Bactericidal ultraviolet light A (UVA) photoactivation of riboflavin potentiated by low levels of hydrogen peroxide in vitro: a potential novel therapy for methicillin sensitive and multidrug resistant-methicillin resistant Staphylococcus aureus keratitis

Abstract

Purpose : To demonstrate the antimicrobial properties of riboflavin (B2) and UVA combination enhanced by an active form of oxygen (O₂) from a low concentration of hydrogen peroxide (H₂O₂) source.

Methods : One methicillin sensitive Staphylococcus aureus (MSSA) Newman strain and five multidrug resistant-methicillin resistant Staphylococcus aureus (MDR-MRSA) strains USA300, CA409, CA127, GA656 and NY315 (10⁷ CFU/ml) were exposed to the triplet of B2 0.1%, H₂O₂ 0.004% and UVA (365 nm wavelength) at a power density of 3 mW/cm² with an 8-mm iris aperture in 12-well culture plates for 15, 20 and 30 min. Additionally, MSSA Newman and MDR-MRSA CA409 strains were exposed to a high flux −accelerated− UVA irradiance: 30 mW/cm² for 3 min and 10 mW/cm² for 10 min. B2 0.5% was also tested for the accelerated process. Photo-reactive solutions containing B2 and/or H₂O₂ 0.004% were prepared. Control groups included: B2 + UVA, B2 + H₂O₂, H₂O₂ + UVA, B2 alone, UVA alone, H₂O₂ alone and an untreated sample. The cell viability was assessed after 24 hours of incubation at 37°C by colony forming unit (CFU) enumeration in units of Log CFU/ml. Experiments were assayed three times. Student's t-test was used for statistical analysis.

Results : Both MSSA and MDR-MRSA strains showed 100% killing (0 CFU) after treatment with the triple combination of B2 0.1% + H₂O₂ 0.004% + UVA at 3 mW/cm² for at least 20 min. Only USA300 and CA127 strains did not show 100% bactericidal activity in 15 min. Treatment with B2 0.1% + UVA at 3 mW/cm² for 30 min resulted in 6.66 ± 0.13 CFU/ml for Newman (p = 0.0004), 7.90 ± 0.03 CFU/ml for USA300 (p = <0.0001), 8.50 ± 0.10 CFU/ml for CA127 (p = 0.0001), 8.46 ± 0.24 CFU/ml for GA656 (p = 0.0008) but killed completely strains CA409 and NY315. The photo-activated combination of B2 0.5% + H₂O₂ 0.004% + UVA (10 mW/cm²) for 10 min had 100% bactericidal activity on all the strains. 30 mW/cm² for 3 min was effective only on CA409.

Conclusions : The photoactivation of a solution containing B2 with low levels of H₂O₂ following specific parameters exerts a significant bactericidal activity on the strains tested through a wide spectrum of biochemical effects which are exposure time-dependent and concentration-dependent.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.