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Visible light-induced killing of bacteria as a function of wavelength: Implication for wound healing
Abstract
Visible light (400-800 nm) at high intensity was previously found to kill bacteria that are frequently found in infected wounds, while low-power white light enhances bacterial proliferation. The phototoxic effect was found to involve induction of reactive oxygen species (ROS) production by the bacteria. The aim of the present study was to identify the most effective wavelengths in the visible range for inducing a bactericidal effect.
ROS production in Staphylococcus aureus and Escherichia coli as a function of wavelengths in the visible range (400-500, 500-800, 415, and 455 nm) was studied using the electron paramagnetic resonance (EPR) spin trapping technique. The phototoxicity of 415 and 455 nm light at different fluencies on the survival of S. aureus and E. coli was assessed by colony count of the bacteria following irradiation.
ROS production following blue (400-500 nm) light illumination was found to be higher than that of red (500-800 nm). Within the blue range, light of 415 nm induced more ROS than 455 nm, which correlated with results obtained for the reduction in colony count of S. aureus and E. coli following illumination using equal intensities of these two wavelengths. At low fluencies, both 415 and 455 nm enhanced proliferation of S. aureus but reduced viability of E. coli.
Intense blue light, preferably at 415 nm, could be used for bacterial eradication. However, it should be noted that low intensity of visible light can be dangerous since it may promote proliferation of the microorganisms.
... In vitro studies suggest that light at wavelengths between 625-1000 nm (from red to infrared) when applied alone does not induce increased growth of bacteria such as S. aureus and P. aeruginosa (Lipovsky et al., 2010;Lee et al., 2011;Baffoni et al., 2012;Costa and Assis, 2012;Araujo et al., 2013;Kim et al., 2013;Pereira et al., 2014). This suggests that such wavelengths do not interact with chromophores known to exist in these infectious agents. ...
... We also showed that exposure to light after seeding the bacteria inoculum in the Petri dish containing agar is not a real problem to evaluate the effect of light if the precautions are taken. In fact, this is the most common method used in these type of studies (Lipovsky et al., 2010;Dai et al., 2013;Kim et al., 2013;Bumah et al., 2015;Sousa et al., 2015;Masson-Meyers et al., 2015;Halstead et al., 2016;Rosa et al., 2016). We also discarded the hypothesis that the type of material containing agar (glass or polystyrene) where the bacteria are growing may interfere on the effect of the photoemission. ...
... Finally, the results presented here are in agreement with several authors who demonstrated the effect of blue light on the bacterial growth (Maclean et al., 2009;Lipovsky et al., 2010;Dai et al., 2013;Kim et al., 2013;Bumah et al., 2015;Masson-Meyers et al., 2015;Halstead et al., 2016;Rosa et al., 2016;Bumah et al., 2017;Ferrer-Espada et al., 2020;Rupel et al., 2019). The use of higher fluencies (157.78 and 284.90 J/cm 2 ) of blue light caused inhibition in all tests performed for both S. aureus and P. aeruginosa. ...
The ability of pathogenic bacteria acquire resistance to the existing antibiotics has long been considered a dangerous health risk threat. Currently, the use of visible light has been considered a new approach to treat bacterial infections as an alternative to antibiotics. Herein, we investigated the antimicrobial effect of two range of visible light, blue and red, on Staphylococcus aureus and Pseudomonas aeruginosa, two pathogenic bacterial commonly found in healthcare settings-acquired infections and responsible for high rate of morbidity and mortality. Bacterial cultures were exposed to blue or red light (470 nm and 660 nm) provided by light-emitting diodes - LED. The fluencies and irradiance used for blue and red light were 284.90 J/cm2, 13.19 mW/cm2 and 603.44 J/cm2, 27.93 mW/cm2 respectively. Different experimental approaches were used to determine the optimal conditions of light application. Only exposure to blue light for 6 hours was able to inhibit about 75% in vitro growth of both bacterial species after 24 hours. The surviving exposed bacteria formed colonies significantly smaller than controls, however, these bacteria were able to resume growth after 48 hours. Blue light was able to inhibit bacterial growth upon inoculation in both saline solution and BHI culture medium. We can conclude that blue light, but not red light, is capable of temporarily retarding the growth of gram negative and gram positive bacteria.
... Some studies suggest that the bacterial inhibition action of blue light exposure is related to an increase of reactive oxygen species (ROS) that mediates harmful actions to the cell (Guffey and Wilborn, 2006;Enwemeka et al., 2008;Lipovsky et al., 2010;Dai et al., 2012;Chui et al., 2012;Kim et al., 2013;Bumah et al., 2015;Masson-Meyers et al., 2015;Rupel et al., 2019). Mouse and human cells exposed to blue light generates ROS production at levels that were not observed when the same type of cells was exposed to red or infrared light (Kushibiki et al., 2013). ...
... The first parameter that needs attention is the wavelength. Different values starting from 405 nm have been used to verify the possible effect against S. aureus (Guffey and Wilborn, 2006;Lipovsky et al., 2010;Bumah et al., 2013Bumah et al., , 2015. Values close to 470 nm are often chosen, showing good antimicrobial suppression results (Guffey and Wilborn, 2006;Enwemeka et al., 2009;Dai et al., 2012;De Lucca et al., 2012;Bumah et al., 2013Bumah et al., , 2015Pileggi et al., 2013;Sousa et al., 2015;Rosa et al., 2016;Cohen-Berneron et al., 2016;Ferrer-Espada et al., 2019;Meurer et al., 2019;Rupel et al., 2019;Schmid et al., 2019). ...
... Irradiance represents the radiation flow per area unity received by a surface (Hadis et al., 2016), it is a variable parameter in different researches, although, in general, the values used are classified as photoemission of low intensity (Huang et al., 2011;Farivar et al., 2014). The protocols adopted in some investigations use 70 to 610 mW/cm (Enwemeka et al., 2008;Lipovsky et al., 2010;Bumah et al., 2013;Sousa et al., 2015;Masson-Meyers et al., 2015;Rosa et al., 2016;Rupel et al., 2019), in other ones, values varies from 10 to 30 mW/cm (Enwemeka et al., 2009;Bumah et al., 2013;Dai et al., 2013;Kim et al., 2013;Zhang et al., 2014;Schmid et al., 2019). However, it is difficult to establish a relation between low intensity irradiation values and bacterial inhibitory effect, because the results show similar inhibition levels. ...
Antibiotic resistance is one of the greatest challenges to treat bacterial infections worldwide, leading to increase in medical expenses, prolonged hospital stay and increased mortality. The use of blue light has been suggested as an innovative alternative to overcome this problem. In this study we analyzed the antibacterial effect of blue light using low emission parameters on Staphylococcus aureus cultures. In vitro bacterial cultures were used in two experimental approaches. The first approach included single or fractionated blue light application provided by LED emitters (470 nm), with the following fluencies: 16.29, 27.16 and 54.32 J/cm2. For the second approach a power LED (470 nm) was used to deliver 54.32 J/cm2 fractionated in 3 applications. Our results demonstrated that bacterial cultures exposed to fractionated blue light radiation exhibited significantly smaller sizes colonies than the control group after 24 h incubation, however the affected bacteria were able to adapt and continue to proliferate after prolonged incubation time. We could conclude that the hypothetical clinical use of low fluencies of blue light as an antibacterial treatment is risky, since its action is not definitive and proves to be ineffective at least for the strain used in this study.
... It was described in previous studies that blue light (visible), with wavelengths of 405-470 nm, when irradiated in certain bacteria such as S. aureus, leads to a photosensitization of endogenous intracellular porphyrins that stimulate the production of reactive oxygen species, predominantly singlet oxygen (1O2), leading the cell to death. (Ashkenazi, Malik, Harth, & Nitzan, 2003;Hamblin & Hasan, 2004;Lipovsky, Nitzan, Gedanken, & Lubart, 2010;Papageorgiou, Katsambas, & Chu, 2000). ...
... Lipovsky et al. and Maclean et al. also achieved significant microbial reduction: 415 nm, 100 mW/cm2, 120 J/cm2 (90% reduction) and 400 nm, 300 mW/cm2, and 450 J/cm2 (3 log reduction), respectively. As the energy density used by both is relatively high, it is believed that the high thermal factor must have been an adjunct to the inhibition of bacterial growth (Lipovsky et al., 2010;Maclean, MacGregor, Anderson, & Woolsey, 2008). ...
One possibility of treatment in Aesthetics for folliculitis is a blue LED, as it acts on microbial control. Studies describe that the blue LED, with 405-470nm wavelength, has a bactericidal effect when irradiated in certain bacteria such as Staphylococcus aureus. This study aimed to evaluate the 450 nm blue LED's efficacy as a modality in aesthetic treatments in the photoinactivation of the planktonic strains of S. aureus and S. epidermidis with power densities of 97, 110, 156, and 200 mW/cm2 in different energy densities. Bacterial suspensions of S. aureus (ATCC 25923) and S. epidermidis (ATCC 12228) were plated in 24-well plates and irradiated with other energy and power densities. After irradiation, each bacterial suspension was diluted in a phosphate buffer solution in a 96-well plate. Aliquots of 10 µL were collected from this dilution and streaked, in triplicate, in Brain Heart Infusion agar plates and incubated for 24h/37 °C. CFU counts were expressed in log10/mL and submitted to ANOVA and Tukey statistical tests. The energy and power densities used were insufficient to cause an antimicrobial effect on S. aureus or S. epidermidis planktonic cultures with a single light application.
... Some studies were conducted on pathogenic E. coli at wavelengths between 450 and 455 nm (Lipovsky et al., 2010;Keshishyan et al., 2015;Lui et al., 2016) ranging from approximately 100-300 J/cm 2 for 1 log inactivation but none of them is close to our results with 1500 J/cm 2 for 1.00 log. Within this study the E. coli strain stands out, although the majority of the data for all strains investigated here already rank at the upper end of visible light inactivation data. ...
... The investigated S. aureus strain however was inactivated by 1.22 log at 160 J/cm 2 of 450 nm, while literature data suggest a 1 log dose of around 300 J/cm 2 at 450 nm at 37 • C (Keshishyan et al., 2015), 400 J/cm 2 at 455 nm (Lipovsky et al., 2010), or 300 J/cm 2 at 460 nm (Decarli et al., 2016) for methicillinsensitive strains. ...
Due to the globally observed increase in antibiotic resistance of bacterial pathogens and the simultaneous decline in new antibiotic developments, the need for alternative inactivation approaches is growing. This is especially true for the treatment of infections with the problematic ESKAPE pathogens, which include Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species, and often exhibit multiple antibiotic resistances. Irradiation with visible light from the violet and blue spectral range is an inactivation approach that does not require any additional supplements. Multiple bacterial and fungal species were demonstrated to be sensitive to this disinfection technique. In the present study, pathogenic ESKAPE organisms and non-pathogenic relatives are irradiated with visible blue and violet light with wavelengths of 450 and 405 nm, respectively. The irradiation experiments are performed at 37 • C to test a potential application for medical treatment. For all investigated microorganisms and both wavelengths, a decrease in colony forming units is observed with increasing irradiation dose, although there are differences between the examined bacterial species. A pronounced difference can be observed between Acinetobacter, which prove to be particularly light sensitive, and enterococci, which need higher irradiation doses for inactivation. Differences between pathogenic and non-pathogenic bacteria of one genus are comparatively small, with the tendency of non-pathogenic representatives being less susceptible. Visible light irradiation is therefore a promising approach to inactivate ESKAPE pathogens with future fields of application in prevention and therapy.
... The prevalence of methicillin-resistant Staphylococcus aureus (MRSA) in infected DFUs is 15%-30% 10 . Excessive administration of inappropriate antibiotics for diabetic foot infections has resulted in concern about the levels of bacterial resistance 11 . ...
... Lipoveski et al. conducted an in vitro study on the antibacterial property of blue light (415 nm, 100 mW, and 30, 60, 120 J/cm 2 ), which showed the inhibitory effect of photobiomodulation on Staphylococcus aureus by reactive oxygen species induction 11 . It is possible that bactericidal effect of photobiomodulation treatment in our study could be attributed to the induction of reactive oxygen species. ...
... The prevalence of methicillin-resistant Staphylococcus aureus (MRSA) in infected DFUs is 15%-30% 10 . Excessive administration of inappropriate antibiotics for diabetic foot infections has resulted in concern about the levels of bacterial resistance 11 . ...
