Antimicrobial and antibiofilm photodynamic therapy against vancomycin resistant Staphylococcus aureus (VRSA) induced infection in vitro and in vivo.

Biofilm mediated infection caused by multi-drug resistant bacteria are difficult to treat since it protects the microorganisms by host defense system, making them resistant to antibiotics and other antimicrobial agents. Combating such type of nosocomial infection, especially in immunocompromised patients, is an urgent need and foremost challenge faced by clinicians. Therefore, antimicrobial photodynamic therapy (aPDT) has been intensely pursued as an alternative therapy for bacterial infections. aPDT leads to the generation of reactive oxygen species (ROS) that destroy bacterial cells in the presence of a photosensitizer, visible light and oxygen. Here, we elucidated a possibility of its clinical application by reducing the treatment time and exposing curcumin to 20 J/cm2 of blue laser light, which corresponds to only 52 s to counteract vancomycin resistant Staphylococcus aureus (VRSA) both in vitro and in vivo. To understand the mechanism of action, the generation of total reactive oxygen species (ROS) was quantified by 2'-7'-dichlorofluorescein diacetate (DCFH-DA) and the type of phototoxicity was confirmed by fluorescence spectroscopic analysis. The data showed more production of singlet oxygen, indicating type-II phototoxicity. Different anti-biofilm assays (crystal violet and congo red assays) and microscopic studies were performed at sub-MIC concentration of curcumin followed by treatment with laser light against preformed biofilm of VRSA. The result showed significant reduction in the preformed biofilm formation. Finally, its therapeutic potential was validated in skin abrasion wistar rat model. The result showed significant inhibition of bacterial growth. Furthermore, immunomodulatory analysis with rat serum was performed. A significant reduction in expression of proinflammatory cytokines TNF-α and IL-6 were observed. Hence, we conclude that curcumin mediated aPDT with 20 J/cm2 of blue laser treatment (for 52 s) could be used against multi-drug resistant bacterial infections and preformed biofilm formation as a potential therapeutic approach.

Antimicrobial Blue Light Inactivation of Microbial Isolates in Biofilms.

BACKGROUND AND OBJECTIVES: Biofilms cause more than 80% of infections in humans, including more than 90% of all chronic wound infections and are extremely resistant to antimicrobials and the immune system. The situation is exacerbated by the fast spreading of antimicrobial resistance, which has become one of the biggest threats to current public health. There is consequently a critical need for the development of alternative therapeutics. Antimicrobial blue light (aBL) is a light-based approach that exhibits intrinsic antimicrobial effect without the involvement of exogenous photosensitizers. In this study, we investigated the antimicrobial effect of this non-antibiotic approach against biofilms formed by microbial isolates of multidrug-resistant bacteria. STUDY DESIGN/MATERIALS AND METHODS: Microbial isolates of Acinetobacter baumannii, Candida albicans, Escherichia coli, Enterococcus faecalis, MRSA, Neisseria gonorrhoeae, Pseudomonas aeruginosa, and Proteus mirabilis were studied. Biofilms were grown in microtiter plates for 24 or 48 hours or in the CDC biofilm reactor for 48 hours and exposed to aBL at 405 nm (60 mW/cm2 , 60 or 30 minutes). The anti-biofilm activity of aBL was measured by viable counts. RESULTS: The biofilms of A. baumannii, N. gonorrhoeae, and P. aeruginosa were the most susceptible to aBL with between 4 and 8 log10 inactivation after 108 J/cm2 (60 mW/cm2 , 30 minutes) or 216 J/cm2 (60 mW/cm2 , 60 minutes) aBL were delivered in the microplates. On the contrary, the biofilms of C. albicans, E. coli, E. faecalis, and P. mirabilis were the least susceptible to aBL inactivation (-0.30, -0.24, -0.84, and -0.68 log10 inactivation, respectively). The same aBL treatment in biofilms developed in the CDC biofilm reactor, caused -1.68 log10 inactivation in A. baumannii and -1.74 and -1.65 log10 inactivation in two different strains of P. aeruginosa. CONCLUSIONS: aBL exhibits potential against pathogenic microorganisms and could help with the significant need for new antimicrobials in clinical practice to manage multidrug-resistant infections. Lasers Surg. Med. © 2019 Wiley Periodicals, Inc.

The impact of photobiomodulation of major salivary glands on caries risk.

