Rose Bengal-Mediated Photodynamic Therapy to Inhibit Candida albicans

Summary

The growing incidence of drug-resistant Candida albicans is a serious health issue worldwide. Antimicrobial photodynamic therapy (aPDT) may offer a strategy to fight against drug-resistant fungal infections. The present protocol describes Rose bengal-mediated aPDT efficacy on a multidrug-resistant C. albicans strain in vitro.

Abstract

Invasive Candida albicans infection is a significant opportunistic fungal infection in humans because it is one of the most common colonizers of the gut, mouth, vagina, and skin. Despite the availability of antifungal medication, the mortality rate of invasive candidiasis remains ~50%. Unfortunately, the incidence of drug-resistant C. albicans is increasing globally. Antimicrobial photodynamic therapy (aPDT) may offer an alternative or adjuvant treatment to inhibit C. albicans biofilm formation and overcome drug resistance. Rose bengal (RB)-mediated aPDT has shown effective cell killing of bacteria and C. albicans. In this study, the efficacy of RB-aPDT on multidrug-resistant C. albicans is described. A homemade green light-emitting diode (LED) light source is designed to align with the center of a well of a 96-well plate. The yeasts were incubated in the wells with different concentrations of RB and illuminated with varying fluences of green light. The killing effects were analyzed by the plate dilution method. With an optimal combination of light and RB, 3-log growth inhibition was achieved. It was concluded that RB-aPDT might potentially inhibit drug-resistant C. albicans.

Introduction

C. albicans colonizes in the gastrointestinal and genitourinary tracts of healthy individuals and can be detected as normal microbiota in about 50 percent of individuals¹. If an imbalance is created between the host and the pathogen, C. albicans is capable of invading and causing disease. The infection can range from local mucous membrane infections to multiple organ failure². In a multicenter surveillance study in the US, around half of the isolates from patients with invasive candidiasis between 2009 and 2017 is C. albicans³. Candidemia can be associated with high morb....

Protocol

1. aPDT system preparation

1. Cut four green light-emitting diodes (LEDs) from a LED strip (see Table of Materials) and align them with four wells of a 96-well plate (Figure 1).
   NOTE: The LEDs were arranged into a 4 x 3 array. The back of the LED was adhered to a heat sink to disperse heat during irradiation.
2. Measure the fluence rate¹¹ of the LED at 540 nm with a light power meter (see Table of Materials). See Supplementary Figure 1 to ensure that the fluence rate of the LED is between 510-560 nm.
3. Put an electric....

Results

Figure 1 shows the aPDT system being used in the present study. Since high temperatures may cause significant cell death, the LED array is cooled by an electric fan, and a heat sink is used during irradiation to maintain a constant temperature at 25 ± 1 °C. The heat effect can be discounted. Having an even light distribution is also an important determining factor for a successful aPDT; therefore, it is critical to align the LED light bulb to the well precisely during illumination........

Discussion

Encouraging results of clinical applications of RB-PDT for fungal keratitis have been reported recently¹⁹. The absorption peak of RB is at 450-650 nm. It is essential to determine the fluence rate of the light source for a successful aPDT. A high fluence (usually >100 J/cm²) is required to treat cancer cells, while a lower fluence is expected to treat infected lesions⁶. A high fluence means a long exposure time which may not be practical in a clinical setting.......

Materials

Name	Company	Catalog Number	Comments
1.5 mL microfuge tube	Neptune, San Diego, USA	#3745.x
5 mL round-bottom tube with cell strainer cap	Falcon, USA	#352235
96-well plate	Alpha plus, Taoyuan Hsien, Taiwan	#16196
Aluminum foil	sunmei, Tainan, Taiwan
Aluminum heat sink	Nanyi electronics Co., Ltd., Tainan, Taiwan	BK-T220-0051-01
Centrifuge	Eppendorf, UK	5415R	disperses heat from the LED array
Graph pad prism software	GraphPad 8.0, San Diego, California, USA		graphing and statistics software
Green light emitting diode (LED) strip	Nanyi electronics Co., Ltd., Tainan, Taiwan	2835
Incubator	Yihder, Taipei, Taiwan	LM-570D (R)	Emission peak wavelength: 525 nm, Viewing angle: 150°; originated from https://www.aliva.com.tw/product.php?id=63
Light power meter	Ophir, Jerusalem, Israel	PD300-3W-V1-SENSOR,
Millex 0.22 μm filter	Merck, NJ, USA	SLGVR33RS
Multidrug-resistant Candida albicans	Bioresource Collection and Research CenterBioresource, Hsinchu, Taiwan	BCRC 21538/ATCC 10231	http://catalog.bcrc.firdi.org.tw/BcrcContent?bid=21538
OD600 spectrophotometer	Biochrom, London, UK	Ultrospec 10
Rose Bengal	Sigma-Aldrich, MO, USA	330000	stock concentration 40 mg/mL = 4%, prepare in PBS, stored at 4 °C
Sterilized glass tube	Sunmei Co., Ltd., Tainan, Taiwan	AK45048-16100
Yeast Extract Peptone Dextrose Medium	HIMEDIA, India	M1363