... Lipoveski et al. conducted an in vitro study on the antibacterial property of blue light (415 nm, 100 mW, and 30, 60, 120 J/cm 2 ), which showed the inhibitory effect of photobiomodulation on Staphylococcus aureus by reactive oxygen species induction 11 . It is possible that bactericidal effect of photobiomodulation treatment in our study could be attributed to the induction of reactive oxygen species. ...
In this study, we sought to investigate the impact of photobiomodulation and adipose-derived stem cells (ADS), alone and in combination, on the maturation step of wound healing in an ischemic infected delayed healing wound model in rats with type 2 diabetes mellitus (DM2). We randomly divided 24 adult male rats into 4 groups (n = 6 per group). DM2 plus an ischemic delayed healing wound were induced in all rats. The wounds were infected with methicillin-resistant Staphylococcus aureus. Group 1 was the control (placebo) group. Group 2 received only photobiomodulation (890 nm, 80 Hz, 0.324 J/cm2, and 0.001 W/cm2). Group 3 received only the allograft ADS. Group 4 received allograft ADS followed by photobiomodulation. On days 0, 4, 8, 12, and 16, we performed microbiological examination (colony forming units, [CFU]), wound area measurement, wound closure rate, wound strength, and histological and stereological examinations. The results indicated that at day 16, there was significantly decreased CFU (Analysis of variance, p = 0.001) in the photobiomodulation + ADS (0.0 ± 0.0), ADS (1350 ± 212), and photobiomodulation (0.0 ± 0.0) groups compared with the control group (27250 ± 1284). There was significantly decreased wound area (Analysis of variance, p = 0.000) in the photobiomodulation + ADS (7.4 ± 1.4 mm2), ADS (11 ± 2.2 mm2), and photobiomodulation (11.4 ± 1.4 mm2) groups compared with the control group (25.2 ± 1.7). There was a significantly increased tensiometeric property (stress maximal load, Analysis of variance, p = 0.000) in the photobiomodulation + ADS (0.99 ± 0.06 N/cm2), ADS (0.51 ± 0.12 N/cm2), and photobiomodulation (0.35 ± 0.15 N/cm2) groups compared with the control group (0.18 ± 0.04). There was a significantly modulated inflammatory response in (Analysis of variance, p = 0.049) in the photobiomodulation + ADS (337 ± 96), ADS (1175 ± 640), and photobiomodulation (69 ± 54) treatments compared to control group (7321 ± 4099). Photobiomodulation + ADS gave significantly better improvements in CFU, wound area, and wound strength compared to photobiomodulation or ADS alone. Photobiomodulation, ADS, and their combination significantly hastened healing in ischemic methicillin-resistant Staphylococcus aureus infected delayed healing wounds in rats with DM2. Combined application of photobiomodulation plus ADS demonstrated an additive effect.
... These bacteria produce porphyrins, which, acting as sensitizer, induce photodynamic reactions, leading to cell death [23]. Therapy based on using blue light is highly effective in fighting bacteria families that colonize wounds, like Staphylococcus aureus and Escherichia coli [24], and bacteria causing periodontal diseases (Staphylococcus epidermidis, Streptococcus pyogenes) [25]. Blue radiation reduces the proliferation of keratinocytes and stimulates their differentiation, and when used in high doses, induces a cytotoxic effect on them. ...
Riboflavin, a water-soluble vitamin B2, possesses unique biological and physicochemical properties. Its photosensitizing properties make it suitable for various biological applications, such as pathogen inactivation and photodynamic therapy. However, the effectiveness of riboflavin as a photosensitizer is hindered by its degradation upon exposure to light. The review aims to highlight the significance of riboflavin and its derivatives as potential photosensitizers for use in photodynamic therapy. Additionally, a concise overview of photodynamic therapy and utilization of blue light in dermatology is provided, as well as the photochemistry and photobiophysics of riboflavin and its derivatives. Particular emphasis is given to the latest findings on the use of acetylated 3-methyltetraacetyl-riboflavin derivative (3MeTARF) in photodynamic therapy.
... In line with these findings, Lipovsky et al, in a laboratory experiment, showed that PBMT (30, 60, 120 J/cm 2 , 100 mW, and 415 nm) inhibits Staphylococcus aureus effects by ROS generation. 44 These antibacterial impacts of PBMT treatment may be attributed to the stimulation of ROS by PBMT. Kouhkheil et al examined the impacts of PBMT (890 nm, 0.2 J/cm 2 , 80 Hz) and human bone marrow mesenchymal stem cell conditioned medium (CM-hBMMSC) (4 injections) alone and together on the wound strength and CFU of an MRSAinfected wound model in DM1 rats. ...
Introduction: Photobiomodulation treatment (PBMT) is a relatively invasive method for treating wounds. An appropriate type of PBMT can produce desired and directed cellular and molecular processes. The aim of this study was to investigate the impacts of PBMT on stereological factors, bacterial count, and the expression of microRNA-21 and FGF2 in an infected, ischemic, and delayed wound healing model in rats with type one diabetes mellitus. Methods: A delayed, ischemic, and infected wound was produced on the back skin of all 24 DM1 rats. Then, they were put into 4 groups at random (n=6 per group): 1=Control group day4 (CG day4); 2=Control group day 8 (CG day8); 3=PBMT group day4 (PGday 4), in which the rats were exposed to PBMT and killed on day 4; 4=PBMT group day8 (PGday8), in which the rats received PBMT and they were killed on day 8. The size of the wound, the number of microbial colonies, stereological parameters, and the expression of microRNA-21 and FGF2 were all assessed in this study throughout the inflammation (day 4) and proliferation (day 8) stages of wound healing. Results: On days 4 and 8, we discovered that the PGday4 and PGday8 groups significantly improved stereological parameters in comparison with the same CG groups. In terms of ulcer area size and microbiological counts, the PGday4 and PGday8 groups performed much better than the same CG groups. Simultaneously, the biomechanical findings in the PGday4 and PGday8 groups were much more extensive than those in the same CG groups. On days 4 and 8, the expression of FGF2 and microRNA-21 was more in all PG groups than in the CG groups (P<0.01). Conclusion: PBMT significantly speeds up the repair of ischemic and MARS-infected wounds in DM1 rats by lowering microbial counts and modifying stereological parameters, microRNA-21, and FGF2 expression.
... In this study, we demonstrated that the use of ADSs can significantly decrease the microbial count compared to the control group. These findings are consistent with Lipovsky et al.'s laboratory experiment, which showed that ADS inhibits Staphylococcus aureus effects by generating ROS (37). The antibacterial effects of ADSs may be attributed to the induction of ROS. ...
Background: Adipose-derived stem cells (ADSCs) have been shown to enhance wound healing in rats with type 1 diabetes (DM1). Objectives: This experimental study aimed to explore how ADSC administration affects bacterial count, wound size, biomechanical and stereological parameters, and the expression of microRNA-21 and FGF2 in a rat model of infected, ischemic, and delayed wound healing in DM1. Methods: Twenty-four male adult Wistar rats weighing less than 250 g were randomly assigned to four groups (n = 6 per group). Type 1 diabetes was induced in all animals, resulting in the development of a delayed, ischemic, and infected wound model. The CGday4 and CGday8 groups served as controls. In the AGday4 group, the animals received allograft h-ADSs and were euthanized on day four after surgery. Similarly, in the AGday8 group, the animals received h-ADSs and were euthanized on day eight after surgery. Microbial colony counts, wound size, stereological parameters, and the expression of microRNA-21 and FGF2 were evaluated in this study during the inflammation (day 4) and proliferation (day 8) stages of wound healing. Results: We demonstrated that h-ADSs significantly reduced microbiological counts compared to the control group on days 4 and 8. Moreover, in the AGday8 group compared to the AGday4 group, this decline in microbiological counts was even more pronounced. Moreover, we observed that the stereological characteristics in the AGday4 and AGday8 groups were significantly superior to those in the CG groups. Additionally, the AGday4 and AGday8 groups exhibited smaller ulcer area sizes compared to the CG groups. Furthermore, the AGday4 and AGday8 groups demonstrated higher expression levels of FGF2 and microRNA-21 than the CG groups on days 4 and 8. Notably, on day 8, the AGday8 group’s outcomes surpassed those of the AGday4 group (P < 0.01). Conclusions: Through lowering microbial counts, modifying stereological parameters, microRNA-21, and FGF2 expression, the administration of hADS dramatically speeds up the healing of MARS-infected and ischemic ulcers in DM1 rats.
... Significantly higher (p < 0.05) microbial reduction, the generation of 3-MBT, and changes in physicochemical parameters were observed in BA beers compared to CA beers, which was attributed to the contribution of increased light penetration depth in the lightercolored BA, as shown with overall lower SRM values. Penetration depth is influenced by light absorbance, the inverse of transmittance [28]. Fluids of lighter color have lower absorbance and higher transmission in the visible light spectrum [29]. ...
Pulsed light (PL) is a novel, non-thermal technology being used to control the microbial spoilage of foods and beverages. Adverse sensory changes, commonly characterized as “lightstruck”, can occur in beers when exposed to the UV portion of PL due to the formation of 3-methylbut-2-ene-1-thiol (3-MBT) upon the photodegradation of iso-α-acids. This study is the first to investigate the effect of different portions of the PL spectrum on UV-sensitive beers (light-colored blonde ale and dark-colored centennial red ale) using clear and bronze-tinted UV filters. PL treatments with its entire spectrum, including the ultraviolet portion of the spectrum, resulted in up to 4.2 and 2.4 log reductions of L. brevis in the blonde ale and centennial red ale beers, respectively, but also resulted in the formation of 3-MBT and small but significant changes in physicochemical properties including color, bitterness, pH, and total soluble solids. The application of UV filters effectively maintained 3-MBT below the limit of quantification but significantly reduced microbial deactivation to 1.2 and 1.0 log reductions of L. brevis at 8.9 J/cm2 fluence with a clear filter. Further optimization of the filter wavelengths is considered necessary to fully apply PL for beer processing and possibly other light-sensitive foods and beverages.
... Therefore, the utilization of endogenous photosensitizers in PDT may be a safer approach for a number of oral black-pigmented bacterial species that naturally synthesize porphyrin. The amount of endogenous porphyrin in oral black-pigmented bacteria was detected to be 267 ng∕ mg in P. intermedia, 47 ng∕ mg in P. nigrescens, 41 ng/mg in P. melaninogenica, and 2.2 ng∕ mg in P. gingivalis [17,35,36]. The visible absorption spectrum of porphyrins has been shown to peak in the range of 405-415 nm, 500-510 nm, 525-535 nm, and 560-570 nm [31]. ...
Purpose:
We investigated whether repeated irradiation with light-emitting diodes (LEDs) at a combination of 470 nm and 525 nm could suppress the progression of experimental periodontitis.
Methods:
A experimental periodontitis model was established in the second, third, and fourth premolars of the mandible in beagle dogs for 2 months. The spontaneous progression of periodontitis was monitored under the specified treatment regimen for 3 months. During this period, the animals were subjected to treatments of either plaque control only (control) or plaque control with LED application (test) at 2-week intervals. The clinical parameters included the probing pocket depth (PPD), gingival recession (GR), and the clinical attachment level (CAL). Histomorphometric analysis was performed using measurements of the length of the junctional epithelium, connective tissue (CT) zone, and total soft tissue (ST).
Results:
There were significant differences in PPD between the control and test groups at baseline and 12 weeks. When the change in PPD was stratified based on time intervals, it was shown that greater differences occurred in the test group, with statistical significance for baseline to 12 weeks, 6 to 12 weeks, and baseline to 6 weeks. There was no significant difference in GR between the control and test groups at any time points. Likewise, no statistically significant differences were found in GR at any time intervals. CAL showed a statistically significant difference between the control and test groups at baseline only, although significant differences in CAL were observed between baseline and 12 weeks and between 6 and 12 weeks. The proportion of CT to ST was smaller for both buccal and lingual areas in the control group than in the test group.
Conclusions:
Repeated LED irradiation with a combination of 470-nm and 525-nm wavelengths may help suppress the progression of periodontal disease.
... This type of technology is already used to treat some infections, and also to promote wound healing, tissue repair and other effects on skin diseases. This feature produces safe radiating power, consumes little power, has a long lifespan, good power and low intensity [14,15]. ...
