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Ashley Behrens, Rebecca Yee, Andreina Tarff, Marisol del Valle Cano, Praveena Gupta, Laura Di Meglio, William May, Arturo Casadevall, Ying Zhang; Bactericidal activity of photoactivated riboflavin, ultraviolet light A, and hydrogen peroxide against stationary phase methicillin-resistant Staphylococcus aureus (MRSA) for potential use in bacterial keratitis. Invest. Ophthalmol. Vis. Sci. 2017;58(8):3897. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Stationary phase bacteria contain persister cells, which are dormant cells that are not susceptible to current antibiotics. While the antimicrobial activity of photoactivated therapies on growing stages of bacteria has been shown to be effective, the antibacterial properties of photochemical therapies on stationary phase bacteria have not been evaluated. Here, we evaluated the bactericidal activity of UVA, riboflavin (B2), and hydrogen peroxide (H2O2) against stationary phase MRSA.
A MRSA Strain USA300, was grown overnight at 37oC with shaking at 220 rpm. In a 12-well plate, the stationary phase culture (107 CFU/ml) was exposed to a combination of UVA (365 nm wavelength) with an 8-mm aperture and a power intensity of 3 mW/cm2 for 30 minutes and a droplet (240 µl) of photo-reactive solution containing B2 (0.1%) + H2O2 (0.004%). Cell viability was assessed by performing colony forming unit (CFU) enumeration after 24h incubation at 37oC. Control groups included B2 + UVA, H2O2 + UVA, B2 + H2O2, B2 only, UVA only, H2O2 only, and no treatment. Experiments were triplicated. Student's t-test was used for statistical analysis.
Stationary phase bacteria treated with B2 + H2O2 + UVA resulted in complete killing with 0 CFU/ml detected as compared to the untreated control which harbored 5.0 ± 3.6 x 108 CFU/ml (p = 0.0004). Two-combination treatments B2 + UVA, B2 + H2O2, and H2O2 + UVA resulted in 1.8 ± 1.0 x 105 CFU/ml, 5.6 ± 2.5 x 108 CFU/ml, and 4.0 ± 1.3 x 103 CFU/ml, respectively (all p < 0.05). Single treatment with B2, UVA, and H2O2 resulted in 5.0 ± 3.4 x 108 CFU/ml, 6.0 ± 1.0 x 108 CFU/ml, and 4.6 ± 1.5 x 108 CFU/ml, respectively (p< 0.005).
Our results suggest that the treatment of B2 (0.1%) + H2O2 (0.004%) + UVA (3 mW/cm2) for 30 minutes has robust bactericidal activity against stationary phase MRSA. This novel treatment may provide means of eradicating severe corneal infections caused by persister bacteria such as those found in biofilm infections. The safety and efficacy of this new treatment is being evaluated in ongoing in-vivo studies.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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