Photodynamic therapy to treat Methicillin-Resistant Staphylococcus Aureus (MRSA) keratitis: An in vitro study

Abstract

 

Purpose

 

To assess the in vitro efficacy of rose bengal (RB) and riboflavin (Ribo) mediated photodynamic therapy (PDT) for the inhibition of a methicillin-resistant Staphylococcus aureus (MRSA) strain.

 

Methods

 

A MRSA type II strain was isolated from the corneal scraping of a patient with confirmed bacterial keratitis. Twenty-four hours prior to experimentation, a parent culture was plated on nutrient agar. Next, a culture of MRSA was transferred into tryptic soy broth and adjusted to a concentration of 1.5x10⁸ colony forming units per mL (cfu/mL). The MRSA suspension was diluted to a concentration of 1.5x10⁷cfu/mL with the appropriate solution and 1mL aliquots were inoculated in triplicate onto nutrient agar plates. The eight groups were: (1) Control (high purity water) (2) UV-A irradiation (3) 0.1% Ribo (4) 0.1% Ribo + UV-A (5) 0.1% RB (6) 0.1% RB + 518nm light (7) 0.03% RB (8) 0.03% RB + 518nm light. All experiments were performed in minimal lighting conditions (4 lux) except for irradiation test plates. The UV-A and green lights are custom built LED sources. The UV-A light, activate Ribo, has a central wavelength of 375nm and an irradiance of 2.91mW/cm² over a 13.8cm² surface. The 518nm light, activate RB, has a central wavelength of 518nm and an irradiance of 2.2mW/cm² over a 28.3cm² surface. Plates were either exposed to UV-A (Ribo) or 518nm (RB) irradiation for 20 minutes. All plates were immediately placed upside down, wrapped in foil, and placed in an incubator at 30°C. Plates were photographed every 24 hours for 6 days.

 

Results

 

Riboflavin without irradiation did not inhibit MSRA growth; however in Ribo with irradiation it did inhibit MRSA growth. 0.1% RB with and without irradiation inhibited the growth of the MRSA. 0.03% RB inhibited MRSA growth with irradiation but did not inhibit MRSA growth in the non-irradiated group. Inhibition in all photosensitizer groups occurred as soon as 24 hours after irradiation.

 

Conclusions

 

Rose Bengal strips of 1.0% concentration are clinically used to detect epithelial defects. Our study demonstrates MRSA can be inhibited with 0.1% RB without irradiation. The RB will be activated even when the patient is exposed to ambient light levels in daily activities. PDT could be an excellent adjunct treatment for MRSA keratitis as it provides a different mechanism of inhibition.  

 

Results of 0.1% Ribo and 0.1% RB

 

 

Results of 0.03% Rose Bengal PDT at 72 hours