Eugenol has already had its pharmacological properties elucidated in previous studies, including antibacterial and antifungal properties. Based on such information, this study aimed to evaluate the antibacterial and modulatory activity of coumarin compounds prepared from dihydroeugenol and to associate them with blue LED light for the same activity. For this study, five of the substances available: compound 1 (C1), 8-methoxy-2-oxo-6-propyl-2H-chromen-3-carboxylic acid, compound (C2), 3-(hydroxy(4-nitrophenyl)methyl)-8- methoxy-6-propyl-2H-chromen-2-one, compound 7 (C3), 8-hydroxy-3-(4-nitrobenzoyl)-6-propyl-2H-chromen-2-one, compound 8 (C4), 3-(4-aminobenzoyl)-8-methoxy-6-propyl-2H-chromen-2-one and Compound 9 (C5), 8-methoxy-3-(4-nitrobenzoyl)-6-propyl-2H-chromen-2-one 2-one. To determine the MIC, the broth microdilution technique was used. The products were evaluated for their potential to modulate the activity of antibiotics. Afterward, the plates were submitted to blue LED light for 20 min. When exposed to LED, C3 exhibited a decrease in MIC for SA ATCC and C5 for EC ATCC, with an average of 645.08 μg/mL for both cases. C2 and C4 exhibited synergism in a greater number of situations. However, C3 showed promising activity against S. aureus. C1 and C2 already acted better against E. coli, with the difference that C1 acted better against these bacteria when associated with LED. In general, the compounds studied here exhibited good antibacterial activity when associated with LED.
... The use of light with a length of 415 nm in this study corroborates the study by Lipovsky et al. [20], who observed a higher production and ROS than in 455 nm light, reducing the colony count of S. aureus and F I G U R E 4 Geometric mean minimum inhibitory concentration (MIC) of the modulation of the compound 8-methoxy-2-oxo-2Hchromen-3-carboxylic acid against Staphylococcus aureus and Escherichia coli with and without exposure to blue LED. **P < 0.01 and ****P < 0.0001 indicate significant difference between groups. ...
The problem of antibiotic resistance by bacteria threatens human health. Therefore, studies in this area seek alternatives to circumvent it. The study with coumarins and eugenol has already proven that these classes of compounds act against bacteria. In this same aspect, exposure to LED also shows a bactericidal effect. Seeking a possible enhancement of this effect, the present work studied coumarins derived from eugenol in association with LED to investigate the bactericidal effect. Four compounds were tested. For this, MICs and modulation with three antibiotics against Escherichia coli and Staphylococcus aureus bacteria were determined. To test the behavior of the activity against exposure to LED, the plates were exposed for 20 minutes to blue light, 415 nm and then incubated at 37°C for 24h. For control, duplicates were made, and one of them did not undergo this exposure. C1 exhibited better activity against S. Aureus, as synergism prevailed under the conditions tested. C3 and C4 were promising against E. coli as they showed synergism in association with the three antibiotics both with and without LED exposure. Thus, the compounds showed bactericidal activity and LED was shown to enhance synergism.
... The Mechanism of Blue Light-Induced Photochemical Damage. As a short-wavelength visible light, blue light is generally defined with a wavelength ranging from 400 to 500 nm [86]. Due to the intraocular refractive system filtering effect, the potentially damaging UVR reaching the retina is limited. ...
Natural visible light is an electromagnetic wave composed of a spectrum of monochromatic wavelengths, each with a characteristic color. Photons are the basic units of light, and their wavelength correlates to the energy of light; short-wavelength photons carry high energy. The retina is a fragile neuronal tissue that senses light and generates visual signals conducted to the brain. However, excessive and intensive light exposure will cause retinal light damage. Within the visible spectrum, short-wavelength light, such as blue light, carries higher energy, and thus the retinal injury, is more significant when exposed to these wavelengths. The damage mechanism triggered by different short-wavelength light varies due to photons carrying different energy and being absorbed by different photosensitive molecules in the retinal neurons. However, photooxidation might be a common molecular step to initiate cell death. Herein, we summarize the historical understanding of light, the key molecular steps related to retinal light injury, and the death pathways of photoreceptors to further decipher the molecular mechanism of retinal light injury and explore potential neuroprotective strategies.
... After formation, internal ROS can be extruded from the cell to the media by active efflux pumps [20]. On the other hand, phototherapy with 405-800 nm light also activates bacterial porphyrins to generate ROS, which cause intracellular damages [21], [22]. We used powerful visible light source with wide spectrum. ...
A lot of human activities have negative impact on water quality and sometimes result in the biological water contamination. Currently used chemical (chlorine, ozone, and etc.) and physical (UV) water disinfection methods have strong environmental disadvantages or suffers from limited efficiency. To overcome these problems, scientists suggest to use photocatalyst activated advanced oxidation processes. One of the most studied photocatalysts which attracts a lot of research interest is titanium dioxide. TiO2 application for the disinfection of water, air or surfaces is increasingly encouraged by researchers. However, to unlock its full potential it is highly desirable to make it suitable for the visible light activation. In the current study the effect of visible light assisted photocatalytic treatment to the outer membrane permeability of Salmonella enterica bacteria and how it changes under different titanium dioxide concentrations was analysed. The results from the treatment of relatively complex Salmonella enterica bacteria organism were compared to the visible light activated TiO2 ability to oxidise considerably simpler objects like methylene blue molecules. The efficiency of TiO2 photocatalytic disinfection process was evaluated using spread plate technique. Membrane permeability of the treated Salmonella enterica bacteria was determined by NPN uptake factor assay. Generation of intracellular reactive oxygen species was evaluated by Dichlorodihydrofluorescein diacetate fluorescence measurements. The key finding of this study was that intense wide spectrum visible light irradiation and TiO2 powder synergistically inactivate S. enterica bacteria and halt its potential to form colonies. High amounts of intracellular reactive oxygen species could be seen as the main suspects for the observed inactivation of S. enterica.
... To assess possible changes in the molecular dynamics induced by luminosity conditions, a preliminary cytosolic proteome comparison has suggested that exposure to light is mostly associated with the ability to sustain oxidative stress. This apparent state of oxidative stress could be owed to the continuous exposure to full-spectrum light, as it has been reported that bacteria irradiated with blue (415-470 nm) and red (620-700 nm) light displayed increased ROS production levels [33]. Moreover, the expression profiles associated with growth in the dark hinted higher overall stress tolerance, stimulation of quorum-sensing and possibly higher secretion of virulence factors in the absence of light, given that the latter were under-expressed in the cytosolic proteome. ...
Pseudomonas aeruginosa can alter its lifestyle in response to changes in environmental conditions. The switch to a pathogenic host-associated lifestyle can be triggered by the luminosity settings, resorting to at least one photoreceptor which senses light and regulates cellular processes. This study aimed to address how light exposure affects the dynamic and adaptability of two P. aeruginosa pneumonia-associated isolates, HB13 and HB15. A phenotypic characterization of two opposing growth conditions, constant illumination and intensity of full-spectrum light and total absence of light, was performed. Given the nature of P. aeruginosa pathogenicity, distinct fractions were characterized, and its inherent pathogenic potential screened by comparing induced morphological alterations and cytotoxicity against human pulmonary epithelial cells (A549 cell line). Growth in the dark promoted some virulence-associated traits (e.g., pigment production, LasA proteolytic activity), which, together with higher cytotoxicity of secreted fractions, supported an increased pathogenic potential in conditions that better mimic the lung microenvironment of P. aeruginosa. These preliminary findings evidenced that light exposure settings may influence the P. aeruginosa pathogenic potential, likely owing to differential production of virulence factors. Thus, this study raised awareness towards the importance in controlling light conditions during bacterial pathogenicity evaluation approaches, to more accurately interpret bacterial responses.
... Esta terapia pode ainda inativar enzimas que são responsáveis por aumentar a permeabilidade celular e pelo metabolismo celular (22). Segundo Lipovskye cols (2010), a inativação fotodinâmica e a fototerapia por meio do uso de aparelhos de LED, vem sendo bastante estudada tendo em vista que estudos realizados frente a cepas de Escherichia coli e Staphylococcus aureus demonstraram que estes métodos são eficazes na erradicação de bactérias (23). ...
Lippia alba Mill., também conhecida como erva cidreira, é um subarbusto aromático que ocorre em todo território brasileiro. Seu óleo essencial possui muitas atividades, mas ainda são escassos estudos do seu uso associado à fototerapia. O objetivo desse estudo foi avaliar a atividade antibacteriana do óleo essencial de Lippia alba (OEL) associado a luzes de LED (Light Emitting Diode) azul, vermelha e amarela contra cepas de Staphylococcus aureus e Escherichia coli. O óleo foi extraído pelo método de hidrodestilação. A atividadeantibacteriana e moduladora foi avaliada pelo método de contato gasoso utilizando o óleo, luzes de LED e antibióticos, os quais foram realizados em triplicata. O OEL possui um potencial antibacteriano frente todas as cepas testadas tendo um resultado mais expressivo na cepa multirresistente de E. coli quando associado a luzes de LED. Na modulação da resistência bacteriana, tanto nas combinações do óleo + antibiótico + luzes de LED como óleo + antibióticos, houve sinergismo frente às linhagens multirresistentes de ambas as cepas testadas. Os resultados obtidos no presente estudo mostraram que o óleo essencial de Lippia alba associado às luzes de LED possui efeito antibacteriano e modulador pelo método de contato gasoso. Estes dados obtidos poderão colaborar com pesquisas futuras que visem o desenvolvimento de novas estratégias terapêuticas no combate de microrganismos resistentes.
... 154,155 VL improves wound healing by reducing the expression of proinflammatory proteins and cytokines, increasing OPN3 mediated keratinocyte differentiation, and preventing bacterial growth. 91,[156][157][158] Hair growth. ...
Approximately fifty percent of sunlight reaching the Earth's surface is visible light (400-700 nm). Other sources of visible light include lasers, light-emitting diodes (LEDs), and flash lamps. Photons from visible light are absorbed by photoreceptive chromophores (e.g., melanin, heme, and opsins), altering skin function by activating and imparting energy to chromophores. Additionally, visible light can penetrate the full thickness of the skin and induce pigmentation and erythema. Clinically, lasers and light devices are used to treat skin conditions by utilizing specific wavelengths and treatment parameters. Red and blue light from LEDs and intense pulsed light (IPL) have been studied as anti-microbial and anti-inflammatory treatments for acne. Pulsed dye lasers are used to treat vascular lesions in adults and infants. Further research is necessary to determine the functional significance of visible light on skin health and wellness without confounding the influence of ultraviolet and infrared wavelengths.
... This antimicrobial effect of LED light, also known as photodynamic inactivation, is due to the activity of endogenous photosensitizers that absorb visible light wavelengths for its activation to form reactive oxygen species, whose action results in biomolecules oxidation and cells lysis (Purushothaman and Mol, 2021). This is the case of porphyrins with blue light, whose antibacterial effect is well known in literature (Lipovsky et al., 2010;Maclean et al., 2014;Wang et al., 2017). However, some results where blue light did not affect microbiological load already exist (Castillejo et al., 2021). ...
The main objective of this study was to evaluate the physiological and quality changes of fresh-cut red chard (Beta vulgaris) and rocket (Diplotaxis tenuifolia) leaves illuminated during storage with monochromatic light emitting diode (LED) lamps, featuring different spectral component (red, green, yellow, white, blue and far-red) and same light intensity (35 μmol m⁻² s⁻¹). As control, storage in darkness was assayed. Biomass, colorimetric and microbiological changes were determined up to 10 d of storage at 5 °C. In addition, total antioxidant activity and bioactive compounds changes along the shelf-life were also monitored. Microbial counts were reduced by yellow and blue light in red chard, and by yellow and green light in rocket. Green and white light enabled to preserve colorimetric indexes and chlorophylls content mostly in rocket and, eventually, increasing carotenoids in red chard. Total antioxidant capacity and total phenols content were stimulated in response to red or blue light application for both species. On the other hand, LED light supply increased weight losses during storage as compared to darkness, although more limitedly in response to yellow and far red light. The study provides solid ground for further exploration on how LED lighting treatment during storage of red chard and rocket may foster product qualitative properties, suggesting that different spectral wavebands may alternatively enhance antioxidant properties and reduce microbiological risks.
... Accordingly, researchers evaluated the bactericidal action of LED devices with wavelengths ranging from 415 to 455 nm against S. aureus and E. coli, and concluded that the wavelength of 415 nm was more effective [31]. It has been demonstrated that the photoinduced antibacterial effect may involve the following mechanisms: Induction of biochemical changes in the bacterial cell membrane; inhibition of the cell respiration through a direct effect on the mitochondria; and induction of DNA damage, which could prevent cell division and inactivate enzymes associated with the cell metabolism [32]. ...
