus acidophilus to PDT grown as multi-species in the biofilm phase versus in dentine carious lesions. A brain-heart infusion culture medium supplemented with 1 % glucose, 2 % sucrose, and 1 % young primary culture of L. acidophilus 108 CFU/mL and S. mutans 108 CFU/mL was used to develop multi-species biofilms and to induce caries on human dentine slabs. Five different concentrations of curcumin (0.75, 1.5, 3.0, 4.0, and 5.0 g/L) were used associated with 5.7 J/cm2 light emission diode. Four different groups were analyzed L-D- (control group), L-D+ (drug group), L+D- (light group), and L+D+ (PDT group). ANOVA/Tukey's tests were conducted to compare groups. A significant reduction (p <0.05) in cell viability was observed in the biofilm phase following photosensitization with all curcumin concentrations tested. To achieve significant bacterial reduction (p <0.05) in carious dentine, it was necessary to utilize 5.0 g/L of curcumin in association with blue light. No significant reduction was found for L-D+, supporting the absence of the drug's dark toxicity. S. mutans and L. acidophilus were susceptible to curcumin in the presence of blue light. However, due to light penetration and drug diffusion difficulties, these microorganisms within dentine carious lesions were less affected than they were in the biofilm phase. © 2013 Springer-Verlag London.

Photodynamic inactivation of biofilm: Taking a lightly colored approach to stubborn infection

Microbial biofilms are responsible for a variety of microbial infections in different parts of the body, such as urinary tract infections, catheter infections, middle-ear infections, gingivitis, caries, periodontitis, orthopedic implants, and so on. The microbial biofilm cells have properties and gene expression patterns distinct from planktonic cells, including phenotypic variations in enzymic activity, cell wall composition and surface structure, which increase the resistance to antibiotics and other antimicrobial treatments. There is consequently an urgent need for new approaches to attack biofilm-associated microorganisms, and antimicrobial photodynamic therapy (aPDT) may be a promising candidate. aPDT involves the combination of a nontoxic dye and low-intensity visible light which, in the presence of oxygen, produces cytotoxic reactive oxygen species. It has been demonstrated that many biofilms are susceptible to aPDT, particularly in dental disease. This review will focus on aspects of aPDT that are designed to increase efficiency against biofilms modalities to enhance penetration of photosensitizer into biofilm, and a combination of aPDT with biofilm-disrupting agents. © 2013 Informa UK Ltd.

Susceptibility of multispecies biofilm to photodynamic therapy using Photodithazine®

This in vitro study evaluated the effect of photodynamic therapy (PDT) on the multispecies biofilm of Candida albicans, Candida glabrata, and Streptococcus mutans. Standardized fungal and bacterial suspensions were cultivated appropriately for each species and inoculated in 96-well microtiter plates for mix-biofilm formation. After 48 h of incubation, the biofilms were submitted to PDT (P + L+) using Photodithazine® (PDZ) at 100, 150, 175, 200, or 250 mg/mL for 20 min and 37.5 J/cm2 of light-emitting diode (LED) (660 nm). Additional samples were treated only with PDZ (P + L-) or LED (P-L+), or neither (control, P-L-). Afterwards, the biofilms were evaluated by quantification of colonies (CFU/mL), metabolic activity (XTT reduction assay), total biomass (crystal violet staining), and confocal scanning laser microscopy (CSLM). Data were analyzed by one-way ANOVA and Tukey tests (p < 0.05). Compared with the control, PDT promoted a significant reduction in colonies viability of the three species evaluated with 175 and 200 mg/mL of PDZ. PDT also significantly reduced the metabolic activity of the biofilms compared with the control, despite the PDZ concentration. However, no significant difference was found in the total biomass of samples submitted or not to PDT. For all analysis, no significant difference was verified among P-L-, P + L-, and P-L+. CSLM showed a visual increase of dead cells after PDT. PDT-mediated PDZ was effective in reducing the cell viability of multispecies biofilm. © 2013 Springer-Verlag London.

Effect of antimicrobial photodynamic therapy on periodontally infected tooth sockets in rats

