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2025 OMIG Abstract

Strain-Specific Impact of Melanin Pathway Inhibition on RB-PDAT Efficacy in Aspergillus Keratitis Isolates

Heather Durkee¹, Suraj Paudyal¹, Sebastian Sanchez Campo¹, Leonardo Gonzalez¹, Brandon Chou¹, Salomon Merikansky^1,2, Juan Carlos Navia¹, Mariela C. Aguilar¹, Harry W. Flynn, Jr^1-3, Jean-Marie Parel^1,3, Guillermo Amescua^1-3, Darlene Miller<sup>1,2

1</sup>Ophthalmic Biophysics Center, Department of Ophthalmology, Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, Miami, Florida; ²Ocular Microbiology Laboratory, Department of Ophthalmology, Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, Miami, Florida; ³Anne Bates Leach Eye Center, Department of Ophthalmology, Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, Miami, Florida

Purpose: Aspergillus keratitis is often associated with poor clinical outcomes and remains difficult to treat, particularly in regions like India where it is common. Alternative therapies such as Rose Bengal photodynamic antimicrobial therapy (RB-PDAT) have shown promise in fungal keratitis but appear less effective against Aspergillus species. This limited efficacy could be due to the melanin of the Aspergillus which protects against oxidative damage. This study aimed to evaluate whether inhibition of the melanin in clinical Aspergillus isolates could enhance the efficacy of RB-PDAT.

Methods: Agar plates to inhibit the two main melanin pathways were prepared according to protocol described by Pal A., 2014. In brief, either kojic acid (DOPA pathway inhibitor) or tricyclazole (DHN pathway inhibitor) were added to potato dextrose agar (PDA) base for a final concentration of 100µg/mL. Four clinical Aspergillus isolates, A. flavus, A. fumigatus, A. glaucus, and A. niger, were cultured twice to reach optimal growth phase and prepared in suspension of 1.5x10E6 CFU/mL. Spore suspensions were mixed with either water (control) or with 0.1% rose bengal solution and rested for 30 minutes. Solutions were aliquoted onto agar plates (250µL) and exposed to either dark or green light (6mW/cm², 5.4 J/cm²). Plates were incubated at 35°C for 72 hours and plates were photographed to assess fungal growth.

Results: The four Aspergillus isolates were most impacted by the DHN pathway inhibitor as evidenced in the reduction in green pigment as compared to the PDA control plates. A. fumigatus had the most significant loss of pigment of the four strains tested. The only strain to show increased inhibition with RB-PDAT with a melanin pathway disrupted was the A. glaucus strain in the presence of the DHN pathway inhibitor (tricyclazole). The RB coloration was also reduced in the tricyclazole plates with A. flavus, suggesting an interaction between RB and melanin pathway.

Conclusions: Inhibiting the DHN melanin pathway reduced pigmentation in Aspergillus isolates, especially A. fumigatus. However, only A. glaucus showed increased RB-PDAT susceptibility, suggesting that melanin may affect RB-PDAT efficacy in a strain-specific manner. Future experimentation will explore higher concentrations of tricyclazole and Kojic acid.