Abstract
Diabetes has been reported to be a serious threat to global health with no socioeconomic or national boundaries, making it among the top 10 causes of death. Diabetic foot ulcers (DFUs) are one of the most common and severe complications of the disease. Aggressive and timeous management of bacterial infections in DFUs using multidisciplinary management approaches can often prevent morbidity and mortality from diabetes. There is a growing rise in antibiotic resistance, hence the need for alternate and combination therapies. Reports of anti-microbial activity and susceptibility to blue light (BL) on several bacterial strains highlight that bacteria are less likely to develop resistance to BL treatment, making it a viable alternative to antibiotic therapy.
Although the exact mechanism of anti-microbial effects of BL has not been fully uncovered, there have been studies that have proposed several biochemical processes resulting in cell death. The aim of this study was to determine the effect of blue laser light at 470 nm on common bacteria affecting diabetic wounds, as well as the effect on fibroblast cells. In doing so, an in vitro study was carried out to determine the optimal energy density at 470 nm for three common bacteria and for fibroblast (normal and diabetic) cells alone.
To determine the anti-bacterial effect of 470 nm, irradiation at various energy densities (0, 5, 10, 30, 55, 100 and 120 J/cm2) was performed on three common bacteria affecting diabetic wounds (Staphylococcus aureus, Pseudomonas aeruginosa, and Streptococcus pyogenes). Effects on bacteria were determined 24 h post-irradiation via colony counting and the BD™ Cell Viability Kit. BJ-5ta cells were utilized in four different cell models (normal, normal wounded, diabetic, and diabetic wounded). Dose response curves (at various energy densities of 5, 10, 30, 55, 100 and 120 J/cm2) were performed on irradiated cell models 24 h post-irradiation to determine cellular morphology, migration rate, viability, and cytotoxicity. An optimum energy density of 10 J/cm2 had been determined from the dose response curve. Thereafter, further assays were done on all cell models to
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determine cellular proliferation, apoptosis, the generation of reactive oxygen species (ROS), and the Live/Dead assay at 24 and 48 h post-irradiation.
Results have highlighted that a single dose of BL (470 nm) significantly decreased colony counts of S. aureus (at 100 and 120 J/cm2) and S. pyogenes (at 10 J/cm2), 24 h post-irradiation. There was also a decrease in viable S. aureus (at 10, 30, and 120 J/cm2) and S. pyogenes (at 5, 10, 30, 55, and 100 J/cm2). P. aeruginosa on the other hand demonstrated stimulatory effects post-irradiation, even at 100 and 120 J/cm2. In the cell models, a decrease in cellular viability, ATP production, and migration rates, and an increase in cellular toxicity (increased LDH) was identified at 100 and 120 J/cm2. Migration rates were higher in the DW cell model as compared to the NW cell model. Further investigations using an energy density of 10 J/cm2, at 24 and 48 h post-irradiation, revealed that the DW cell model demonstrated a significant increase in the percentage of S phase (DNA synthesis) cells, indicating active cell proliferation. Oxidative stress (ROS) in the D and DW models, 48 h post-irradiation (10 J/cm2), showed a significant decrease in oxidative stress. Fluorescent microscopy also revealed that the majority of cells were viable at 24 h and 48 h post-irradiation (10 J/cm2).
In conclusion, a single-dose of BL (470 nm) at 10 J/cm2 is able to inhibit S. aureus and S. pyogenes, thereby providing an anti-microbial effect, while still being able to achieve human fibroblast preservation and proliferation. The in vitro nature of these types of studies may limit the ability to generalise to real life clinical scenarios, however, the results have highlighted the possibility of the implementation of further therapeutic interventions with clinical relevance for bacterial eradication in the fight to limit the severity of diabetic foot complications.