JoVE Logo
Faculty Resource Center

Sign In

Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Immunology and Infection

Rose Bengal-Mediated Photodynamic Therapy to Inhibit Candida albicans

Published: March 24th, 2022

DOI:

10.3791/63558

* These authors contributed equally

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.

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.

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....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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 Materi.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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........

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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].

....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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

  1. Naglik, J. R., Challacombe, S. J., Hube, B. Candida albicans secreted aspartyl proteinases in virulence and pathogenesis. Microbiology and Molecular Biology Reviews. 67 (3), 400-428 (2003).
  2. Pappas, P. G., et al. Clinical practice guideline for the management of candidiasis: 2016 update by the Infectious Diseases Society of America. Clinical Infectious Diseases. 62 (4), 1-50 (2016).
  3. Ricotta, E. E., et al. Invasive candidiasis species distribution and trends, United States, 2009-2017. Journal of Infectious Diseases. 223 (7), 1295-1302 (2021).
  4. Koehler, P., et al. Morbidity and mortality of candidaemia in Europe: an epidemiologic meta-analysis. Clinical Microbiology and Infection. 25 (10), 1200-1212 (2019).
  5. Toda, M., et al. Population-based active surveillance for culture-confirmed candidemia - four sites, United States, 2012-2016. Morbidity and Mortality Weekly Report Surveillance Summaries. 68 (8), 1-15 (2019).
  6. Lee, C. N., Hsu, R., Chen, H., Wong, T. W. Daylight photodynamic therapy: an update. Molecules. 25 (21), 5195 (2020).
  7. Wainwright, M. Photodynamic antimicrobial chemotherapy (PACT). Journal of Antimicrobial Chemotherapy. 42 (1), 13-28 (1998).
  8. Wong, T. W., et al. Indocyanine green-mediated photodynamic therapy reduces methicillin-resistant staphylococcus aureus drug resistance. Journal of Clinical Medicine. 8 (3), 411 (2019).
  9. Kim, M. M., Darafsheh, A. Light sources and dosimetry techniques for photodynamic therapy. Photochemistry and Photobiology. 96 (2), 280-294 (2020).
  10. Wong, T. W., Sheu, H. M., Lee, J. Y., Fletcher, R. J. Photodynamic therapy for Bowen's disease (squamous cell carcinoma in situ) of the digit. Dermatologic Surgery. 27 (5), 452-456 (2001).
  11. Wong, T. W., et al. Photodynamic inactivation of methicillin-resistant Staphylococcus aureus by indocyanine green and near infrared light. Dermatologica Sinica. 36 (1), 8-15 (2018).
  12. Stasko, N., et al. Visible blue light inhibits infection and replication of SARS-CoV-2 at doses that are well-tolerated by human respiratory tissue. Scientific Reports. 11 (1), 20595 (2021).
  13. Crosbie, J., Winser, K., Collins, P. Mapping the light field of the Waldmann PDT 1200 lamp: potential for wide-field low light irradiance aminolevulinic acid photodynamic therapy. Photochemistry and Photobiology. 76 (2), 204-207 (2002).
  14. Feenstra, R. P., Tseng, S. C. Comparison of fluorescein and rose bengal staining. Ophthalmology. 99 (4), 605-617 (1992).
  15. Demidova, T. N., Hamblin, M. R. Effect of cell-photosensitizer binding and cell density on microbial photoinactivation. Antimicrobial Agents and Chemotherapy. 49 (6), 2329-2335 (2005).
  16. Shahid, H., et al. Duclauxin derivatives from fungi and their biological activities. Frontiers in Microbiology. 12, 766440 (2021).
  17. Arendrup, M. C., Park, S., Brown, S., Pfaller, M., Perlin, D. S. 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. Antimicrobial Agents and Chemotherapy. 55 (5), 1891-1895 (2011).
  18. Mukaremera, L., Lee, K. K., Mora-Montes, H. M., Gow, N. A. R. Candida albicans yeast, pseudohyphal, and hyphal morphogenesis differentially affects immune recognition. Frontiers in Immunology. 8, 629 (2017).
  19. Hung, J. H., et al. Recent advances in photodynamic therapy against fungal keratitis. Pharmaceutics. 13 (12), 2011 (2021).
  20. Martinez, J. D., et al. Rose Bengal photodynamic antimicrobial therapy: a pilot safety study. Cornea. 40 (8), 1036-1043 (2021).

This article has been published

Video Coming Soon

JoVE Logo

Privacy

Terms of Use

Policies

Research

Education

ABOUT JoVE

Copyright © 2024 MyJoVE Corporation. All rights reserved