The aim of the present study was to evaluate the antimicrobial effect of antimicrobial photodynamic therapy (aPDT) in alveolar treatment of areas with induced periodontitis. Thirty male Wistar rats were subjected to ligature-induced periodontal disease (PD) in the first left inferior molars, while the right side molars did not receive ligatures. After 7 days of PD evolution, ligatures were removed from the left side, and the first left and right mandibular molars were extracted. Afterwards, animals were divided into groups according to the following treatments: control (C)-no treatment; mechanical debridement (MD)-mechanical debridement and irrigation with saline solution; and aPDT-mechanical debridement, irrigation with toluidine blue O (TBO), and 1 min of laser irradiation (GaAlAs, 660 nm, 30 mW, 32 J/cm2, 60 s). Ligatures were removed and samples of the alveolar content after extraction and after each treatment were collected for microbial processing by real-time polymerase chain reaction with specific primers for Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, and Treponema denticola. Data were submitted to statistical analysis by multiple comparison tests (McNemar test; p < 0.05). T. denticola was not found in the collected samples. A. actinomycetemcomitans and P. gingivalis were found in ligature samples. Tooth socket samples without periodontitis induction presented lesser microbial charge than samples with induced periodontitis (p < 0.05). aPDT significantly reduced A. actinomycetemcomitans levels on the left side (p < 0.05). It was concluded that aPDT was an effective antimicrobial treatment for tooth sockets in areas affected by induced periodontitis. © 2013 Springer-Verlag London.

Photodynamic potential of curcumin and blue LED against streptococcus mutans in a planktonic culture

Background: The photodynamic therapy (PDT) involves the use of light of specific wavelength to activate a nontoxic photosensitizing agent or dye in the presence of oxygen for eradication of target cells. In dentistry, this therapy is used to suppress the growth of microorganisms involved directly with dental decay and periodontitis process. There are evidences that curcumin dye is able to control microbial activity when illuminated with specific wavelength. The purpose of this study was to evaluate the in vitro efficacy of PDT using curcumin dye (Cur-C) in combination with a blue LED (L) device on a planktonic model of Streptococcus mutans ( S. mutans). Methods: Suspensions (0.5mL) containing S. mutans at 1×107CFUmL-1 were prepared and divided into 4 groups: Group C-L- (control: no treatment and 1 experimental condition), Group C+L- (curcumin at 3 different concentrations: 2000; 4000 and 8000μM and 3 experimental conditions), Group C-L+ (LED at 3 different dosages: 24, 48 and 72Jcm-2 and 3 experimental conditions), and Group C+L+ (PDT group: curcumin at respective concentrations combined to LED dosages and 9 experimental conditions). Samples of each experimental condition were cultured in Petri dishes of BHI agar. Incubation in micro-aerophilia at 37°C for 48h was performed for subsequent visual counting of CFU/mL. Data were transformed into log10 and analyzed by two-way ANOVA and Tukey's test at p<0.05. Results: Group C. +. L+, in specific experimental conditions, demonstrated a log bacterial reduction 70% higher than Group C. -. L-. Both groups C. -. L+ and C. +. L- presented a slight decrease in log bacterial counting. Conclusion: This in vitro method was able to reduce the number of S. mutans in a planktonic suspension. © 2013 Elsevier B.V.

Comparison of the effect of rose bengal- and eosin Y-mediated photodynamic inactivation on planktonic cells and biofilms of Candida albicans

Candida albicans is an opportunistic yeast that can cause oral candidosis through the formation of a biofilm, an important virulence factor that compromises the action of antifungal agents. The objective of this study was to compare the effect of rose bengal (RB)- and eosin Y (EY)-mediated photodynamic inactivation (PDI) using a green light-emitting diode (LED; 532 ± 10 nm) on planktonic cells and biofilms of C. albicans (ATCC 18804). Planktonic cultures were treated with photosensitizers at concentrations ranging from 0.78 to 400 μM, and biofilms were treated with 200 μM of photosensitizers. The number of colony-forming unit per milliliter (CFU/mL) was compared by analysis of variance and Tukey's test (P ≤ 0.05). After treatment, one biofilm specimen of the control and PDI groups were examined by scanning electron microscopy. The photosensitizers (6.25, 25, 50, 200, and 400 μM of EY, and 6.25 μM of RB or higher) significantly reduced the number of CFU/mL in the PDI groups when compared to the control group. With respect to biofilm formation, RB- and EY-mediated PDI promoted reductions of 0.22 log10 and 0.45 log10, respectively. Scanning electron microscopy showed that the two photosensitizers reduced fungal structures. In conclusion, EY- and RB-mediated PDI using LED irradiation significantly reduced C. albicans planktonic cells and biofilms. © 2013 Springer-Verlag London.

Photodynamic inactivation of clinical isolates of Candida using Photodithazine

This study evaluated the photodynamic inactivation (PDI) mediated by Photodithazine® (PDZ) against 15 clinical isolates of Candida albicans, Candida glabrata and Candida tropicalis. Each isolate, in planktonic and biofilm form, was exposed to PDI by assessing a range of PDZ concentrations and light emitting diode fluences. Cell survival of the planktonic suspensions was determined by colony forming units (CFU ml-1). The antifungal effects of PDI against biofilms were evaluated by CFU ml-1 and metabolic assay. Data were analyzed by non-parametric tests (α = 0.05). Regardless of the species, PDI promoted a significant viability reduction of planktonic yeasts. The highest reduction in cell viability of the biofilms was equivalent to 0.9 log10 (CFU ml-1) for C. albicans, while 1.4 and 1.5 log10 reductions were obtained for C. tropicalis and C. glabrata, respectively. PDI reduced the metabolic activity of biofilms by 62.1, 76.0, and 76.9% for C. albicans, C. tropicalis, and C. glabrata, respectively. PDZ-mediated PDI promoted significant reduction in the viability of Candida isolates. © 2013 Taylor & Francis.