This study aimed to evaluate the antibacterial and antibiotic-enhancing effects of the essential oil obtained from Ocimum gratissimum L. (OEOg) alone or in association with light-emitting diodes (LED) lights. The essential oil was obtained by hydrodistillation and its chemical composition analysed by gas chromatography coupled to mass spectrometry. The antibacterial and antibiotic-enhancing activities against multiresistant strains of Staphylococcus aureus and Escherichia coli were evaluated by the gaseous contact method. The analysis of the photoinductive effect on the antibacterial activity of the OEOg and antibiotics was assessed through exposure to different LED lights (red, blue and yellow). The phytochemical analysis identified five compounds, including eugenol, as the major constituent. The OEOg caused a significant inhibition of the halo, indicating a direct antibacterial effect. Exposure to the LED lights significantly enhanced the activity of the OEOg against E. coli. On the other hand, the action of the essential oil against S. aureus was enhanced by exposure to both blue and yellow lights. The effects of LED light exposure on the activity of conventional antibiotics varied significantly according to the drug and the bacterial strain. However, most combinations of LED lights and the OEOg presented synergistic effects against resistant bacterial strains, indicating enhanced antibacterial activity. Thus, these in vitro findings suggest that both OEOg and LED lights have promising antibacterial effects. Nevertheless, further research is required to evaluate in vivo the potential of these therapies for the treatment of infectious skin diseases.
... A key limitation in the use of the popular ratiometric probe, pHluorin, is that the illumination wavelengths used to excite the fluorophore promote the well-known blue light effect-incident light at 405 nm has bactericidal effects on a variety of species [42][43][44][45][46]. This could interfere with pH measurements by affecting cell physiology [45,47]. ...
Fluorescent proteins that modulate their emission intensities when protonated serve as excellent probes of the cytosolic pH. Since the total fluorescence output fluctuates significantly due to variations in the fluorophore levels in cells, eliminating the dependence of the signal on protein concentration is crucial. This is typically accomplished with the aid of ratiometric fluorescent proteins such as pHluorin. However, pHluorin is excited by blue light, which can complicate pH measurements by adversely impacting bacterial physiology. Here, we characterized the response of intensity-based, pH-sensitive fluorescent proteins that excite at longer wavelengths where the blue light effect is diminished. The pH-response was interpreted in terms of an analytical model that assumed two emission states for each fluorophore: a low intensity protonated state and a high intensity deprotonated state. The model suggested a scaling to eliminate the dependence of the signal on the expression levels as well as on the illumination and photon-detection settings. Experiments successfully confirmed the scaling predictions. Thus, the internal pH can be readily determined with intensity-based fluorophores with appropriate calibrations irrespective of the fluorophore concentration and the signal acquisition setup. The framework developed in this work improves the robustness of intensity-based fluorophores for internal pH measurements in E. coli, potentially extending their applications.
... In a study of a culture system probe with PBM (415 nm, 100 mW and 30, 60, 120 J/cm 2 Clinical Care/Education/Nutrition by stimulation of ROS synthesis. 48 Consistent with this finding, we hypothesized that the bactericidal effect of the PBM-treated groups in this probe might be due to the induction of ROS by PBM. ...
Objective
We assessed the therapeutic effects of photobiomodulation (PBM) and adipose-derived stem cell (ADS) treatments individually and together on the maturation step of repairing of a delayed healing wound model in rats with type 1 diabetes mellitus (DM1).
Research design and methods
We randomly assigned 24 rats with DM1 to four groups (n=6 per group). Group 1 was the control (placebo) group. In group 2, allograft human ADSs were transplanted. Group 3 was subjected to PBM (wavelength: 890 nm, peak power output: 80 W, pulse frequency: 80 Hz, pulsed duration: 180 ns, duration of exposure for each point: 200 s, power density: 0.001 W/cm ² , energy density: 0.2 J/cm ² ) immediately after surgery, which continued for 6 days per week for 16 days. Group 4 received both the human ADS and PBM. In addition, we inflicted an ischemic, delayed healing, and infected wound simulation in all of the rats. The wounds were infected with methicillin-resistant Staphylococcus aureus (MRSA).
Results
All three treatment regimens significantly decreased the amount of microbial flora, significantly increased wound strength and significantly modulated inflammatory response and significantly increased angiogenesis on day 16. Microbiological analysis showed that PBM+ADS was significantly better than PBM and ADS alone. In terms of wound closure rate and angiogenesis, PBM+ADS was significantly better than the PBM, ADS and control groups.
Conclusions
Combination therapy of PBM+ADS is more effective that either PBM or ADS in stimulating skin injury repair, and modulating inflammatory response in an MRSA-infected wound model of rats with DM1.
... In the clinic, PBM can be used as an adjuvant therapy to improve clinical outcomes by selecting the correct parameters and indications [28]. Blue LED has a broad spectrum of antibacterial effects [29]. Visible blue light promotes increases in human keratinocyte and endothelial cell differentiation markers [12]. ...
This study aimed to evaluate the effects of low-energy blue LED irradiation on the osteogenic differentiation of stem cells from the apical papilla (SCAPs). SCAPs were derived from human tooth root tips and were irradiated with 0 (control group), 1 J/cm2, 2 J/cm2, 3 J/cm2, or 4 J/cm2 blue light in osteogenic induction medium. Cell proliferation was analyzed using the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay. Osteogenic differentiation activity was evaluated by monitoring alkaline phosphatase (ALP), alizarin red staining, and real-time polymerase chain reaction (RT-PCR). The results of the MTT assay indicated that SCAPs in the LED groups exhibited a lower proliferation rate than those in the control group, and there were statistically differences between the 2 J/cm2, 3 J/cm2, and 4 J/cm2 groups and the control group (P < 0.05). The results of the ALP and alizarin red analyses showed that blue LED promoted osteogenic differentiation of the SCAPs. And 4 J/cm2 blue light upregulates the expression levels of the osteogenic/dentinogenic genes ALP, dentin sialophosphoprotein (DSPP), dentin matrix protein-1 (DMP-1), and osteocalcin (OCN) in SCAPs. Our results confirmed that low-energy blue LED at 1 J/cm2, 2 J/cm2, 3 J/cm2, and 4 J/cm2 could inhibit the proliferation of SCAPs and promotes osteogenic differentiation of SCAPs. Further in vitro studies are required to explore the mechanisms of the effects by low-energy blue LED.
... In a study of a culture system probe with PBM (415 nm, 100 mW and 30, 60, 120 J/cm 2 Clinical Care/Education/Nutrition by stimulation of ROS synthesis. 48 Consistent with this finding, we hypothesized that the bactericidal effect of the PBM-treated groups in this probe might be due to the induction of ROS by PBM. ...
Combination therapy of PBM+ADS is more effective that either PBM or ADS in stimulating skin injury repair, and modulating inflammatory response in an MRSA-infected wound model of rats with DM1.
The genus Bacillus is present everywhere in the environment, it has environmental, industrial, and medical signifi cance. This heterogeneous group of bacteria shares unique characteristics, one of these is the resistance to physical and chemical agents. Laser light is one of the most interesting physical agents with a wide range of applications in all scientifi c fi elds. It has the ability to alter bacterial growth by triggering inhibitory or stimulatory mechanisms. In this study, laser parameters were varied to evaluate the eff ect of each one on the viability of Bacillus species. CW or chopped (532 nm) laser light at diff erent irradiation periods were examined. Results showed that tested bacterial species’ response to green laser light varied. Bacterial viability was dropped with increasing chopping frequency and exposure time. In conclusion, high repetition rate and prolonged exposure time increase the lethal eff ects of this type of laser
Cell sheets and spheroids are cell aggregates with excellent tissue-healing effects. However, their therapeutic outcomes are limited by low cell-loading efficacy and low extracellular matrix (ECM). Preconditioning cells with light illumination has been widely accepted to enhance reactive oxygen species (ROS)-mediated ECM expression and angiogenic factor secretion. However, there are difficulties in controlling the amount of ROS required to induce therapeutic cell signaling. Here, we develop a microstructure (MS) patch that can culture a unique human mesenchymal stem cell complex (hMSCcx), spheroid-attached cell sheets. The spheroid-converged cell sheet structure of hMSCcx shows high ROS tolerance compared to hMSC cell sheets owing to its high antioxidant capacity. The therapeutic angiogenic efficacy of hMSCcx is reinforced by regulating ROS levels without cytotoxicity using light (610 nm wavelength) illumination. The reinforced angiogenic efficacy of illuminated hMSCcx is based on the increased gap junctional interaction by enhanced fibronectin. hMSCcx engraftment is significantly improved in our novel MS patch by means of ROS tolerative structure of hMSCcx, leading to robust wound-healing outcomes in a mouse wound model. This study provides a new method to overcome the limitations of conventional cell sheets and spheroid therapy.
Background:
The carbon dioxide (CO2 ) fractional laser resurfacing has become one of the hottest therapies for dermatoses. However, complications such as skin swelling, prolonged erythema, post-inflammatory hyperpigmentation, and scar formation remain. Low-level laser (LLL) therapy is accepted to promote skin wound healing and regeneration, decrease inflammation and pain, and modulate immunoreaction with low-dose laser of different wavelength. 532 nm laser therapy is commonly used to remove pigmented spots and to tender skin, but not utilized in wound care.
Objective:
We aimed to determine the efficacy of the low-level 532 nm green laser in wound healing after CO2 fractional laser.
Methods:
Six adult male mice (C57BL/6, eight weeks old) were prepared for animal experiments. The dorsum of each mouse was divided into four parts that respectively received designed treatments, as controlled (group Ctrl), 532 nm low-level laser (LLL)-treated (group GL), CO2 fractional laser-treated (group FL), and CO2 fractional laser followed by three times 532 nm LLL-treated (group FG). Hematoxylin-eosin staining (H&E), Masson-trichrome staining, CD31 immunohistochemical staining were performed to evaluate the efficacy of wound healing after treated by different irradiations. Western blotting was used to detect the expression of related proteins. Mouse skin fibroblasts (MSFs) were treated with LLL using a wavelength of 532 nm once. Cellular responses were observed and analyzed after 48 hours. Cell viability and migration of different groups were assessed by scratch and the Cell Counting Kit-8 (CCK8) assays, respectively.
Results:
Collagen remodeling and epidermis thickness were significantly enhanced in group FG than that in group FL in morphology. Besides, CD31 immunohistochemical staining indicated prominently increased angiogenesis in both groups FL and FG than non-irradiation group. The expression of extracellular matrix (ECM)-related protein (Col1, Col3 and MMP1) showed a remarkable improvement in wound healing in group FG than that in group FL. Irradiated MSFs showed a better migration ability compared with non-irradiated controls. LLL enhanced the secretion function of MSFs on Collagen I and III.
Conclusions:
Low-level green laser promotes wound healing after CO2 fractional laser by improving the integrity of skin barrier and allowing for scarless healing. Therefore, low-level green laser therapy might serve as a sequential therapy of invasive laser surgery to ensure a better wound care.
Strawberry (Fragaria Ananassa sp.) is non-climacteric, aggregate fruit with a rich source of anthocyanin and vitamin C. However, strawberry has short storage life and is very susceptible to decay and physiological deterioration process. Therefore, emitting the post-harvest LED light to strawberries during cold storage has been studied. This work aimed to evaluate different LED light treatment effects to retain post-harvest quality and microbial quality of the strawberry. The post-harvest parameters that have been used are weight loss, colour, firmness, total soluble solids, titratable acidity, and pH. The microbial parameter such as Escherichia coli count, Salmonella count, total plate count, and mold count would also has been assessed. Strawberries were irradiated under three different LED light types (white, blue and red). Blue and red LED lights have different wavelengths, which are 470 nm and 630 nm. The strawberry fruits were stored at 5ºC in a cold room and study parameter were assessed in 10 days. There were statistically significant differences (p<0.05) between the treatment of weight loss, firmness, pH, and total soluble solid. The best treatment for this experiment is blue LED light which showed the lowest weight loss, retained the colour and delayed firmness loss of the strawberry. This study also proved that blue LED light could reduce the growth of E.coli. In conclusion, LED light produces minimal heat, which improves food safety and preserves post-harvest quality of strawberry.
Built environments play a key role in the transmission of infectious diseases. Ventilation rates, air temperature, and humidity affect airborne transmission while cleaning protocols, material properties and light exposure can influence viability of pathogens on surfaces. We investigated how indoor daylight intensity and spectrum through electrochromic (EC) windows can impact the growth rate and viability of indoor pathogens on different surface materials (polyvinyl chloride [PVC] fabric, polystyrene, and glass) compared to traditional blinds. Results showed that tinted EC windows let in higher energy, shorter wavelength daylight than those with clear window and blind. The growth rates of pathogenic bacteria and fungi were significantly lower in spaces with EC windows compared to blinds: nearly 100% growth rate reduction was observed when EC windows were in their clear state followed by 41%–100% reduction in bacterial growth rate and 26%–42% reduction in fungal growth rate when EC windows were in their darkest tint. Moreover, bacterial viabilities were significantly lower on PVC fabric when they were exposed to indoor light at EC‐tinted window. These findings are deemed fundamental to the design of healthy modern buildings, especially those that encompass sick and vulnerable individuals.
