Blue light absorbing pigment in Streptococcus agalactiae does not potentiate the antimicrobial effect of pulsed 450 nm light.

INTRODUCTION: Recently, it was shown that Group B Streptococcus (GBS) COH1 strain, which has granadaene-an endogenous chromophore known to absorb blue light-is not susceptible to 450 nm pulsed blue light (PBL) inactivation unless the bacterium is co-cultured with exogenous porphyrin. PURPOSE: To confirm or refute the finding, we studied the effect of blue light on NCTC, another strain of GBS with more granadaene than COH1, to determine if the abundance of granadaene-and by implication more absorption of blue light-fosters GBS susceptibility to PBL. METHODS: We irradiated cultures of the bacterium with or without protoporphyrin, coproporphyrin, flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), nicotinamide adenine dinucleotide (NAD) or NADH. After 24-h incubation, bacterial colonies were enumerated, log10 CFU/mL computed, and descriptive and inferential data analyzed and compared. RESULTS: (1) The rich amount of granadaene in NCTC did not enhance its susceptibility to antimicrobial pulsed blue light (PBL). (2) Adding exogenous porphyrin fostered NCTC susceptibility to irradiation, resulting in 100% bacterial suppression. (3) Exogenous FMN or FAD, which strongly absorb 450 nm light, did not promote the antimicrobial effect of PBL, neither did exogenous NAD or NADH, two weak blue light-absorbing photosensitizers. CONCLUSION: These results strengthen our previous assertion that an endogenous chromophore with the capacity to absorb and transform light energy into a biochemical process that engenders bacterial cell death, is essential for 450 nm PBL to suppress GBS.

The viability of human cells irradiated with 470-nm light at various radiant energies in vitro.

Blue light is known to be antimicrobial, but its effect on normal cutaneous and subcutaneous cells remains unclear. Therefore, we studied the effect of 470-nm light on the viability of adult and neonatal human dermal fibroblasts, Jurkat T-cells, and THP-1 monocytes in vitro. Each culture was irradiated with 0, 3, 55, or 110 J/cm2 of 470-nm light and subjected to trypan blue assay to ascertain viability. Further, MTT, neutral red, and fluorescence assays of fibroblasts were performed, and cell morphology visualized using bright field and fluorescence microscopy. At each dose and in each of the four cell lines, there was no significant difference in cell concentration between irradiated and non-irradiated cultures, even though irradiation with 55 J/cm2 or 110 J/cm2 slightly decreased cell count. Light microscopy showed progressive morphological changes in the fibroblasts as energy fluence increased from 55 to 110 J/cm2. Irradiation at 3 J/cm2 produced a slight but non-significant increase in the viability of Jurkat T-cells and THP-1 monocytes. In contrast, at 110 J/cm2 radiant exposure, irradiation slightly decreased the viability of all four cells. While 3 J/cm2 appears stimulatory, our finding that 110 J/cm2 produces a slight decrease in viability and engenders morphological changes in fibroblasts, suggesting that such high doses should be avoided in blue light treatments.

The importance of porphyrins in blue light suppression of Streptococcus agalactiae.

