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Anders Backman, Raymond Goodrich, Karim Makdoumi; Blue Light /Riboflavin-mediated Elimination of Methicillin-resistant Staphylococcus aureus, Using an In Vitro Model of Keratitis Treatment.. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):268.
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© ARVO (1962-2015); The Authors (2016-present)
Antibiotic resistant bacteria could cause keratitis treatment failures. Blue light has better properties than the used 365nm light, used in collagen crosslinking (CXL) and suggested therapy of keratitis. We wanted to investigate the in vitro effect of two different Blue lights/Riboflavin-mediated photo-oxidative stress on a pathogenic bacteria with antibiotic resistance properties in a crosslinking (CXL) treatment model.
Staphylococcus aureus(MRSA)(CCUG41586/ATCC43300, Methicillin resistant), were cultured and suspended in PBS to around 4 x 105/mL. The suspension (10 µL) was mixed with 50 µL of riboflavin (RF)(Sigma) solution in RPMI (GIBCO) to 0.01% RF. This was pipetted (15 µL) onto 7 mm wells on microscope slides (CEL-30-2325BLHTC, Thermo Scientific), forming a 0.4 mm layer, exposed 9-10 min to 28.4J or 5.4J of blue light; 412 nm (TRIA-Beauty lamp Mod: SPBL, CA,USA) or 450 nm ( prototype lamp ,TERUMO BCT, Denver, Colorado). The effect on a deeper fluid layer (1.14mm) was tested in a 40 mL well similarly. Samples were withdrawn (5 µL) from the wells and diluted in PBS and spread on Blood agar plates, incubated, and the CFU- counts were calculated. The mean results (n=8) were compared to simultaneous controls with and without light treatment(T-test).
MRSA was efficiently eliminated similarly by 412nm or 450nm+ RF at high dose (99%/98%). A lower dose of 412nm/450nm +RF reduced MRSA much less significantly (55%/44%)(P˂0.0002). The effect of light only treatment reduced the CFU equally efficient at high dose (79 and 81%). A low dose of light only (412 or 450nm) had no significant effect (P= 0.1). Blue light of 412nm was effective on 1.14mm layers and reduced bacteria at high dose+/-RF (93%/83%) and low dose +RF (67%)(P˂0.00001).
MRSA could be reduced almost completely by exposure to Blue light (412 or 450nm) at high dose (28J) +/- RF, in this treatment model. The safe CXL dose of 5.4J (365nm) applied with 412nm +RF (10 min) reduced bacteria by 55%-67%. A longer exposure (CXL =30 min.) and lower irradiation setting would probably increase the efficacy(x 1.5) as seen previously. This could perhaps also be a possible way to improve treatment of keratitis in cases involving antibiotic resistant bacteria, perhaps with superior effect compared to standard CXL in deeper infections. Further studies are needed to evaluate this.
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