A large fraction of nosocomial infections is associated with medical devices that are deemed life-threatening in immunocompromised patients. Medical device-related infections are a result of bacterial colonization and biofilm formation on the device surface that affects >1 million people anually in the US alone. Over the past few years, light-based antimicrobial therapy has made substantial advances in tackling microbial colonization. Taking the advantage of light and antibacterial properties of nitric oxide (NO), for the first time, a robust, biocompatible, anti-infective approach to design a universal disposable catheter disinfection insert (DCDI) that can both prevent bacterial adhesion and disinfect indwelling catheters in situ is reported. The DCDI is engineered using a photo-initiated NO donor molecule, incorporated in polymer tubing that is mounted on side glow fiber optic connected to an LED light source. Using a smartphone application, the NO release from DCDI is photoactivated via white light resulting in tunable physiological levels of NO for up to 24 h. When challenged with microorganisms S. aureus and E. coli, the NO-releasing DCDI statistically reduced microbial attachment by >99% versus the controls with just 4 h of exposure. The DCDI also eradicated ∼97% of pre-colonized bacteria on the CVC catheter model demonstrating the ability to exterminate an established catheter infection. The smart, mobile-operated novel universal antibacterial device can be used to both prevent catheter infections or can be inserted within an infected catheter to eradicate the bacteria without complex surgical interventions. The therapeutic levels of NO generated via illuminating fiber optic can be the next generation biocompatible solution for catheter-related blood-stream infections.
Human dental pulp stem cells (hDPSCs) have attracted tremendous attention in tissue regeneration engineering due to their excellent multidirectional differentiation potential. Photobiomodulation (PBM) using low-level light-emitting diodes (LEDs) or lasers has been proved to promote the osteogenesis of mesenchymal stem cells. However, the effect of LEDs on osteogenic differentiation of hDPSCs has little published data. In this work, the effect of blue LEDs with different energy densities of 2, 4, 6, 8, 10 J/cm² on osteogenic differentiation of hDPSCs was examined by using in vitro ALP staining, ALP activity, mineralization, and real-time PCR. The results showed that compared with the control group, osteogenic differentiation was significantly enhanced in blue LEDs treated groups. As the energy density increased, the level of osteogenesis initially increased and then decreased reaching the highest level at 6 J/cm². Transient receptor potential vanilloid 1 (TRPV1), a Ca²⁺ ion channel, was believed to be a potential player in osteogenesis by photobiomodulation. By immunofluorescence assay, calcium influx assay, PCR, and ALP staining, it was shown that blue LEDs irradiation can increase the activity of TRPV1 and intracellular calcium levels similarly to the agonist of TRPV1 capsaicin. Additionally, pretreatment with capsazepine, a selective TRPV1 inhibitor, was able to abrogate the osteogenic effect of blue LEDs. In conclusion, these findings proposed that blue LEDs can promote the osteogenesis of hDPSCs within the appropriate range (4-8 J/cm²) during culture of osteogenic medium, and TRPV1/Ca²⁺ may be an essential signaling pathway involved in blue LEDs-induced osteogenesis, providing new insights for the use of hDPSCs in tissue regeneration engineering.
The effect of sunlight and salinities (10; 20; 39 and 60 psu) on the survival of V. parahaemolyticus strains carrying either (thermostable direct hemolysin) tdh, the (thermostable related hemolysin) trh, and both or none of them were studied in water microcosms stabilized at 20°C using plate count agar and acridine orange direct viable count. All V. parahaemolyticus strains exposed to sunlight rapidly lose their culturability and evolve into a viable, but non-culturable state (VBNC). However, the tdh positive strains remain more culturable than the non-virulent or trh positive strain, but statically insignificant. At tested salinities, the survival time was higher at 10, 20 and 60 psu compared to that observed in seawater (39 psu).
In seawater under dark condition, Vibrio strains remain culturable for up to 200 days with a significant difference between strains (p< 0.05). Furthermore, the non-pathogenic strain survives longer than the virulent ones. At different salinities, a better adaptation is observed at 10 and 20 psu compared to 39 and 60 psu. Resuscitations essays performed on VBNC bacteria in a nutrient broth at 20 and 37°C does not show any revivification.
The use of light emitting diode (LED)-based photodynamic therapies in the treatment of periodontitis is increasing because these modalities are effective, safe, and painless. They are not subject to acquired drug resistance or environmental issues and are associated with no complications when used appropriately. These light sources have also been used in combination with pharmacological measures to synergize their effects and optimize therapeutic outcomes. This review focuses on optical devices used in treating periodontitis and delineates the current applications of various methods, including their utility and efficacy. The application of LEDs in periodontology is described.
In this paper, the photodynamic effect of a ternary nanocomposite (TiO2-Ag/graphene) on Escherichia coli bacteria and two human cell lines: A375 (melanoma) and HaCaT (keratinocyte) after exposure to different wavelength domains (blue, green or red-Light Emitting Diode, LED) was analyzed. The results obtained through bioassays were correlated with the morphological, structural and spectral data obtained through FT-IR, XPS and UV–Vis spectroscopy, powder X-Ray diffractometry (XRD) and STEM/EDX techniques, leading to conclusions that showed different photodynamic activation mechanisms and effects on bacteria and human cells, depending on the wavelength. The nanocomposite proved a therapeutic potential for blue light-activated antibacterial treatment and revealed a keratinocyte cytotoxic effect under blue and green LEDs. The red light–nanocomposite duo gave a metabolic boost to normal keratinocytes and induced stasis to melanoma cells. The light and nanocomposite combination could be a potential therapy for bacterial keratosis or for skin tumors.
Background: Periodontal disease affects 20–50% of population worldwide, posing a global health challenge. It has been reported to be more prevalent among adults. Actinobacillus actinomycetemcomitans (A. actinomycetemcomitans) is an important organism associated with localized juvenile periodontitis. Photodynamic therapy (PDT) has been widely utilized for the treatment of periodontal disease; however, the effect of laser (light) profile on the antibacterial efficacy of PDT remains to be established. The quantitative measurement of laser profile is required to confirm the in vitro efficacy of PDT.
Aim: In the present study, a low cost PDT system comprising of six copper tube waveguides (CTW) was developed to provide more uniform irradiation of the culture plate.
Methods: The antibacterial effect of PDT, in combination with 200 μg/mL methylene blue (MB) as photosensitizer and 60 sec of irradiation, was studied on A. actinomycetemcomitans and Streptococcus mutans (S. mutans). In the present case, 660 nm laser guided with unpolished CTW, polished CTW, and optical fiber waveguide (OFW) provided radiant exposure of 0.86, 1.38, and 1.36 J/cm², respectively, for a 24‐well culture plate.
Results: The designed PDT system provided antimicrobial efficacy of 98% and 91% for A. actinomycetemcomitans and S. mutans, respectively, which was significantly higher as compared to OFW guided PDT.
Conclusion: The results of the study highlighted the importance of laser profile as a key parameter that determines the survival rate of bacteria at the edge of the culture plate. Thus, the dose of PDT at the margin of optical profile is important for antibacterial activity for in vitro evaluation.
Objective:
To determine effective treatment strategies against bacterial infections of chronic wounds, we tested different blue light (BL)-emitting light-emitting diode arrays (420, 455, and 480 nm) against wound pathogens and investigated in parallel BL-induced toxic effects on human dermal fibroblasts.
Background:
Wound infection is a major factor for delayed healing. Infections with Pseudomonas aeruginosa and Staphylococcus aureus are clinically relevant caused by their ability of biofilm formation and their quickly growing antibiotics resistance. BL has demonstrated antimicrobial properties against various microbes.
Methods:
Determination of antibacterial and cell toxic effects by colony-forming units (CFUs)/biofilm/cell viability assays, and live cell imaging.
Results:
A single BL irradiation (180 J/cm2), of P. aeruginosa at both 420 and 455 nm resulted in a bacterial reduction (>5 log10 CFU), whereas 480 nm revealed subantimicrobial effects (2 log10). All tested wavelengths of BL also revealed bacteria reducing effects on Staphylococcus epidermidis and Escherichia coli (maximum 1-2 log10 CFU) but not on S. aureus. Dealing with biofilms, all wavelengths using 180 J/cm2 were able to reduce significantly the number of P. aeruginosa, E. coli, and S. epidermidis. Here, BL420nm achieved reductions up to 99%, whereas BL455nm and BL480nm were less effective (60-83%). Biofilm-growing S. aureus was more BL sensitive than in the planktonic phase showing a reduction by 63-75%. A significant number of cell toxic events (>40%) could be found after applying doses (>30 J/cm2) of BL420nm. BL455nm showed only slight cell toxicity (180 J/cm2), whereas BL480nm was nontoxic at any dose.
Conclusions:
BL treatment can be effective against bacterial infections of chronic wounds. Nevertheless, using longer wavelengths >455 nm should be preferred to avoid possible toxic effects on skin and skin cells. To establish BL therapy for infected chronic wounds, further studies concerning biofilm formation and tissue compatibility are necessary.
It is of particular interest to develop new antibacterial agents with low risk of drug resistance development and low toxicity toward mammalian cells to combat pathogen infections. Although gaseous signaling molecules (GSMs) such as nitric oxide (NO) and formaldehyde (FA) have broad-spectrum antibacterial performance and the low propensity of drug resistance development, many previous studies heavily focused on nanocarriers capable of delivering only one GSM. Herein, we developed a micellar nanoparticle platform that can simultaneously deliver NO and FA under visible light irradiation. An amphiphilic diblock copolymer of poly(ethylene oxide)-b-poly(4-((2-nitro-5-(((2-nitrobenzyl)oxy)methoxy)benzyl)(nitroso)amino)benzyl methacrylate) (PEO-b-PNNBM) was successfully synthesized through atom transfer radical polymerization (ATRP). The resulting diblock copolymer self-assembled into micellar nanoparticles without premature NO and FA leakage, whereas they underwent phototriggered disassembly with the corelease of NO and FA. We showed that the NO- and FA-releasing micellar nanoparticles exhibited a combinatorial antibacterial performance, efficiently killing both Gram-negative (e.g., Escherichia coli) and Gram-positive (e.g., Staphylococcus aureus) bacteria with low toxicity to mammalian cells and low hemolytic property. This work provides new insights into the development of GSM-based antibacterial agents.
Metal-free CO-releasing micelles can be selectively taken up by Gram-positive bacteria, exerting a narrow-spectrum bactericidal activity toward only Gram-positive bacteria under red light irradiation. The CO-releasing micelles synergistically eradicate MRSA pathogens and accelerate MRSA-infected wound healing.
Abstract
Carbon monoxide (CO) is an important gaseous signaling molecule. The use of CO-releasing molecules such as metal carbonyls enables the elucidation of the pleiotropic functions of CO. Although metal carbonyls show a broad-spectrum antimicrobial activity, it remains unclear whether the bactericidal property originates from the transition metals or the released CO. Here, we develop nonmetallic CO-releasing micelles via a photooxygenation mechanism of 3-hydroxyflavone derivatives, enabling CO release under red light irradiation (e.g., 650 nm). Unlike metal carbonyls that non-specifically internalize into both Gram-positive and Gram-negative bacteria, the nonmetallic micelles are selectively taken up by S. aureus instead of E. coli cells, exerting a selective bactericidal effect. Further, we demonstrate that the CO-releasing micelles can cure methicillin-resistant S. aureus (MRSA)-infected wounds, simultaneously eradicating MRSA pathogens and accelerating wound healing.
Carbon monoxide (CO) has been known as an important gaseous signaling molecule. The use of CO‐releasing molecules (CORMs) such as metal carbonyls offers the possibility to elucidate the pleiotropic physiological functions of CO. Although metal carbonyls show a broad‐spectrum antimicrobial activity, it remains unclear whether the bactericidal property originates from the transition metals or the released CO. Herein, we develop nonmetallic micellar nanoparticles for localized CO delivery via a photooxygenation mechanism of 3‐hydroxyflavone derivatives, enabling CO release under red light irradiation (e.g., 650 nm). In sharp contrast to metal carbonyls that non‐specifically internalize into both Gram‐positive and Gram‐negative bacteria and show a broad‐spectrum antibacterial activity, the nonmetallic micelles were selectively taken up by Staphylococcus aureus ( S. aureus ) instead of Escherichia coli ( E. coil ) cells, exerting a selective bactericidal effect toward S. aureus . Further, we demonstrate that the metal‐free micelles can cure methicillin‐resistant S. aureus (MRSA)‐infected wounds in a full‐thickness skin wound model by taking advantage of the localized release CO, simultaneously eradicating MRSA pathogens and accelerating wound healing.
