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

Abstract

Purpose : 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.

Methods : A MRSA Strain USA300, was grown overnight at 37^oC 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 37^oC. 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.

Results : 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).

Conclusions : 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.