It is well documented that blue light absorption by bacterial chromophores triggers downstream production of reactive oxygen species (ROS), which in turn results in bacterial cell death. To elucidate the importance of chromophores in the bactericidal effect of blue light, and to determine whether blue light absorption per se or the presence of porphyrins known to engender ROS is crucial in blue light treatment, we studied the effect of 450 nm pulsed light on Streptococcus agalactiae, also known as Group B Streptococcus (GBS) strain COH1. GBS does not synthesize porphyrins but has a blue light-absorbing chromophore, granadaene. We irradiated planktonic cultures of GBS with or without exogenous chromophore supplementation using either protoporphyrin IX (PPIX), coproporphyrin III (CPIII), Nicotinamide adenine dinucleotide (NAD), reduced nicotinamide adenine dinucleotide (NADH), Flavin adenine dinucleotide (FAD), or Flavin mononucleotide (FMN). Quantification of surviving bacterial colonies, presented as percent survival and CFU/mL (log10), showed that (1) 450 nm blue light does not suppress the growth of GBS, even though its endogenous chromophore, granadaene, absorbs light in the 450 nm spectrum. (2) The addition of either of the two exogenous porphyrins, PPIX or CPIII, significantly suppressed GBS, indicating the importance of porphyrins in the antimicrobial action of blue light. (3) Adding exogenous FMN or FAD, two known absorbers of 450 nm light, minimally potentiated the bactericidal effect of blue light, again confirming that mere absorption of blue light by chromophores does not necessarily result in bacterial suppression. (4) Irradiation of GBS with or without NAD+ or NADH supplementation-two weak absorbers of 450 nm light-minimally suppressed GBS, indicating that a blue light-absorbing chromophore is essential for the bactericidal action of blue light. (5) Collectively, these findings show that in addition to the presence of a blue light-absorbing chromophore in bacteria, a chromophore with the right metabolic machinery and biochemical structure, capable of producing ROS, is necessary for 450 nm blue light to suppress GBS.

Light as a potential treatment for pandemic coronavirus infections: A perspective.

The recent outbreak of COVID-19, which continues to ravage communities with high death tolls and untold psychosocial and catastrophic economic consequences, is a vivid reminder of nature's capacity to defy contemporary healthcare. The pandemic calls for rapid mobilization of every potential clinical tool, including phototherapy-one of the most effective treatments used to reduce the impact of the 1918 "Spanish influenza" pandemic. This paper cites several studies showing that phototherapy has immense potential to reduce the impact of coronavirus diseases, and offers suggested ways that the healthcare industry can integrate modern light technologies in the fight against COVID-19 and other infections. The evidence shows that violet/blue (400-470 nm) light is antimicrobial against numerous bacteria, and that it accounts for Niels Ryberg Finsen's Nobel-winning treatment of tuberculosis. Further evidence shows that blue light inactivates several viruses, including the common flu coronavirus, and that in experimental animals, red and near infrared light reduce respiratory disorders, similar to those complications associated with coronavirus infection. Moreover, in patients, red light has been shown to alleviate chronic obstructive lung disease and bronchial asthma. These findings call for urgent efforts to further explore the clinical value of light, and not wait for another pandemic to serve as a reminder. The ubiquity of inexpensive light emitting lasers and light emitting diodes (LEDs), makes it relatively easy to develop safe low-cost light-based devices with the potential to reduce infections, sanitize equipment, hospital facilities, emergency care vehicles, homes, and the general environment as pilot studies have shown.

Optimizing the bactericidal effect of pulsed blue light on Propionibacterium acnes - A correlative fluorescence spectroscopy study.

Propionibacterium acnes infection is the eighth most prevalent disease, affecting 80% of people worldwide. Resistance to antibiotics has been on the rise; over 40% of acne infections now resist commonly used topical and oral anti-acnes antibiotics, making treatment difficult. In our effort to refine blue light as an alternative safe clinically effective treatment, we determined if 100% bacterial suppression is attainable at ultralow irradiances and radiant energies, and explored the relationship between bacterial suppression and fluorescence during treatment. P. acnes were irradiated in vitro repeatedly three times per day at 3- or 4-hour intervals over three or more days, using 3 or 5 J/cm2 radiant energy of 450 nm pulsed blue light (PBL) at irradiances as low as 2 mW/cm2. In another series of experiments, we measured changes in P. acnes fluorescence as bacteria were repeatedly irradiated at various radiant exposures over three to four days. Our results showed that (1) 33% PBL, applied three times per day at 3-hour intervals each day over a three-day period at 2 mW/cm2 irradiance and 5 J/cm2 radiant exposure, resulted in100% bacterial suppression (7 log10 reduction), (2) the absorbed 450 nm light caused P. acnes to fluoresce predominantly in the red spectrum, with the fluorescence diminishing correlatively as treatment was repeated at 3-hour intervals and rising significantly during long periods of no treatment, and (3) treatment at 3-hour intervals gave better results than treatment at 4-hour intervals.

