Name: rose bengal-mediated photodynamic therapy to inhibit candida albicans
Uploaded: 2022-03-24T14:00:00
Description: Watch this Scientific Journal Video about Rose Bengal-Mediated Photodynamic Therapy to Inhibit Candida albicans at JoVE.com

This work has received funding from the Center of Applied Nanomedicine, National Cheng Kung University from the Featured Areas Research Center Program within the framework of the Higher Education Sprout Project by the Ministry of Education (MOE), and Ministry of Science and Technology, Taiwan [MOST 109-2327-B-006-005] to TW Wong. J.H. Hung acknowledges funding from National Cheng Kung University Hospital, Taiwan [NCKUH-11006018], and [MOST 110-2314-B-006-086-MY3].

1. aPDT system preparation
<ol>
	<li>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.</li>
	<li>Measure the fluence rate11 of the LED at 540 nm with a light power meter (see Table of Materi...

<ol>
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W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photodynamic+inactivation+of+methicillin-resistant+Staphylococcus+aureus+by+indocyanine+green+and+near+infrared+light.">Photodynamic inactivation of methicillin-resistant Staphylococcus aureus by indocyanine green and near infrared light.</a> Dermatologica Sinica. 36 (1), 8-15 (2018).</li><li>Stasko, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visible+blue+light+inhibits+infection+and+replication+of+SARS-CoV-2+at+doses+that+are+well-tolerated+by+human+respiratory+tissue.">Visible blue light inhibits infection and replication of SARS-CoV-2 at doses that are well-tolerated by human respiratory tissue.</a> Scientific Reports. 11 (1), 20595 (2021).</li><li>Crosbie, J., Winser, K., Collins, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+the+light+field+of+the+Waldmann+PDT+1200+lamp:+potential+for+wide-field+low+light+irradiance+aminolevulinic+acid+photodynamic+therapy.">Mapping the light field of the Waldmann PDT 1200 lamp: potential for wide-field low light irradiance aminolevulinic acid photodynamic therapy.</a> Photochemistry and Photobiology. 76 (2), 204-207 (2002).</li><li>Feenstra, R. 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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+CLSI+M44-A2+disk+diffusion+and+associated+breakpoint+testing+of+caspofungin+and+micafungin+using+a+well-characterized+panel+of+wild-type+and+fks+hot+spot+mutant+Candida+isolates.">Evaluation of CLSI M44-A2 disk diffusion and associated breakpoint testing of caspofungin and micafungin using a well-characterized panel of wild-type and fks hot spot mutant Candida isolates.</a> Antimicrobial Agents and Chemotherapy. 55 (5), 1891-1895 (2011).</li><li>Mukaremera, L., Lee, K. K., Mora-Montes, H. M., Gow, N. A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Candida+albicans+yeast,+pseudohyphal,+and+hyphal+morphogenesis+differentially+affects+immune+recognition.">Candida albicans yeast, pseudohyphal, and hyphal morphogenesis differentially affects immune recognition.</a> Frontiers in Immunology. 8, 629 (2017).</li><li>Hung, J. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+photodynamic+therapy+against+fungal+keratitis.">Recent advances in photodynamic therapy against fungal keratitis.</a> Pharmaceutics. 13 (12), 2011 (2021).</li><li>Martinez, J. 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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 &#62;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...

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.5 mL microfuge tube</td>
			<td>Neptune, San Diego, USA</td>
			<td>#3745.x</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL round-bottom tube with cell strainer cap</td>
			<td>Falcon, USA</td>
			<td>#352235</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plate</td>
			<td>Alpha plus, Taoyuan Hsien, Taiwan</td>
			<td>#16196</td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminum foil</td>
			<td>sunmei, Tainan, Taiwan</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminum heat sink</td>
			<td>Nanyi electronics Co., Ltd., Tainan, Taiwan</td>
			<td>BK-T220-0051-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf, UK</td>
			<td>5415R</td>
			<td>disperses heat from the LED array</td>
		</tr>
		<tr>
			<td>Graph pad prism software</td>
			<td>GraphPad 8.0, San Diego, California, USA</td>
			<td></td>
			<td>graphing and statistics software</td>
		</tr>
		<tr>
			<td>Green light emitting diode (LED) strip</td>
			<td>Nanyi electronics Co., Ltd., Tainan, Taiwan</td>
			<td>2835</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Yihder, Taipei, Taiwan</td>
			<td>LM-570D (R)</td>
			<td>Emission peak wavelength: 525 nm, Viewing angle: 150&#176;; originated from https://www.aliva.com.tw/product.php?id=63</td>
		</tr>
		<tr>
			<td>Light power meter</td>
			<td>Ophir, Jerusalem, Israel</td>
			<td>PD300-3W-V1-SENSOR,</td>
			<td></td>
		</tr>
		<tr>
			<td>Millex 0.22 &#956;m filter</td>
			<td>Merck, NJ, USA</td>
			<td>SLGVR33RS</td>
			<td></td>
		</tr>
		<tr>
			<td>Multidrug-resistant Candida albicans</td>
			<td>Bioresource Collection and Research CenterBioresource, Hsinchu, Taiwan</td>
			<td>BCRC 21538/ATCC 10231</td>
			<td>http://catalog.bcrc.firdi.org.tw/BcrcContent?bid=21538</td>
		</tr>
		<tr>
			<td>OD600 spectrophotometer</td>
			<td>Biochrom, London, UK</td>
			<td>Ultrospec 10</td>
			<td></td>
		</tr>
		<tr>
			<td>Rose Bengal</td>
			<td>Sigma-Aldrich, MO, USA</td>
			<td>330000</td>
			<td>stock concentration 40 mg/mL = 4%, prepare in PBS, stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Sterilized glass tube</td>
			<td>Sunmei Co., Ltd., Tainan, Taiwan</td>
			<td>AK45048-16100</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast Extract Peptone Dextrose Medium</td>
			<td>HIMEDIA, India</td>
			<td>M1363</td>
			<td></td>
		</tr>
	</tbody>
</table>

rose bengal-mediated photodynamic therapy to inhibit candida albicans

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.

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 &#177; 1 &#176;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....

Watch this Scientific Journal Video about Rose Bengal-Mediated Photodynamic Therapy to Inhibit Candida albicans at JoVE.com 

Rose Bengal-Mediated Photodynamic Therapy to Inhibit Candida albicans (Video) | JoVE 

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.

Rose Bengal-Mediated Photodynamic Therapy to Inhibit Candida albicans

Invasive Candida albicans infection is a significant opportunistic fungal infection in humans because it is one of the most common colonizers of the gut, ...

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