Dental caries is a complex multifactorial chronic infectious disease guided by several risk or protective factors. Saliva has an important role in caries and the remineralization process. Caries risk assessment is defined as the probability of new caries lesion development or the existing lesion progression in a given time period. Caries diagnostics and risk factor assessment are followed by targeted elimination of risk factors and less conservative but abundant preventive therapeutic measures. The aim of our prospective randomized study was to elucidate on how photobiomodulation of major salivary glands with polychromatic light or LED light affects caries risk factors in high caries-risk patients. Thirty-six patients were assigned to one of the following three experimental groups: the first, irradiated with polarized polychromatic light (40 mW/cm2, wavelengths 480-3400 nm); the second, a continuous LED light (16 mW/cm2, wavelengths 625, 660, 850 nm); the third, same LED light in a pulsed mode. The fourth group was the control, for which a non-therapeutic visible light was used. Light was administered extra-orally bilaterally above the parotid and submandibular glands for 10 min and intra-orally above the sublingual glands for 5 min, 3 times a week, for 4 consecutive weeks. Each patient's caries risk was assessed according to Cariogram before and after therapy. Caries risk factors were determined from samples of saliva before therapy, two weeks after it commenced, at the end of therapy, and four weeks after the end of therapy. At the end of treatment, the following findings were obtained: In the group irradiated with polarized polychromatic light and in the group irradiated with continuous LED light, the Streptococcus mutans and Lactobacillus counts decreased and salivary buffering capacity increased (p < 0.05). In the group irradiated with pulsed LED light, Streptococcus mutans counts decreased and unstimulated salivary flow and salivary buffering capacity increased (p < 0.05). In all three experimental groups, caries risk was lower (p < 0.05). In the placebo control group, there were no statistically significant differences between parameters before and after therapy. We concluded that photobiomodulation of major salivary glands in high caries-risk patients can reduce the cariogenic bacteria in saliva and improve some salivary parameters, thus reducing caries risk.

The earthworm Dendrobaena veneta (Annelida): A new experimental-organism for photobiomodulation and wound healing.

Photobiomodulation (PBM) is a manipulation of cellular behavior using non-ablative low intensity light sources. This manipulation triggers a cascade of metabolic effects and physiological changes resulting in improved tissue repair, of benefit in the treatment of tissue injury, degenerative or autoimmune diseases. PBM has witnessed an exponential increase in both clinical instrument technology and applications. It is therefore of benefit to find reliable experimental models to test the burgeoning laser technology for medical applications. In our work, we proposed the earthworm Dendrobaena veneta for the study of non-ablative laser-light effects on wound healing. In our preliminary work, D. veneta has been shown to be positively affected by PBM. New tests using D. veneta were set up to evaluate the effectiveness of a chosen 808 nm-64 J/cm2-1W-CW laser therapy using the AB2799 hand-piece with flat-top bean profile, on the wound healing process of the earthworm. Effective outcome was assimilated through examining the macroscopic, histological, and molecular changes on the irradiated posterior-segment of excised-earthworms with respect to controls. Three successive treatments, one every 24 hours, were concluded as sufficient to promote the wound healing, by effects on muscular and blood vessel contraction, decrement of bacteria load, reduction of inflammatory processes and tissue degeneration. D. veneta was demonstrated to be a reliable experimental organism that meets well the 3Rs principles and the National Science Foundation statement. Through their genetic and evolutionary peculiarity, comparable to those of scientifically accredited models, D. veneta allows the effect of laser therapies by multidisciplinary methods, at various degree of complexity and costs to be investigated.

The Effect of Diode Laser on Planktonic Enterococcus faecalis in Infected Root Canals in an Ex Vivo Model.

OBJECTIVE: This study examined the bactericidal effect of diode laser irradiation against intracanal Enterococcus faecalis. METHODS AND MATERIALS: m total of 128 extracted single-rooted and single-canal teeth were treated with ProTaper instruments (Dentsply Maillefer, Ballaigues, Switzerland). A total of 120 root canals were inoculated with E. faecalis for 21 days, and the samples were randomly divided into five groups: Group 1 (n = 24) samples were irrigated with only saline solution (positive controls); Group 2 (n = 24) was treated with only 5.25% sodium hypochlorite; Group 3 (n = 24) was irrigated with saline solutions activated by diode laser; Group 4 (n = 24) was treated with 5.25% sodium hypochlorite activated by diode laser; and Group 5 (n = 24) was irrigated with saline solution with methylene blue dye activated by the diode laser Fox (Sweden & Martina, Padova, Italy); additionally, eight teeth were not contaminated and their canals were irrigated with saline solution and used as a negative control. The Uro-Quick system was used to determine the microbial residual charge. The data were analyzed using Pearson's chi-square test (p < 0.001). RESULTS: A statistically significant reduction in bacterial count was observed in Group 2 and Group 4 (p < 0.001). There were no statistically significant differences among the other groups (p > 0.001). CONCLUSIONS: Evidence indicates that the diode laser was not more effective than sodium hypochlorite in reducing free bacteria.