Pulsed 450 nm blue light significantly inactivates Propionibacterium acnes more than continuous wave blue light.

Infection with Propionibacterium acnes is ubiquitous, and drug resistant strains have been on the rise as the use of pharmaceutical antimicrobials continues to engender the emergence of further resistant strains. In previous studies, we showed that treatment with blue light serves as an alternative to pharmaceutical intervention. As a part of our ongoing effort to improve the antimicrobial efficacy of blue light, we studied the effect of pulsed 450 nm light on P. acnes in vitro and compared two pulsed rates with continuous wave irradiation. We irradiated cultures of P. acnes at various irradiances and radiant energies either singly or repeatedly at various time intervals, using printed micro-LEDs, with the goal of finding the lowest combination of irradiance and radiant energy that would yield 100% bacterial suppression. Our results show that treatment with 33% pulsed light gave the best result compared to 20% pulsed wave or continuous wave. Timing irradiation to coincide with the replication cycle of P. acnes produced a significantly better antimicrobial effect. Furthermore, repeated irradiation at 3-h or 4-h interval enabled significant bacterial suppression even at lower irradiances; thus, making single irradiation at high irradiances unnecessary. Moreover, combining repeated irradiation with appropriate duration of treatment and 33% irradiation pulse rate gave optimal 100% [7 log10] bacterial suppression.

Pulsed 450 nm blue light suppresses MRSA and Propionibacterium acnes in planktonic cultures and bacterial biofilms.

In our recent study, we showed that pulsed blue light (PBL) suppresses the growth of Propionibacterium acnes more than continuous wave (CW) blue light in vitro, but it is not known that other bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA), respond similarly to PBL. The high potency of PBL relative to CW blue light makes it a suitable antimicrobial for suppressing bacterial growth in biofilms as well. Therefore, we determined if MRSA-a deadly bacterium of global concern-is susceptible to 450 nm PBL irradiation in vitro, and ascertained whether the bactericidal effect of PBL on planktonic P. acnes culture can be replicated in biofilms of P. acnes and MRSA. In three series of experiments, we irradiated P. acnes and MRSA respectively, either in planktonic cultures, forming biofilms or formed biofilms. Compared to controls, the results showed 100% bacterial suppression in planktonic cultures of MRSA irradiated with 3 mW/cm2 irradiance and 7.6 J/cm2 radiant exposure three times at 30-minute intervals, and also in P. acnes cultures irradiated with 2 mW/cm2 irradiance 5 J/cm2 radiant exposure thrice daily during each of 3 days. Irradiation of biofilms with the same irradiances and radiant exposures that gave 100% bacterial suppression in planktonic cultures resulted in disruption and disassembly of the architecture of MRSA and P. acnes biofilms, more so in forming biofilms than formed biofilms. The antimicrobial effect on each bacterium was minimal in forming biofilms, and even less in formed biofilms. Increasing radiant exposure slightly from 7.6 J/cm2 to 10.8 J/cm2 without changing any other parameter, yielded more disruption of the biofilm and fewer live MRSA and P. acnes, suggesting that 100% bacterial suppression is possible with further refinement of the protocol. In both planktonic cultures and biofilms, PBL suppressed MRSA more than P. acnes.

Blue light does not impair wound healing in vitro.