Antimicrobial photodynamic inactivation is currently one of the most promising trends in the modern bactericidal protocols. Under the conditions defined in our studies, we found that in vitro photosensitization of A. baumannii with 5-ALA as a precursor of protoporphyrin IX (photosensitizer) reduces the concentration of viable cells in planktonic cultures, and this process can be strongly enhanced by pentamidine. Diode lasers with the peak-power wavelength of ʎ = 405 nm (radiation intensity of 26 mW cm-2) and ʎ = 635 nm (radiation intensity of 55 mW cm-2) were used in this study. It was found that a blue laser light (energy fluence of 64 J cm-2; no external photosensitizer) in the presence of pentamidine resulted in a reduction of CFU of 99.992% compared to 99.97% killing without pentamidine. When a red laser light was used in the experiments (energy fluence of 136 J cm-2; no external photosensitizer), the mortality rate was 99.98% in the presence of pentamidine compared to 99.93% of those killed without the addition of this drug. The lethal effect with 5-ALA was achieved under blue light fluence of 16 J cm-2 (in the presence of pentamidine) and 32 J cm-2 (without pentamidine). In the case of laser light of 635 nm, the lethal effect with 5-ALA was attained with energy fluence of 51 J cm-2 (with pentamidine) and 102 J cm-2 (without pentamidine). The possible roles of pentamidine in enhancing photodynamic inactivation of A. baumannii have been discussed.
In this work we adapt rare earth ions doped NaYF4 nanoparticles coated by silicon oxide shell (NaYF4:20%Yb,0.2%Tm@SiO2) for biological and medical applications (for example: imaging of cancer cells and therapy at the nano-dimension). Wide upconversion emission range under 980 nm excitation allows to use the nanoparticles for the cancer cells (4T1) photodynamic therapy (PDT) without any photosensitizer. The reactive oxygen species (ROS) are generated by Tm/Yb ions upconversion emission (blue and UV light). The in-vitro PDT was tested on 4T1 cells incubated with the NaYF4:20%Yb,0.2%Tm@SiO2 nanoparticles and irradiated with NIR light. After 24 hours, the cell viability decreased to below 10% demonstrating a very good treatment efficiency. High modification susceptibility of the SiO2-shell allows attachment of biological molecules (specific antibodies). In this work we attached anti-human IgG antibody to silane-PEG-NHS modified NaYF4:20%Yb,0.2%Tm@SiO2 nanoparticles and specifically marked membrane model by bio-conjugation. Thus it could be possible to perform a selective search (high quality optical method with very low level of organic background) and eventual damage of the targeted cancer cells. The studies focus on therapeutic properties of NaYF4:20%Yb,0.2%Tm@SiO2 nanoparticles and demonstrate, upon biological functionalization, their potentials for targeting therapy.
The direct application of light for photo-disinfection potentially provides a safe and novel modality to inhibit or eliminate cariogenic bacteria residing upon and within dentine. This study aimed to both; characterize the pattern of transmission of 405 nm light through molar dentine at different tooth locations, as well as, determining the irradiation parameters that are antibacterial for Streptococcus mutans under various growth conditions, including lawns, planktonic cultures, and biofilms. To determine the amount of light (405 nm) transmitted at different anatomical tooth locations; irradiance values were recorded after blue light (470–4054 mW/cm²) had traversed through occlusal, oblique, and buccal dentine sections; and three thicknesses - 1, 2 and 3 mm were investigated. To determine tubular density; scanning electron micrographs from 2 mm outer (dentine-enamel junction) and inner (pulp) dentine sections were analysed. For photo-disinfection studies; S. mutans was irradiated using the same 405 nm wavelength light at a range of doses (110–1254 J/cm²) in both biofilm and planktonic cultures. The inhibitory effect of the irradiation on bacterial lawns was compared by measuring zones of inhibition; and for planktonic cultures both spectrophotometric and colony forming unit (CFU) assays were performed. A live/dead staining assay was utilised to determine the effect of irradiation on bacterial viability in mature biofilms. Data indicated that increasing dentine thickness decreased light transmission significantly irrespective of its orientation. Occlusal and oblique samples exhibited higher transmission compared with buccal dentine. Oblique dentine 405 nm light transmission was comparable with that of occlusal dentine independent of section thickness. An increased tubule density directly positively correlated with light transmission. Irradiation at 405 nm inhibited S. mutans growth in both biofilm and planktonic cultures and a dose response relationship was observed. Irradiation at doses of 340 and 831 J/cm² led to significant reductions in bacterial growth and viability; as determined by CFU counting and live/dead staining. Data suggests that phototherapy approaches utilising a 405 nm wavelength have therapeutic potential to limit cariogenic bacterial infections both at the surface and within dentine.
Abstract
Introduction: Cavity disinfection is necessary to prevent a progressive infection of the crown dentin and pulp. Increasing intolerance and resistance to antiseptics and antibiotics as well as the controversy over the effects of those on the dental hard tissue and composite have prompted the investigation of alternative treatment options. The objective of this pilot study is to evaluate the antibacterial potential of a diode laser with a wavelength of 445 nm in the cavity preparation using the bacterium Streptococcus salivarius associated with caries in conjunction with the characteristics and influences of dentin on light transmission.
Methods: The bactericidal effect of the laser irradiation was determined in culture experiments by using caries-free human dentin samples on bacteria-inoculated agar. For this, dentin discs
(horizontally cut coronal dentin) of 500 µm and 1000 µm thicknesses were produced and irradiated with the laser with irradiation parameters of 0.7-1 W in a cw-mode and exposure times of between
5-30 s. Based on the different sample thicknesses, the penetration depth effect of the irradiation was ascertained after the subsequent incubation of the bacteria-inoculated agar. Additional influential
parameters on the irradiation transmission were investigated, including surface moisture, tooth color as well as the presence of a smear layer on the dentin surface.
Results: The optical transmission values of the laser radiation for dentin were significantly dependent on the sample thickness (P = 0.006) as well as its moisture content (P = 0.013) and were independent of the presence of a smear layer. There was a 40% reduction in bacteria after the radiography of the 500-µm-thick dentin samples, which was shown as the lowest laser dose (443 J/cm2).
Conclusion: These findings indicate that the diode laser with light emission at a wavelength of 445 nm is interesting for the supportive cavity disinfection within the scope of caries therapy and show
potential for clinical applications.
Dentistry is a field that has been evaluated to act beyond the teeth. Treatment of diseases such as gingivitis, neuralgia, and others is common nowadays. The constant demand created by the highest life expectations of men and women requires constantly changing procedures. Geriatric dentistry is, today, a specialty among the general dental activities. This constant evolution is directing dentistry to offer patients much more than the conventional curative procedures, also migrating to rehabilitation and to aesthetic restoration to promote full harmonization. Based on modern technologies, with special attention to those promoted by biophotonics, orofacial rehabilitation and harmonization is becoming a reality. In this chapter, we describe a collection of procedures and technologies that has allowed orofacial biophotonics-based rehabilitation and harmonization to become a new specialty for modern dentistry.
Background:
The Photobiomodulation (PBM) effect has been applied to various clinical therapy for a long time. However, the mechanism related to the PBM effect in terms of wavelengths has been lack of in-depth study, except that ultraviolet radiation has attracted much attention due to its strong cell killing effect.
Purpose:
To clarify the principle behind PBM and the main mechanism of improvement.
Methods:
To carry on this study, we created light equipment using three LED chips, which emit 390 nm ultraviolet radiation, 415 nm blue light and 660 nm red light, respectively. We choose human fibroblasts (HF) to be irradiated by three different wavelengths for PBM test. In this study, we used cell counting kit (CCK-8) test to show the cell proliferation roughly and reported on a systematic RNA sequencing (RNA-seq) analysis at transcriptional expression levels from HF, which accepted PBM of different wavelengths of light.
Results:
We found that 415 nm blue light inhibited cell proliferation and 660 nm red light stimulated cell proliferation while 390 nm ultraviolet radiation has little influence on cell proliferation. Furthermore, RNA-seq results showed that CSF1R, PPP3CC, ITGAL, ITGAM, IL2RB and several other differentially expressed genes (DEGs) are involved in the cell proliferation. Relative DEGs values for matrix metalloproteinases (MMPs) gene family has shown a great difference in blue and red light radiation especially on MMP25, MMP9, MMP21 and MMP13.
Conclusion:
Taken together, the results provide a valuable resource to describe the variation of HFs under PBM of different light at gene level.
The approach of this timely review considers the current literature that is focused on the interface nanostructure/cell-wall microorganism to understand the annihilation mechanism. Morphological studies use optical and electronic microscopes to determine the physical damage on the cell-wall and the possible cell lysis that confirms the viability and microorganism death. The key parameters of the tailoring the surface of the photoactive nanostructures such as the metal functionalization with bacteriostatic properties, hydrophilicity, textural porosity, morphology and the formation of heterojunction systems, can achieve the effective eradication of the microorganisms under natural conditions, ranging from practical to applications in environment, agriculture, and so on. However, to our knowledge, a comprehensive review of the microorganism/nanomaterial interface approach has rarely been conducted. The final remarks point the ideal photocatalytic way for the effective prevention/eradication of microorganisms, considering the resistance that the microorganism could develop without the appropriate regulatory aspects for human and ecosystem safety.
We investigate the p-type doping difficulty in ZnO by first-principles total-energy calculations. The dopants being considered are group-I elements Li, Na, and K and group-V elements N, P, and As. We find that substitutional group-I elements are shallow acceptors, while substitutional group-V elements such as P and As are deep acceptors. The AX centers that convert acceptors into deep donors are found to be unstable except for P and As. Without compensation by intrinsic defects, the most likely cause for doping difficulty is the formation of interstitials for group-I elements and antisites for group-V elements. Among all the dopants studied here, N is a relatively better candidate for p-type ZnO.
The civilian, commercial, and defense sectors of most advanced industrialized nations are faced with a tremendous set of environmental problems related to the remediation of hazardous wastes, contaminated groundwaters, and the control of toxic air contaminants. Problems with hazardous wastes at military installations are related in part to the disposal of chemical wastes in lagoons, underground storage tanks, and dump sites. Typical wastes of concern include heavy metals, aviation fuel, military-vehicle fuel, solvents and degreasing agents, and chemical byproducts from weapons manufacturing. In the civilian sector, the elimination of toxic and hazardous chemical substances such as the halogenated hydrocarbons from waste effluents and previously contaminated sites has become a major concern. General classes of compounds of concern include: solvents, volatile organics, chlorinated volatile organics, dioxins, dibenzofurans, pesticides, PCB's, chlorophenols, asbestos, heavy metals, and arsenic compounds. Advanced physicochemical processes such as semiconductor photocatalysis are intended to be both supplementary and complementary to some of the more conventional approaches to the destruction or transformation of hazardous chemical wastes such as high-temperature incineration, amended activated sludge digestion, anaerobic digestion, and conventional physicochemical treatment. 441 refs.
Background
Infections with MRSA remain a growing public health concern, prompting the need to explore alternative treatments instead of the on-going effort to develop stronger drug-based therapies. We studied the effect of 405 nm blue light on two strains of MRSA—US-300 strain of CA-MRSA and the IS853 strain of HA-MRSA—in vitro.Methods
We cultured and plated each strain, following which bacteria colonies were irradiated with 0, 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 25, 30, 35, 40, 45, 50, 55, or 60 J cm−2 energy densities—just once—using a Solaris® superluminous diode (SLD) device. Specimens were incubated at 35°C for 24 hours. Then, digital images obtained were quantified to obtain colony counts and the aggregate area occupied by bacteria colonies.ResultsBlue light irradiation produced a statistically significant dose-dependent reduction in both the number and the aggregate area of colonies formed by each bacteria strain (P<0.001). Maximum eradication of the US-300 (92.1%) and the IS-853 colonies (93.5%) was achieved within 9.2 and 8.4 minutes of exposure, respectively. The longer the irradiation the more bacteria were eradicated. However, the effect was non-linear as increases of energy densities between 1.0 and 15 J cm−2 resulted in more bacteria death than similar increases between 15 and 60 J cm−2.Conclusion
At low doses, blue light photo-destroys HA-MRSA and CA-MRSA in vitro; raising the prospect that phototherapy may be an effective clinical tool in the on-going effort to stem MRSA infections. Lasers Surg. Med. 40:734–737, 2008. © 2008 Wiley-Liss, Inc.