The disinfection of impression materials by using microwave irradiation and hydrogen peroxide.

STATEMENT OF PROBLEM: Microwave irradiation and immersion in solutions have been recommended for denture disinfection. However, the effect of dry conditions and impression materials has not been completely evaluated. PURPOSE: The purpose of this study was to evaluate the effectiveness of microwave irradiation and hydrogen peroxide for the disinfection of dental impression materials. MATERIAL AND METHODS: Specimens (diameter 10 mm, thickness 2 mm) were made with polyvinyl siloxane. Experimental groups were treated with hydrogen peroxide (group H), microwave irradiation (group M), and a combination of both hydrogen peroxide and microwave irradiation (group MH) for 1 minute, 2 minutes, and 3 minutes. The control group was untreated. The total sample size was 120. The specimens were divided into 2 groups, those exposed to Streptococcus mutans and those exposed to Escherichia coli. The disinfection effect and physical properties (contact angle, compatibility with gypsum, strain in compression, tear strength) were evaluated. RESULTS: All 3 groups (H, M, MH) were effective in reducing the number of colony forming units (CFU) per unit volume (mL) for both S mutans and E coli compared with the control. The most significant reduction in the CFU/mL of both bacteria was noted in the MH group and was used to compare either treatment alone (P<.05). No statistically significant difference was noted between the control and treatment groups in terms of all of the physical properties tested (P>.05). CONCLUSIONS: Microwave irradiation was identified as a useful disinfection method against S mutans and E coli, especially when combined with H2O2, without adversely affecting the physical properties of dental impression materials.

Photodynamic therapy versus ultrasonic irrigation: interaction with endodontic microbial biofilm, an ex vivo study.

INTRODUCTION: Photodynamic therapy was introduced as an adjuvant to conventional chemo-mechanical debridement during endodontic treatment to overcome the persistence of biofilms. The aim of this study was to evaluate the ability of photodynamic therapy (PDT) to disrupt an experimental microbial biofilm inside the root canal in a clinically applicable working time. MATERIALS AND METHODS: Thirty extracted teeth were prepared and then divided in three groups. All samples were infected with an artificially formed biofilm made of Enterococcus faecalis, Streptococcus salivarius, Porphyromonas gingivalis and Prevotella intermedia bacteria. First group was treated with Aseptim Plus® photo-activated (LED) disinfection system, second group by a 650 nm Diode Laser and Toluidine blue as photosensitizer, and the third group, as control group, by ultrasonic irrigation (PUI) using EDTA 17% and NaOCl 2.6% solutions. The working time for all three groups was fixed at 3 min. Presence or absence of biofilm was assessed by aerobic and anaerobic cultures. RESULTS: There was no statistically significant difference between results obtained from groups treated by Aseptim Plus® and Diode Laser (P<0.6267). In cultures of both groups there was a maximal bacterial growth. The group that was treated by ultrasonic irrigation and NaOCl and EDTA solutions had the best results (P<0.0001): there was a statistically significant reduction of bacterial load and destruction of microbial biofilm. CONCLUSION: Under the condition of this study, Photodynamic therapy could not disrupt endodontic artificial microbial biofilm and could not inhibit bacterial growth in a clinically favorable working time.

Antimicrobial action of minocycline microspheres versus 810-nm diode laser on human dental plaque microcosm biofilms.

