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2020 Harry Hirsch Leiter Award Recipient:

 The Efficacy of Riboflavin Versus Rose Bengal Photodynamic Antimicrobial Therapy on South Florida Nocardia Keratitis Isolates
Ethan Adre, BS1,2, Heather Durkee, MS2, Alejandro ArboledaMD, MS2, Jorge MaestrePhD3,
Guillermo Amescua, MD2,4,5, Darlene Miller, DHSc, MPH3-5, Jean-Marie Parel, PhD2,4,5
1University of Miami Miller School of Medicine; 2Ophthalmic Biophysics Center;
3Ocular Microbiology Laboratory; 4Anne Bates Leach Eye Center; 5Bascom Palmer Eye Institute, Miami, FL

Purpose: Novel approaches to cure Nocardia keratitis with photodynamic antimicrobial therapy (PDAT) have not yet been examined. PDAT utilizes light-activated reactive oxygen species to initiate cell death. Riboflavin is one of the most established conduits for PDAT and the efficacy of rose bengal (RB) as a photosensitizer has also been established in treating other types of bacterial keratitis. This study seeks to compare the in vitro growth inhibition of Nocardia keratitis isolates using riboflavin and RB PDAT.

Methods: Three Nocardia strains (Nocardia amikacinitolerans, Nocardia farcinica and Nocardia asteroides) were isolated from patients with confirmed NK. Each isolate was cultured and prepared in suspension to a concentration of 1.5 x 108 CFU/mL using a 0.5 McFarland standard. In minimal lighting conditions of <1 Lux, 0.1% solutions of RB and riboflavin were made by mixing 1mg of each photosensitizer per 1ml of high-purity water. The bacterial suspensions were then mixed with either water (control and irradiation only groups), RB, or riboflavin solutions (photosensitizer dark, PDAT) groups for a final bacterial concentration of 1.5 x 107 CFU/mL. Each condition was tested in triplicate and 1 mL aliquots of each respective suspension were plated in 5% sheep’s blood agar. Using two custom PDAT light sources of with power of 6mW/cm2, RB and riboflavin PDAT plates were irradiated with a 525nm or 375nm light source for 15-minutes for a total fluence of 5.4 J/cm2. All plates were incubated in a 35ºC non-CO2 incubator for 96 hours and photographed. Nocardia growth was evaluated using Labview-based software.

Results: All strains of Nocardia exposed to either 375 or 525 nm light alone demonstrated no inhibition. All strains of Nocardia mixed with 0.1% riboflavin in the dark without radiation demonstrated no inhibition. All strains of Nocardia mixed with 0.1% RB in the dark without radiation demonstrated no inhibition. All strains of Nocardia tested with 0.1% riboflavin mixed in the dark and irradiated for 15-minutes demonstrated no inhibition of growth. All strains of Nocardia tested with 0.1% RB in the dark and irradiated for 15-minutes demonstrated significant inhibition of growth.

Conclusion: Amongst all strains, 0.1% RB PDAT was most effective when compared with 0.1% riboflavin PDAT. The outstanding success of a novel approach to Nocardia inhibition with in vitro 0.1% RB PDAT supports its potential as a new alternative or complementary NK treatment.

Disclosure: N

Support: Edward D. and Janet K. Robson Foundation; Florida Lions Eye Bank and Beauty of Sight Foundation; Gifts from Drs. H. W. Flynn Jr, K. R. Olsen, M. E. Hildebrandt, R. Urs and A. Furtado; NIH Center Grant P30EY14801; unrestricted funds from Research to Prevent Blindness to the department of Ophthalmology; Henri and Flore Lesieur Foundation (J.-M. Parel)

 

 

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