Photodynamic Therapy in Pythium insidiosum - An In Vitro Study of the Correlation of Sensitizer Localization and Cell Death

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

The influence of photodynamic therapy parameters on the inactivation of Candida spp: in vitro and in vivo studies

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Safety assessment of oral photodynamic therapy in rats

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Photodynamic Therapy for Pododermatitis in Penguins

Pododermatitis is currently one of most frequent and important clinical complications in seabirds kept in captivity or in rehabilitation centers. In this study, five Magellanic penguins with previous pododermatitis lesions on their footpad were treated with photodynamic therapy (PDT). All PDT treated lesions successfully regressed and no recurrence was observed during the 6-month follow-up period. PDT seems to be an inexpensive and effective alternative treatment for pododermatitis in Magellanic penguins encouraging further research on this topic. (C) 2014 Wiley Periodicals, Inc.

Biofilms of Candida albicans serotypes A and B differ in their sensitivity to photodynamic therapy

Candida albicans is classified into different serotypes according to cell wall mannan composition and cell surface hydrophobicity. Since the effectiveness of photodynamic therapy (PDT) depends on the cell wall structure of microorganisms, the objective of this study was to compare the sensitivity of in vitro biofilms of C. albicans serotypes A and B to antimicrobial PDT. Reference strains of C. albicans serotype A (ATCC 36801) and serotype B (ATCC 36802) were used for the assays. A gallium-aluminum-arsenide laser (660 nm) was used as the light source and methylene blue (300 mu M) as the photosensitizer. After biofilm formation on the bottom of a 96-well microplate for 48 h, each Candida strain was submitted to assays: PDT consisting of laser and photosensitizer application (L + P+), laser application alone (L + P-), photosensitizer application alone (L-P+), and application of saline as control (L-P-). After treatment, biofilm cells were scraped off and transferred to tubes containing PBS. The content of the tubes was homogenized, diluted, and seeded onto Sabouraud agar plates to determine the number of colony-forming units (CFU/mL). The results were compared by analysis of variance and Tukey test (p < 0.05). The two strains studied were sensitive to PDT (L + P+), with a log reduction of 0.49 for serotype A and of 2.34 for serotype B. Laser application alone only reduced serotype B cells (0.53 log), and the use of the photosensitizer alone had no effect on the strains tested. It can be concluded that in vitro biofilms of C. albicans serotype B were more sensitive to PDT.

Antimicrobial photodynamic therapy in rat experimental candidiasis: evaluation of pathogenicity factors of Candida albicans

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Tooth Discoloration Induced by Endodontic Phenothiazine Dyes in Photodynamic Therapy

Objective: This study sought to assess if discoloration of tooth structures occurs after photodynamic therapy (PDT) and to determine the efficacy of a protocol to remove the photosensitizers. Background data: PDT has been used in root canal treatment to enhance cleaning and disinfection of the root canal system. PDT uses a low power laser in association with a dye as a photosensitizer. Photosensitizers can induce staining of the dental structures, resulting in an unaesthetic appearance. Methods: Forty teeth were randomly divided into four groups according to the photosensitizer used and pre-irradiation time: 0.01% methylene blue for 5 min (MB5); 0.01% methylene blue for 10 min (MB 10); 0.01% toluidine blue for 5 min (TB5); and 0.01% toluidine blue for 10 min (TB 10). Specimens were irradiated with a 660 nm diode laser with a 300 mu m diameter optical fiber, at 40 mW power setting for 3 min. Immediately after, the photosensitizers were removed with Endo-PTC cream +2.5% sodium hypochlorite (NaOCl). The shade was measured by a Vita Easyshade spectrophotometer based on the CIELAB color system (L*a*b* values) at three different experimental times: before PDT (T0), immediately after PDT (T1), and after removal of the photosensitizer (T2). Results: The results showed a decrease in the averages of the L*a*b* coordinate values after PDT (T1) in all the groups, when compared with the number at T0, with a significant statistical difference in group MB10. After photosensitizer removal (T2), all the values of the coordinates increased with significant statistical differences (p < 0.05) between T1 and T2 in L* and a*. Conclusions: It can be concluded that both methylene blue and toluidine blue dyes cause tooth discoloration, and that Endo-PTC cream associated with 2.5% NaOCl effectively remove these dyes, regardless of the pre-irradiation time used for PDT.