BACKGROUND: The purpose of this study is to investigate the antimicrobial effects of minocycline hydrochloride microspheres versus infrared light at 810 nm from a diode laser on multispecies oral biofilms in vitro. These biofilms were grown from dental plaque inoculum (oral microcosms) and were obtained from six systemically healthy individuals with generalized chronic periodontitis. METHODS: Multispecies biofilms were derived using supra- and subgingival plaque samples from mesio-buccal aspects of premolars and molars exhibiting probing depths in the 4- to 5-mm range and 1- to 2-mm attachment loss. Biofilms were developed anaerobically on blood agar surfaces in 96-well plates using a growth medium of prereduced, anaerobically sterilized brain-heart infusion with 2% horse serum. Minocycline HCl 1 mg microspheres were applied on biofilms on days 2 and 5 of their development. Biofilms were also exposed on days 2 and 5 of their growth to 810-nm light for 30 seconds using a power of 0.8 W in a continuous-wave mode. The susceptibility of microorganisms to minocycline or infrared light was evaluated by a colony-forming assay and DNA probe analysis at different time points. RESULTS: At all time points of survival assessment, minocycline was more effective (>2 log10 colony-forming unit reduction) than light treatment (P <0.002). Microbial analysis did not reveal susceptibility of certain dental plaque pathogens to light, and it was not possible after treatment with minocycline due to lack of bacterial growth. CONCLUSION: The cumulative action of minocycline microspheres on multispecies oral biofilms in vitro led to enhanced killing of microorganisms, whereas a single exposure of light at 810 nm exhibited minimal and non-selective antimicrobial effects.

The efficiency of root canal disinfection using a diode laser: in vitro study.

AIMS: The aim of this study is to verify the disinfection of diode laser, following chemo-mechanical procedures against Enterococcus fecalis. MATERIALS AND METHODS: Crowns of 30 extracted premolar teeth were sectioned at the cemento- enamel junction. The canals were shaped using step-back technique to K-file #40. The teeth were randomly assigned to three groups and placed into nutrient broth containing bacterial suspension of Enterococcus fecalis. Group A received no laser radiation. Specimens of group B and C were treated with diode laser (Sirona) with energy set at 1.5 and 3 W, respectively. After laser irradiation, the teeth were placed in vials, which contained 2 mL of the nutrient broth. The vials were incubated at 37°C for 24 h. Grown colonies were identified by standard methods. STATISTICAL ANALYSIS USED: Statistical analysis used was the nonparametric Kruskal-Wallis test, with comparison using the Bonferroni methods of means. RESULTS: Higher mean CFU/mL is recorded in Group A (without laser disinfection) followed by Group B (with 1.5 W laser disinfection) and Group C (with 3 W laser disinfection), respectively. The difference in CFU/mL between the three groups is found to be statistically significant ( P < 0.001). CONCLUSIONS: The results of this research show that the 980 nm diode laser can eliminate bacteria that has immigrated into dentin, thus being able to increase the success rate in endodontic therapy.

Decontamination efficacy of photon-initiated photoacoustic streaming (PIPS) of irrigants using low-energy laser settings: an ex vivo study.

AIM: To assess ex vivo, the antibacterial effectiveness of photon-initiated photoacoustic streaming (PIPS) of irrigants using an Er:YAG laser equipped with a newly designed, stripped and tapered tip in extracted teeth with infected root canals. METHODOLOGY: One hundred and forty-eight single-rooted extracted teeth were prepared to a size 25, 0.06 taper. The specimens were sterilized, and all teeth except ten (negative control group) were inoculated with Enterococcus faecalis and incubated in a CO(2) chamber at 37 °C for 15 days in Eppendorf tubes filled with trypticase soy broth medium changed every 2 days. Infected teeth were then randomly divided into four test groups (n = 32 for each): pulsed erbium/YAG laser at nonablative settings for 30 s with sterile bi-distilled water (Group A) or 5% sodium hypochlorite (NaOCl) (Group B); without laser-activated sterile bi-distilled water irrigation for 30 s (Group C) or 5% NaOCl irrigation for 30 s (Group D); the positive control group received no treatment in infected teeth (n = 10). Colony-forming units (CFUs) were counted from bacteriologic samples taken before (S1) and after treatment (S2). Data were analysed by Kruskal-Wallis and post hoc Dunn's multiple comparison tests. RESULTS: CFU counts were significantly lower in 5% NaOCl groups with or without laser activation than in sterile bi-distilled water without laser activation group (P < 0.001). Moreover, there was a significant difference between bi-distilled water groups with or without laser activation (P < 0.001). Sodium hypochlorite with laser activation group had the greatest CFU reduction, which was significantly greater than that evident in bi-distilled water groups with or without laser activation (P < 0.001). There were no significant differences between 5% NaOCl groups with or without laser activation (P > 0.05). None of the four groups generated negative samples predictably. CONCLUSIONS: Under the conditions of this ex vivo study, there were no significant differences in bacterial reduction between the laser and NaOCl or NaOCl alone groups. [Correction added after online publication, 18th April 2012: The following statement has been deleted: 'Thus, the use of a laser did not improve microbial killing over and above use of NaOCI alone.'].

