Inhibitory Effect of Disodium and Lactone Rose Bengal on the Growth of Candida spp. Isolates In Vitro

Abstract

Purpose : Effective therapies to manage infectious keratitis are limited and rose bengal photodynamic antimicrobial therapy (RB-PDAT) is an emerging adjunct treatment (AJO 2019 PMID: 31493402). However, the antifungal activity of the photosensitizers on yeast species is unknown. We report the in vitro fungicidal effect of research (RB-1) versus clinical grade (RB-2) photosensitizers for RB-PDAT on Candida spp. Growth curve analysis was performed to assess the impact of non-irradiated rose bengal disodium (RBD) and lactone (RBL) on the metabolic activity of Candida spp. prior to photoactivation.

Methods : Candida albicans (CA, n=2) and Candida parapsilosis (CP, n=2) isolates were inoculated in tryptic soy broth (TSB) at 1.5 × 10⁷ CFU/mL then 0.1% RB-1 or 0.1% RB-2 in 0.9% sodium chloride. Aliquots were plated on Sabourad-Dextrose agar in 3 groups: 1) Control (no photosensitizer/no irradiation); 2) Photosensitizer/No Irradiation; 3) Photosensitizer/Irradiation with 525 nm green LED light for 15 minutes at 5.4 J/cm2. After 72 hours of incubation at 37 degrees C, plates were photographed. Subsequently, three groups of 8 Candida spp. isolates (CA n=3, CP n=3, standard strains n=2, ATCC 90028, ATCC 22019) were prepared at 1.5 × 10³ CFU/mL and inoculated in 1) TSB only 2) 0.01% RBD and 3) 0.01% RBL. 200μl samples were loaded onto a 96-well plate in triplicate and growth kinetics were measured on a Fluostar Omega instrument over 48 hours.

Results : Fungicidal activity (>95% elimination) was observed in all strains treated with RB-PDAT, whereas organisms that did not receive RB-PDAT grew confluent colonies with zero growth inhibition. Growth curve kinetics of Candida spp. prior to photoactivation showed no differences in ATCC CA, ATCC CP, CP-2 and CP-3 organisms in RBD. CA-2, CA-3, CA-4, and CP-1 in RBD versus RBL, showed increased growth in RBL at the stationary phase.

Conclusions : Candida spp. isolates recovered from patients treated with disodium RB were susceptible to in vitro RB-PDAT irradiation. A comparison of Candida spp. growth curves in non-irradiated RBD versus RBL showed no difference in optical density with either photosensitizer, suggesting that the solutions alone are insufficient to inhibit fungal proliferation.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.

 

Figure 1: Candida albicans (CA) and Candida parapsilosis (CP) treated with RB-PDAT in NaCl.

 

Figure 2: Growth kinetics of Candida spp. in TSB versus non-irradiated 0.01% RBD and 0.01% RBL.