Nanostructured ZnO photo catalyst was synthesized by precipitation method and was applied in conjunction with 355nm pulsed
laser irradiation for effective disinfection of the water contaminated with Escherichia coli micro organism. The morphological studies using X-Ray Diffractometer (XRD) and Transmission Electron Microscope (TEM) were
carried out on the synthesized nano-ZnO, and these studies indicated that the catalyst has the crystallographic structure
of hexagonal wurtzite and has the grain size of around 20–40nm. The bacteria decay rate constants were estimated for nine
different concentrations of nano-ZnO in infected water. The parametric optimization was carried out, and we could reach the
decay rate constant as high as 0.24min,−1 which is higher than micro-structured ZnO and the familiar TiO2 photo catalysts under similar experimental condition.
KeywordsSynthesis of nano-structured ZnO–Laser induced photo catalysis–Water disinfection–Waste water treatment–
E. Coli
–Health effects
In recent years nano-metaloxides which easily penetrate into the cells with special interest due to their higher chemical reactivity as compared to that of similar materials in the bulk form. Of particular interest are nano-TiO(2) and ZnO, which have been widely used for their bactericidal and anticancerous properties.
The aim of the present study was to examine the bactericidal properties of nano-TiO(2) and ZnO combined with visible light on S. aureus and S. epidermitis, known for their high prevalence in infected wounds.
Using the technique of electron-spin resonance (ESR) coupled with spin trapping, we examined the ability of TiO(2) and ZnO nanoparticle suspensions in water to produce reactive oxygen species (ROS) with and without visible light irradiation. The possibility of exciting these nanoparticles with visible light in order to enhance their antimicrobial activity was also tested.
Electron-spin resonance measurements revealed that ZnO and TiO(2) nanoparticles are able to produce ROS in water suspension. A remarkable enhancement of ROS production was found following illumination with blue light. In addition, illumination significantly enhanced the antibacterial activity of the nanoparticles.
The results suggest that nanoparticles combined with visible light can be used for sterilization purposes and may be effective for treating infected wounds.
The antibacterial effect of zinc oxide (ZnO) nanoparticles on Campylobacter jejuni was investigated for inhibition and inactivation of cell growth. The results showed that C. jejuni was extremely sensitive to treatment with ZnO nanoparticles. The MIC of ZnO nanoparticles for C. jejuni was determined to be 0.05 to 0.025 mg/ml, which is 8- to 16-fold lower than that for Salmonella enterica serovar Enteritidis and Escherichia coli O157:H7 (0.4 mg/ml). The action of ZnO nanoparticles against C. jejuni was determined to be bactericidal, not bacteriostatic. Scanning electron microscopy examination revealed that the majority
of the cells transformed from spiral shapes into coccoid forms after exposure to 0.5 mg/ml of ZnO nanoparticles for 16 h,
which is consistent with the morphological changes of C. jejuni under other stress conditions. These coccoid cells were found by ethidium monoazide-quantitative PCR (EMA-qPCR) to have a
certain level of membrane leakage. To address the molecular basis of ZnO nanoparticle action, a large set of genes involved
in cell stress response, motility, pathogenesis, and toxin production were selected for a gene expression study. Reverse transcription-quantitative
PCR (RT-qPCR) showed that in response to treatment with ZnO nanoparticles, the expression levels of two oxidative stress genes
(katA and ahpC) and a general stress response gene (dnaK) were increased 52-, 7-, and 17-fold, respectively. These results suggest that the antibacterial mechanism of ZnO nanoparticles
is most likely due to disruption of the cell membrane and oxidative stress in Campylobacter.
In a previous study, we showed that 405-nm light photo-destroys methicillin-resistant Staphylococcus aureus (MRSA). The 390-420 nm spectral width of the 405-nm superluminous diode (SLD) source may raise safety concerns in clinical practice, because of the trace of ultraviolet (UV) light within the spectrum.
Here we report the effect of a different wavelength of blue light, one that has no trace of UV, on two strains of MRSA--the US-300 strain of CA-MRSA and the IS-853 strain of HA-MRSA--in vitro.
We cultured and plated each strain, and then irradiated each plate with 0, 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 25, 30, 35, 40, 45, 50, 55, or 60 J/cm2 of energy a single time, using a 470-nm SLD phototherapy device. The irradiated specimens were then incubated at 35 degrees C for 24 h. Subsequently, digital images were made and quantified to obtain colony counts and the aggregate area occupied by bacteria.
Photo-irradiation produced a statistically significant dose-dependent reduction in both the number and the aggregate area of colonies formed by each strain (p < 0.001). The higher the dose the more bacteria were killed, but the effect was not linear, and was more impressive at lower doses than at higher doses. Nearly 30% of both strains was killed with as little as 3 J/cm2 of energy. As much as 90.4% of the US-300 and the IS-853 colonies, respectively, were killed with an energy density of 55 J/cm2. This same dose eradicated 91.7% and 94.8% of the aggregate area of the US-300 and the IS-853 strains, respectively.
At practical dose ranges, 470-nm blue light kills HA-MRSA and CA-MRSA in vitro, suggesting that a similar bactericidal effect may be attained in human cases of cutaneous and subcutaneous MRSA infections.
This paper reviews studies on the basic principles of biostimulation of wound healing by various low-energy lasers. It looks at the mechanism of action of biostimulation as well as the lasers effect on cell proliferation, collagen synthesis, and would healing.
ZnO nanoparticles were modified by polyaniline (PANI) using ‘in situ’ chemical oxidative polymerization method. The morphology, structure, and light absorption properties of PANI-ZnO composites were examined by powder X-ray diffraction (XRD), transmission electron microscopy (TEM) and UV–vis spectra. UV–vis spectra reveal that PANI-ZnO composites showed stronger absorption than neat ZnO under the whole range of visible light. New characteristic peaks were found in PANI-ZnO composites according to the X-ray diffraction patterns after hybridization of PANI and ZnO, which indicating that there was a strong interaction between PANI and ZnO nanoparticles. The resulting PANI-ZnO composites exhibit significantly higher photocatalytic activity than that of neat ZnO for degradation of anthraquinone dye (reactive brilliant blue KN-R) aqueous solution under visible light irradiation (λ > 420 nm).
Herein we obtain a novel incorporating style of polyaniline (PANI)/TiO2 composites (Ns) via a designed two-step route. In comparison with conventional incorporating style (Cs), one-dimensional (1D) PANI is introduced in the step of TiO2 crystal growth instead of after crystallization. During the hydrothermal process, 1D PANI can act as an effective template to stabilize TiO2 particles at nanoscale through interfacial chemical bonds. In this way, the transition of some newly formed anatase crystals to rutile phase is partly suppressed. Also, the as-prepared composites show obvious light response in a wide range of 230–900 nm due to the sensitizing effect of PANI. Ns exhibit the improved visible photocatalytic property with scarce decrease of UV photoactivity. Overall, this work would provide new insights into the fabrication of conducting polymer/semiconductor composites with desired nanostructure as high performance photocatalysts and would facilitate their applications in environmental purification and solar energy conversion.
Visible light responsible TiO2 nano-particles (TiO2 NPs) were prepared through sol–gel method using tetrabutyl titanate and biomolecule cystine as raw materials. The resulting materials were characterized by X-ray diffraction (XRD), ultraviolet–visible diffuse reflectance spectroscopy (UV–vis DRS), surface photovoltage spectroscopy (SPS) and electric field induced surface photovoltage spectroscopy (EFISPS). The XRD results showed that the addition of cystine could effectively retard the phase transformation of TiO2 from anatase to rutile and the growth of crystallite sizes. DRS results indicated that the light absorption edge of TiO2 was significantly red-shifted to visible region with the addition of biomolecule cystine. SPS results exhibited that the separation efficiency of the photoinduced charge carriers of TiO2 were effectively improved. In addition, EFISPS indicated that TiO2 nano-particles modification with biomolecule cystine were still n-type semiconductor.
Graphite oxide (GO)–TiO2 nanocomposite was prepared by a facile hydrothermal process and was characterized by X-ray powder diffraction, Transmission electron microscopy, UV–vis diffusion reflectance spectroscopy, X-ray photoelectron spectroscopy, and Raman spectroscopy. TiO2 particles with average particle size of ∼20 nm in the nanocomposites are attached to the surface of GO and/or intercalated into the interlayer of GO. The obtained GO–TiO2 was used as photocatalyst for H2 production under visible light (λ ≥ 420 nm) irradiation, and an optimal photocatalytic H2 production rate of 380 μmol h−1 can be obtained over 2 wt% GO–TiO2. The encouraging results presented here demonstrate that GO can serve as visible-light-driven photocatalyst and photosensitizer to expand the photoresponsive range of TiO2 to visible light for H2 production. The possible mechanism for H2 production was proposed for better understanding the visible-light-driven photocatalytic behaviour of the GO–TiO2 nanocomposite.
Although the antibacterial activity of ceramics has been reported in many literatures, there are none that report on the significant relation between the crystallite size and antibacterial activity. The influence of the crystallite size on antibacterial activity of MgO and ZnO powder samples was investigated in the present study. The crystallinity of powder samples was determined by X-ray diffraction measurement. From Williamson–Hall plots calculated from obtained diffraction peaks, it was found that differences in crystallinity were dependent on the crystallite size.Antibacterial activity of the powder samples was examined by the colony count method, using Escherichia coli and Staphylococcus aureus. The powder samples with a small crystallite size showed greater antibacterial activity than those with a large crystallite size. We found that the crystallite size affects the antibacterial activity of MgO and ZnO powder samples.
The effects of preparation methods, humidity, and calcination temperatures on the behavior of nanoscale TiO2 photocatalysts were investigated in this study. Application of these photocatalysts in the gas-phase decomposition of 1-butene demonstrated that the nanoscale catalysts, prepared by the acid-assisted sol–gel technique, showed higher photocatalytic reactivity than commercially available Degussa P-25 TiO2, while lower photoactivity was obtained on TiO2 catalysts prepared without adding acid to titanium isopropoxide. Our experimental results revealed that the oxidation rates of 1-butene decreased exponentially with increasing water concentrations in the flowing stream. However, a trace amount of water vapor was indispensable in maintaining the stability of the catalysts. Proper calcination temperature (300°C) could promote the resistance of catalysts against the poisoning effects of humidity. Higher calcination temperature (400°C) unfavorably lowered the photoactivity due to phase transformations occurring at such a temperature. Amorphous and rutile-typed TiO2 showed less photocatalytic reactivity. XRD patterns and BET measurements indicated that moderate surface areas (from 100 to 160 m2/g), appropriate crystallite sizes (5∼6 nm), and crystallinity of anatase were beneficial to the photoactivity of TiO2 catalysts. In situ FTIR studies indicated that catalyst surfaces contained large amounts of chemisorbed water and hydroxyl groups, which are considered to be active sites in photocatalytic reactions. The accumulation of carbonate species on active sites resulted in the deactivation of TiO2 catalysts under dry conditions. Quantum size effects were thought to be responsible for the high photoactivity achieved on the nanoscale TiO2 catalysts prepared by sol–gel methods.
Photosensitization of the TiO2 nanoparticles with carotenoids leads to the formation of superoxide anion and singlet oxygen on red light irradiation. It has been shown that carotenoids facilitate generation of superoxide radical anion O-2(.-) in irradiated TiO2 colloids. At low carotenoid concentrations (<3 x 10(-5) M) the rate constant of O-2(.-) production exceeds that obtained in the absence of carotenoid. EPR experiments with 2,2,6,6-tetramethyl-4-piperidone and spin trapping with alpha-phenyl-N-tert-butyl nitrone and 5,5-dimethyl-pyrroline-N-oxide showed that both superoxide anion and singlet oxygen can be produced in irradiated TiO2 suspensions in toluene and CH2Cl2. The PBN-O-2(.-) and DMPO-O-2(.-) spin adducts were detected by the 9 GHz EPR/ENDOR spin trapping technique. The dismutation of superoxide radical anion was proposed to generate singlet oxygen through intermediate formation of HO2. radicals. The PBN-HO2. spin adduct was identified by the 9 GHz EPR/ENDOR and 95 GHz EPR spin trapping technique.