Er:YAG laser treatment in supportive periodontal therapy.

OBJECTIVE: To assess clinical and microbiological outcomes of an Er:YAG laser in comparison with sonic debridement in the treatment of persistent periodontal pockets in a prospective randomized controlled multicentre study design. MATERIAL AND METHODS: A total of 78 patients in supportive periodontal therapy with two residual pockets were included, 58 were available for the whole follow-up period. Root surfaces were instrumented either with a sonic scaler (Sonicflex(®) 2003 L) or with an Er:YAG laser (KEY Laser(®) 3). Clinical attachment levels (CAL), Probing depths (PD), Plaque control record (PCR) and Bleeding on probing (BOP) were assessed at baseline, 13 and 26 weeks after treatment. In addition, microbiological analysis was performed employing a DNA diagnostic test kit (micro-IDent(®) Plus). RESULTS: Probing depths and CAL were significantly reduced in both groups over time (p < 0.05), without significant differences between the groups (p > 0.05). BOP frequency values decreased significantly within both groups (p < 0.05), with no difference between the laser and the sonic treatment (p > 0.05). PCR frequency values did not change during the observation period (p > 0.05). Microbiological analysis failed to expose any significant difference based on treatment group or period. CONCLUSION: Employing both sonic and laser treatment procedures during supportive periodontal care, similar clinical and microbiological outcomes can be expected.

Evaluation of Nd:YAG and Er:YAG irradiation, antibacterial photodynamic therapy and sodium hypochlorite treatment on Enterococcus faecalis biofilms.

AIM: To compare the antimicrobial efficacy of two-high power lasers (Nd:YAG and Er:YAG) and two commercial antimicrobial photodynamic therapy (aPDT) systems with that of sodium hypochlorite (NaOCl) action on Enterococcus faecalis biofilms grown on dentine discs. METHODOLOGY: Enterococcus faecalis biofilms were grown on dentine discs in a microtiter plate, incubated for 24 h and subjected to the following treatments: aPDT (Denfotex and Helbo system), Er:YAG laser irradiation (2940 nm, 50 mJ or 100 mJ, 15 Hz, 40 s), Nd:YAG laser irradiation (1064 nm, 2 W, 15 Hz, 40 s) and immersion in 2.5% (w/v) NaOCl for 1, 5, 10 and 30 min. Surviving bacteria were harvested, and the number of CFU per disc was determined by plate counting. RESULTS: Significant reductions (anova, P ≤ 0.05) in viable counts were observed for aPDT (Helbo) (2 log(10) reduction), Er:YAG irradiation using 100 mJ pulses (4.3 log(10) reduction) and all NaOCl treatments (>6 log(10) reduction). NaOCl (2.5%) for 5 min effectively eliminated all bacteria. aPDT (Denfotex), Er:YAG irradiation using 50 mJ pulses and Nd:YAG treatment caused a reduction in the viable counts of <1 log(10) unit; these results were not significantly different from the untreated controls. CONCLUSION: Within the limitations of this particular laboratory set-up, NaOCl was the most effective in E. faecalis biofilm elimination, while Er:YAG laser treatment (100 mJ pulses) also resulted in high reductions in viable counts. The use of both commercial aPDT systems resulted in a weak reduction in the number of E. faecalis cells. Nd:YAG irradiation was the least effective.

Bactericidal activity of erbium, chromium:yttrium-scandium-gallium-garnet laser in root canals.

The aim of this study was to investigate the effectiveness of the erbium, chromium:yttrium-scandium-gallium-garnet (Er,Cr:YSGG) laser by measuring its bactericidal effect inside root canals experimentally colonized with Enterococcus faecalis. We also determined the optimal conditions for the Er,Cr:YSGG laser to achieve the maximal bactericidal effect. An Er,Cr:YSGG Waterlase laser was used, and its antimicrobial effect was compared with that of sodium hypochlorite (NaOCl) at various concentrations as widely used in clinics. This laser emits photons at a wavelength of 2.78 microm. It is a pulsed laser operating at 20 Hz (20 pulses/s). Significant differences between measurements in the different groups (P < 0.05) were observed, depending on time and power used. The use of NaOCl 5% was the most effective procedure, with NaOCl 0.5% being the least effective; however, laser treatment was as effective as NaOCl 5% when applied at 2 W for 60 s.