Photochemical antimicrobial therapy for keratitis with an improved riboflavin conjugate (BPerox) for highly resistant bacteria in vitro

Abstract

Purpose : The combination of riboflavin (RF) and ultraviolet light A (UVA) is an antimicrobial strategy centered on electron transfer and generation of reactive oxygen. Hydrogen peroxide acts as an oxidizing and a reducing agent that could maximize RF redox potential. The purpose of this study is to evaluate the bactericidal efficacy of BPerox, a novel light-sensitive phototherapy molecule.

Methods : Carbapenemase producing Klebsiella pneumonia (CPKP), fluoroquinolones-resistant Acinetobacter baumannii (FRAB) and multidrug-resistant Escherichia coli (MDREC) (10⁷ CFU/ml) were exposed in 12-well culture plates to BPerox, a light sensitive compound containing RF + hydrogen peroxide 0.004%. Two treatment plans according to the dose were established: A) BPerox 0.1% - 0.004% photoactivated by 3 mW/cm² for 30 min and B) BPerox 0.5% - 0.004% photoactivated by 10 mW/cm² UVA for 10 min, both at a wavelength of 365 nm through an 8-mm optic aperture. In addition, wild type Pseudomonas aeruginosa (PA) (10⁵ CFU/ml) biofilms were grown on soft hydrogel contact lenses, and after 12-h incubation, planktonic cells were removed by three washes in phosphate buffered saline (PBS). BPerox-UVA regimens A and B were administered and sonication was applied after 12-h incubation to separate the biofilm from the lenses surface. For all bacteria, serially diluted samples were plated and incubated overnight to enumerate cell survival (CFU/ml). Controls and triplicate values were obtained. Data was analyzed using unpaired t-test.

Results : BPerox-UVA treatment plans produced complete bacterial sterilization (0 CFU/ml) on CPKP, FRAB and MDREC. Treatments A and B for RF + UVA and hydrogen peroxide +UVA resulted in at least 10⁶ CFU/ml (p ≤ 0.0005). The untreated sample and single treatments with BPerox, RF, UVA and hydrogen peroxide resulted in at least 10⁸ CFU/ml (p ≤ 0.0001). BPerox-UVA photoactivation consistently killed all (0 cell/ml) PA biofilms by using treatment doses A and B, and controls showed consistently more bacteria 10⁶-10⁹ CFU/ml (p ≤ 0.0004).

Conclusions : BPerox photochemical therapy exhibited 100% killing activity against highly resistant bacteria in vitro and was capable of disrupting the bacterial biofilm architecture.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.