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 morbidity rates, mortality, prolonged hospital stay⁴. US Centers of Disease Control and Prevention reported that about 7% of all Candida blood samples tested are resistant to the antifungal drug fluconazole⁵. The emergence of drug-resistant Candida species raises the concern to develop an alternative or adjuvant therapy to antimycotic agents.

Antimicrobial photodynamic therapy (aPDT) involves activating a specific photosensitizer (PS) with light at the peak absorption wavelength of the PS⁶. After excitation, the excited PS transfers its energy or electrons to the nearby oxygen molecules and returns to the ground state. During this process, reactive oxygen species and singlet oxygen are formed and cause cell damage. aPDT has been widely used to kill microorganisms since the 1990s⁷. One of the benefits of aPDT is that multiple organelles are damaged in a cell by singlet oxygen and/or reactive oxygen species (ROS) during irradiation; thus, resistance to aPDT has not been found till today. Moreover, a recent study reported that the bacteria that survived after aPDT became more sensitive to antibiotics⁸.

The light sources used in aPDT include lasers, metal halogen lamps with filters, near-infrared light, and light-emitting diode (LED)^9,10,11,12. The laser provides a high light power, usually larger than 0.5 W/cm², that allows for the delivery of a high light dose in a very short time. It has been widely used in cases where a longer treatment time is inconvenient such as aPDT for oral infections. The drawback of a laser is that its spot size of illumination is small, ranging from a few hundred micrometers to 10 mm with a diffuser. Moreover, laser equipment is expensive and needs specific training to operate. On the other hand, the irradiation area of a metal halogen lamp with filters is relatively larger¹³. However, the lamp is too hefty and expensive. LED light sources have become mainstream of aPDT in the dermatologic field because it is small and less expensive. The irradiation area can be relatively large with an array arrangement of the LED light bulb. The whole face can be illuminated at the same time⁹. Nevertheless, most, if not all, LED light sources available today are designed for clinical use. It may not be suitable for experiments in a lab because it is space-occupying and expensive. We developed an inexpensive LED array that is very small and can be cut and assembled from a LED strip. The LEDs can be fitted into different arrangements for different experimental designs. Different conditions of aPDT can be completed in a 96-well plate or even a 384-well plate in one experiment.

Rose bengal (RB) is a colored dye widely used to enhance visualization of corneal damages in human eyes¹⁴. RB-mediated aPDT has shown killing effects on Staphylococcus aureus, Escherichia coli, and C. albicans with roughly comparable efficiency to that of Toluidine blue O¹⁵. This study demonstrates a method to validate the effect of RB-aPDT on multidrug-resistant C. albicans.

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 fan alongside the plate during irradiation to maintain the constant temperature (25 ± 1 °C)¹¹. See Supplementary Figure 2 to ensure that the constant temperature of the medium is maintained during irradiation.

2. Culturing of the yeast form of  C. albicans

NOTE: A multidrug-resistant C. albicans (BCRC 21538/ATCC 10231), resistant to most antifungals, including fluconazole, is used for the experiments¹⁶.

1. Determine the antimycotic drug sensitivity with a disk diffusion method following previously published report¹⁷.
2. Grow C. albicans in yeast, hyphae, and pseudohyphae forms depending on the micro-ecological environments¹⁸.
   ​NOTE: The hyphae and pseudohyphae forms are difficult for an accurate calculation. The yeast form can be calculated precisely under a microscope or with flow cytometry. The temperature during cell growth determines its morphology. At room temperature (25 °C), nearly all cells are yeast form. A 4 h short incubation of C. albicans at 30 °C did not affect its yeast morphology.

3. aPDT on planktonic  C. albicans

1. Isolate a single colony of C. albicans from an agar plate with a sterile loop and add it to a 3 mL yeast extract peptone dextrose (YPD) medium (see Table of Materials) in a sterilized glass tube.
   1. Incubate the tube at 25 ± 1 °C overnight (14-16 h) in an incubator with a rotation speed of 155 rpm to expand C. albicans and maintain the fungus in yeast form for accurate quantification.
2. Dilute the overnight culture with medium to an OD₆₀₀ value of around 0.5 at 30° C and rotate at a speed of 155 rpm for 4 h to achieve a log growth phase of C. albicans.
3. Dilute the log phase culture again with fresh YPD medium to an OD₆₀₀ value of 0.65 (around 1 x 10⁷ colony forming units, CFU/mL). Confirm the final concentration by serial dilution method on an agar plate⁸.
4. Prepare a stock solution (4%) of Rose bengal (RB) by dissolving the powder in 1x PBS. Filter and sterilize it with a 0.22 µm filter and store it at 4 °C in the dark. The final working concentration of RB is 0.2%.
5. Add 111 µL of 2% RB to 1 mL of log-phase C. albicans in a 1.5 mL microcentrifuge tube and co-culture at different time points (0, 15, and 30 min) at room temperature to understand the absorption of RB in the cells (Figure 2).
6. Wash the co-culture three times with 1 mL of 1x PBS with centrifugation at 16,100 x g for 2.5 min at room temperature.
   NOTE: aPDT contains four different conditions: absolute control (no light exposure, no RB), dark control (no light but incubates with RB), light control (exposes to light without RB), aPDT (exposes to light in the presence of RB).
7. Resuspend the C. albicans in 1 mL of 1x PBS and allocate them into three different wells in a 96 well plate for each condition. Align the wells with the LED array after washing.
8. In light exposed groups, turn on the electric fan and light.
   ​NOTE: A different fluence (J/cm²) can be achieved by exposing the wells to varying time periods. For example, a 16.7 min light exposure comes to 10 J/cm² with a 10 mW/cm² LED light bulb.
9. After irradiation, add 20 µL of the co-culture solution from one well to a 1.5 mL centrifuge tube containing 180 µL of 1x PBS to prepare a 10x dilution. Further, dilute ten times, subsequently following the same method.
10. Drop three drops of 20 µL of each serial dilution onto one quadrant of a YPD agar plate to achieve countable colonies the plate. Calculate the CFU/mL by multiplying the colonies with the dilution factors³.

4. Statistical analysis

1. Analyze the collected data using a graphing and statistics software (see Table of Materials).
2. Depict data by mean ± standard error of the mean. Perform a two-way ANOVA analysis of variance⁸ to evaluate significant differences between the different test conditions.
3. Perform Tukey's multiple comparison tests for pairwise comparisons⁸. For each different treatment, perform at least three independent experiments. Consider p-value < 0.05 statistically significant.

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