Metal-ion-doped TiO2 nanoparticles were prepared with sol-gel method. The photocurrents of these metal-ion-doped TiO2 electrodes were measured at different electrode potentials and different excitation wavelengths, and different photoelectrochemical behaviors were observed. The phenomenon of p–n conversion was observed for Fe3+- doped TiO2 electrode; however, the characteristic of pure n-type semiconductor was observed for lanthanide ion (such as La3+, Nd3+, Pr3+, Sm3+ and Eu3+), Zn2+, Cd2+, Cr3+ and Co2+-doped TiO2 electrodes. The effect of the content of Zn2+ on photocurrent was investigated and the result showed that the incident photon conversion efficiency (IPCE) was the largest when the content of Zn2+ was 0.5%.
A Photosensitized electrode was fabricated through a novel polymerization of pyrrole initiated by TiO2 nanoparticles under UV irradiation. Conjugated polypyrrole (PPy) continuously grows on the surface of the TiO2 film, resulting in fully covered film with the stably-bound PPy layer. The film structures were analysed using Raman and X-ray photoelectron spectroscopy (XPS). It was found that the film electrode exhibits improved photoresponse including the flat band potential, photocurrent and separation of charges. The lowest unoccupied molecular orbital (LUMO) level of the PPy is higher in energy than the LUMO of PPy in electrodeposited electrodes, providing a stronger driving force for the electron injection into the TiO2.
A novel phosphor-doped titania in an anatase phase colored yellow was prepared by a simple modified sol-gel method with hypophosphorous acid as precursor. The as-prepared P-doped titania shows a narrower band gap than pure titania and an absorption tail in the visible range, which results in its effective photocatalytic degradation of 4-chlorophenol under visible-light (> 420 nm) irradiation.
This review discusses salient features of interfacial electron transfer reactions in colloidal semiconductor solutions and thin films and their application for solar light energy conversion and photocatalytic water purification. This research is interdisciplinary and is situated at the limit between colloid science, and electrochemistry, and semiconductor physics. The section on colloidal semiconductors discusses material science aspects, optical properties, electronic properties, and charge carrier reactions in colloidal semiconductor solutions. The section on nanocrystalline semiconductor electrodes discusses some basic properties and preparation procedures of nanocrystalline TiO[sub 2] films, energetics and operations of the nanoporous solar cell, observations and importance of electronic states in the band gap region, photoinduced processes in nanocrystalline semiconductor electrodes, the electric field in a nanocrystalline electrode, and kinetic rate constants in the photosensitized solar cell. 156 refs.
Titanium dioxide (TiO2) doped with sulfur (S) was synthesized by oxidation annealing of titanium disulfide (TiS2). According to the x-ray diffraction patterns, TiS2 turned into anatase TiO2 when annealed at 600 °C. The residual S atoms occupied O-atom sites in TiO2 to form Ti�S bonds. The S doping caused the absorption edge of TiO2 to be shifted into the lower-energy region. Based on the theoretical analyses using ab initio band calculations, mixing of the S 3p states with the valence band was found to contribute to the band gap narrowing. © 2002 American Institute of Physics.
Zinc oxide nanocrystallites down to 1.5 nm in diameter were made by spray combustion of Zn/Si precursors. These crystallites exhibit a quantum size effect: blueshift of light absorption with decreasing crystallite size. X-ray diffraction, high-resolution transmission electron microscopy, and nitrogen adsorption showed that the addition of controlled amounts of silica prevented the growth and stabilized the ZnO crystals. The blue shift of the ultraviolet-vis absorption edge with decreasing ZnO crystal size closely followed a correlation between optical band gap and crystallite size from the literature. The band-gap energy of these ZnO quantum dots increased with silica content in the spray and particles consistently. The as-prepared quantum dots were stable and did not require any post processing. © 2002 American Institute of Physics.
Irradiation of selectedEscherichia colidefective strains with red-light induces a stimulation of the cell growth rate. Such effect is wavelength-dependent and is accompanied by a transient increase of the cell volume and some enzymic activities. The presence of oxygen appears to be essential for the occurrence of a significant photostimulatory effect. The results obtained upon irradiation in the presence of quenchers (tryptophan, histidine, azide) or enhancers (deuterium oxide) of singlet oxygen (1O2) strongly suggest that this activated oxygen derivative is generated by excitation of endocellular chromophores (possibly cytochromes). The reaction of1O2with nearby cellular targets could induce a sublethal cell damage which in turn promotes an accelerated cell metabolism.
Nanoparticles of ZnO have been shown to have marked antibacterial and anticancerous activities. The toxic effect of ZnO has been found to exist due to a reaction of the ZnO surface with water. In the present work electron-spin resonance measurements revealed that aqueous suspensions of small nanoparticles of ZnO produce increased levels of reactive oxygen species, namely hydroxyl radicals and singlet oxygen. Interestingly, a remarkable enhancement of the oxy radicals, was detected when the ZnO water suspension had been irradiated with blue (400−500 nm) light. The characterization of the mechanism of light-induced oxy radicals formation by ZnO nanoparticles would contribute to its use as a sterilization agent.
In this paper, mesoporous nitrogen-doped TiO 2 microspheres were prepared by a template-free solvothermal method. The nitrogen-doped TiO 2 mesoporous spheres show higher visible-light photocatalytic activity than the undoped TiO 2. The dual role of urea helps the formation of a mesoporous structure and the doping of nitrogen into TiO 2 to be completed simultaneously during the solvothermal process. The amount of urea shows the crucial effect on the mesoporous structure and nitrogen doping in TiO 2.
The author provides this introduction as many students do not have an opportunity to work in surface chemistry. This present paper will review from a pedagogical point of view some of the fundamentals of semiconductors as they related to catalysis on semiconductors and as they relate to catalysis on semiconductor surfaces. Keywords (Audience): Upper-Division Undergraduate
A systematic study of metal ion doping in quantum (Q)-sized (2-4 nm) TiO_2 colloids is performed by measuring their photoreactivities and the transient charge carrier recombination dynamics. The presence of metal ion dopants in the TiO_2 crystalline matrix significantly influences photoreactivity, charge carrier recombination rates, and interfacial electron-transfer rates. The photoreactivities of 21 metal ion-doped colloids are quantified in terms of both the conduction band electron reduction of an electron acceptor (CCl_4 dechlorination) and the valence band hole oxidation of an electron donor (CHCl_3 degradation). Doping with Fe^(3+), Mo^(5+), Ru^(3+), Os^(3+), Re^(5+), V^(4+), and Rh^(3+) at 0.1-0.5 at.% significantly increases the photoreactivity for both oxidation and reduction while Co^(3+) and Al^(3+) doping decreases the photoreactivity. The transient absorption signals upon laser flash photolysis (λ_(ex) = 355 nm) at λ = 600 nm are extended up to 50 ms for Fe^(3+)-, V^(4+)-, Mo^(5+)-, and Ru^(3+)-doped TiO_2 while the undoped Q-sized TiO_2 shows a complete "blue electron" signal decay within 200 μs. Co^(3+)- and Al^(3+)-doped TiO_2 are characterized by rapid signal decays with a complete loss of absorption signals within 5 μs. The quantum yields obtained during CW photolyses are quantitatively correlated with the measured transient absorption signals of the charge carriers. Photoreactivities
are shown to increase with the relative concentration of trapped charge carriers. The photoreactivity of doped TiO_2 appears to be a complex function of the dopant concentration, the energy level of dopants within the TiO_2 lattice, their d electronic configuration, the distribution of dopants, the electron donor concentration, and the light intensity.
The incorporation of controlled amounts of nitrogen into group IVB metal oxide nanoparticles through a wet-chemical procedure under ambient conditions was presented and the effects of doping on their optical and photocatalytic properties was analyzed. The results show that the N binding energies for the metal oxide nanoparticles are centered at 399.4, 398.6 and 399.0 eV, while N-doping levels can be controlled by the amount of acid and base added during hydrolysis. The absorption of the metal oxides is red-shift with an increase in N doping, which shows that the metal oxides enhance the visible-light absorption of the N-doped nanoparticles. The results also show that the N-doped metal oxide nanoparticles show enhanced photocatalytic performance as compared to pure anatase nanoparticles.
Oxidation and reduction rates are increased when the photocatalyst TiO_2 is doped with metal ions. The photochemical oxidation of CHCl_3 and the photochemical reduction of CCl_4 were examined in the presence of 21 different doped TiO_2 catalysts. Dopant metal ions with closed shells were found to have little effect on reactivity. The authors present their results in the form of a periodic chart.
Titanium dioxide nanoparticles were modified by polyaniline (PANI) using ‘in situ’ chemical oxidative polymerization method in hydrochloric acid solutions. Powder X-ray diffraction (XRD), transmission electron microscopy (TEM), Fourier-transform infrared spectra (FT-IR), X-ray photoelectron spectroscopy spectrum (XPS) and UV–vis spectra were carried out to characterize the composites with different PANI contents. The photocatalytic degradation of phenol was chosen as a model reaction to evaluate the photocatalytic activities of the modified catalysts. Results show that TiO2 nanoparticles are deposited by PANI to mitigate TiO2 particles agglomeration. The modification does not alter the crystalline structure of the TiO2 nanoparticles according to the X-ray diffraction patterns. UV–vis spectra reveal that PANI-modified TiO2 composites show stronger absorption than neat TiO2 under the whole range of visible light. The resulting PANI-modified TiO2 composites exhibit significantly higher photocatalytic activity than that of neat TiO2 on degradation of phenol aqueous solution under visible light irradiation (λ ≥ 400 nm). An optimum of the synergetic effect is found for an initial molar ratio of aniline to TiO2 equal to 1/100.
The inactivation of model microbes in aqueous matrix by visible light photocatalysis as mediated by ZnO nanorods was investigated. ZnO nanorods were grown on glass substrate following a hydrothermal route and employed in the inactivation of gram-negative Escherichia coli and gram-positive Bacillus subtilis in MilliQ water. The concentration of Zn(2+) ions in the aqueous matrix, bacterial cell membrane damage, and DNA degradation at post-exposure were also studied. The inactivation efficiencies for both organisms under light conditions were about two times higher than under dark conditions across the cell concentrations assayed. Anomalies in supernatant Zn(2+) concentration were observed under both conditions as compared to control treatments, while cell membrane damage and DNA degradation were observed only under light conditions. Inactivation under dark conditions was hence attributed to the bactericidal effect of Zn(2+) ions, while inactivation under light conditions was due to the combined effects of Zn2+ ions and photocatalytically mediated electron injection. The reduction of pathogenic bacterial densities by the photocatalytically active ZnO nanorods in the presence of visible light implies potential ex situ application in water decontamination at ambient conditions under sunlight.
Objective
To determine whether laser light can improve healing of skin wounds by killing wound bacteria while simultaneously accelerating host tissue activity.Materials and Methods
Wounds on the rat dorsum were irradiated or sham-irradiated three times weekly from days 1 to 19 using 635 or 808 nm diode lasers at 1 or 20 J/cm2. Wound area and bacterial growth were evaluated three times weekly. Histological analysis was performed on days 8 and 19. Immunohistochemical analysis was performed on day 19.ResultsWounds that were irradiated using 635 nm light at 1 J/cm2 healed similarly to controls. Wounds that were irradiated using 808 nm (1 and 20 J/cm2, P≤0.05) and 635 nm light (20 J/cm2, P≤0.05) were worse in wound maximum area at day 3 compared with controls. Further, 635 nm light at 20 J/cm2 delayed wound closure at day 19 (ASL = 0.0127). Bacterial colonization of wounds was altered using 635 nm (20 J/cm2, P<0.0001) and 808 nm light (1 J/cm2, P<0.0001; 20 J/cm2, P = 0.02). In particular, the presence of normal skin flora decreased (P<0.0001–0.0002) and, when using 808 nm light, the presence of S. aureus increased (P = 0.0001). There was histological evidence of advanced repair using 635 nm at 1 J/cm2 at day 8 (ASL<0.04). In contrast, markers of acute repair were increased and of late repair were decreased at day 19 using 635 nm at 20 J/cm2 (ASL<0.04).Conclusions
This study demonstrates that while clearing wounds of certain bacteria is feasible it does not necessarily translate into a healing advantage. When normal flora are disturbed, environmental organisms more readily colonize the wound surface. It is not clear when using 808 nm light whether the loss of normal flora in the wound alone is responsible for the proliferation of S. aureus or whether the light adds to the effect by stimulating S. aureus growth. Lasers Surg. Med. 41:372–381, 2009. © 2009 Wiley-Liss, Inc.