Irradiation with red or near infrared light promotes tissue repair, while treatment with blue light is known to be antimicrobial. Consequently, it is thought that infected wounds could benefit more from combined blue and red/infrared light therapy; but there is a concern that blue light may slow healing. We investigated the effect of blue 470nm light on wound healing, in terms of wound closure, total protein and collagen synthesis, growth factor and cytokines expression, in an in vitro scratch wound model. Human dermal fibroblasts were cultured for 48h until confluent. Then a linear scratch wound was created and irradiated with 3, 5, 10 or 55J/cm(2). Control plates were not irradiated. Following 24h of incubation, cells were fixed and stained for migration and fluorescence analyses and the supernatant collected for quantification of total protein, hydroxyproline, bFGF, IL-6 and IL-10. The results showed that wound closure was similar for groups treated with 3, 5 and 10J/cm(2), with a slight improvement with the 5J/cm(2) dose, and slower closure with 55J/cm(2) p<0.001). Total protein concentration increased after irradiation with 3, 5 and 10J/cm(2), reaching statistical significance at 5J/cm(2) compared to control (p<0.0001). However, hydroxyproline levels did not differ between groups. Similarly, bFGF and IL-10 concentrations did not differ between groups, but IL-6 concentration decreased progressively as fluence increased (p<0.0001). Fluorescence analysis showed viable cells regardless of irradiation fluence. We conclude that irradiation with blue light at low fluence does not impair in vitro wound healing. The significant decrease in IL-6 suggests that 470nm light is anti-inflammatory.

The relative antimicrobial effect of blue 405 nm LED and blue 405 nm laser on methicillin-resistant Staphylococcus aureus in vitro.

It has long been argued that light from a laser diode is superior to light from a light-emitting diode (LED) in terms of its effect on biological tissues. In order to shed light on this ongoing debate, we compared the antimicrobial effect of light emitted from a 405-nm LED with that of a 405-nm laser on methicillin-resistant Staphylococcus aureus (MRSA) at comparable fluences. We cultured 5 × 10(6) CFU/ml MRSA on tryptic soy agar and then irradiated culture plates once, twice, or thrice with either LED or laser light using 40, 54, 81, or 121 J/cm(2) fluence at 15-, 30-, or 240-min time interval between irradiation. Cultures were incubated immediately after irradiation at 37 °C for 24 h before imaging and counting remnant bacterial colonies. Regardless of the device used, LED or laser, irradiation at each fluence resulted in statistically significant bacterial growth suppression compared to non-irradiated controls (p < 0.0001). The antimicrobial effect of both light sources, LED and laser, was not statistically different at each fluence in 35 of the 36 experimental trials. Bacterial growth suppression achieved with either source of light increased with repeated irradiation, particularly at the 15- or 30-min treatment time interval. Thus, we conclude that the antimicrobial effect of 405-nm laser and 405-nm LED on MRSA is similar; neither has a superior antimicrobial effect when compared to the other.

The bactericidal effect of 470-nm light and hyperbaric oxygen on methicillin-resistant Staphylococcus aureus (MRSA).

It has been shown that, in vitro, hyperbaric oxygen (HBO) suppresses 28 % bacterial growth, while 470-nm blue light alone suppresses up to 92 % methicillin-resistant Staphylococcus aureus (MRSA) in one application in vitro. Therefore, we determined if combined 470-nm light (55 J/cm(2)) and HBO will yield 100 % bacterial suppression in experimental simulation of mild, moderate or severe MRSA infection. We cultured MRSA at 3 × 10(6), 5 × 10(6), 7 × 10(6), 8 × 10(6), or 12 × 10(6) CFU/ml and treated each concentration in four groups as follows: (1) control (no treatment) (2) photo-irradiation only, (3) photo-irradiation then HBO, (4) HBO only, and (5) HBO then photo-irradiation. Bacteria colonies were then quantified. The results showed that at each bacterial concentration, HBO alone was significantly less effective in suppressing MRSA than photo-irradiation or combined HBO and photo-irradiation (p < 0.0001). Similarly, at no bacterial concentration did combined HBO and 470-nm light treatment yield a statistically better result than 470-nm light alone (p > 0.05), neither did HBO treatment either before or after irradiation make a difference. Furthermore, at no bacterial concentration was 100 % MRSA suppression achieved. Indeed, the maximum bacterial suppression attained was in the mild infection model (3 × 10(6) CFU/ml), with blue light producing 97.3 ± 0.2 % suppression and HBO + 55 J/cm(2) yielding 97.5 ± 2.5 % suppression. We conclude that (1) HBO and 470-nm light individually suppress MRSA growth; (2) 470-nm blue light is more effective in suppressing MRSA than HBO; and (3) HBO did not act synergistically to heighten the bactericidal effect of 470-nm light.