Induction of Petite Colonies in Candida glabrate via Rose Bengal-Mediated Photodynamic Therapy

Summary

The significance of petite colonies in Candida spp. drug resistance has not been fully explored. Antimicrobial photodynamic therapy (aPDT) offers a promising strategy against drug-resistant fungal infections. This study demonstrates that rose bengal-mediated aPDT effectively deactivates Candida glabrata and induces petite colonies, presenting a unique procedure.

Abstract

Facing a 40% mortality rate in candidemia patients, drug-resistant Candida and their petite mutants remain a major treatment challenge. Antimicrobial photodynamic therapy (aPDT) targets multiple fungal structures, unlike antibiotics/antifungals, potentially thwarting resistance. Traditional methods for inducing petite colonies rely on ethidium bromide or fluconazole, which can influence drug susceptibility and stress responses. This study investigated the application of green light (peak 520 nm) and rose bengal (RB) photosensitizer to combat a drug-resistant Candida glabrata isolate. The findings revealed that aPDT treatment significantly inhibited cell growth (≥99.9% reduction) and effectively induced petite colony formation, as evidenced by reduced size and loss of mitochondrial redox indicator staining. This study provides initial evidence that aPDT can induce petite colonies in a multidrug-resistant C. glabrata strain in vitro, offering a potentially transformative approach for combating resistant fungal infections.

Introduction

Fungal infections, particularly those caused by Candida albicans and increasingly drug-resistant Candida glabrata, pose a serious global threat¹. These infections can be deadly, especially for hospitalized patients and those with weakened immune systems. Rising antifungal resistance threatens the control of invasive candidiasis, a severe fungal infection with high mortality, especially from Candida albicans². Resistant strains hinder effective treatment, potentially increasing both complexity and death rates. In Alameda County, California, USA, C. glabrata has become the most prevalent....

Protocol

1. Culturing of C. glabrata

NOTE: A multidrug-resistant C. glabrata (C2-1000907) that is resistant to most antifungal agents, including fluconazole, is used for the experiments. The experimental conditions may need to be adapted to the specific strain, as variations may exist among different strains. All experiments used log-phase Candida grown at 25 °C (mimicking natural infection) for consistency. C. glabrata's lack of hyphae simplifies quan.......

Discussion

This study unveils PDT as the first reported method to induce petite colony formation in Candida, surpassing the established effects of ethidium bromide and fluconazole. This novel observation necessitates further exploration to unravel its implications for both fungal eradication by decreasing virulence and the emergence of resistance mechanisms.

RB-mediated PDT effectively inhibits the growth of C. glabrata, suggesting a potential alternative treatment approach for Candida infection.......

Materials

Name	Company	Catalog Number	Comments
0.22 μm filter	Merck, Taipei, Taiwan	Millex, SLGVR33RS
1.5 mL microfuge tube	Neptune, San Diego, USA	#3745
20% Triphenyltetrazolium chloride (TTC)	Sigma-Aldrich, MO, USA	T8877
5 mL polypropylene round bottom tube	Corning, AZ, USA	352059
5 mL round-bottom tube with cell strainer cap	Corning, AZ, USA	Falcon, #352235
96-well plate	Alpha plus, Taoyuan Hsien, Taiwan	#16196
Agar	BRS, Tainan, Taiwan	AG012
Blank disk	Advantec, Tokyo, Japan	49005040
Centrifuge	Eppendorf, UK	5415R
Ethidium bromide solution	Sigma-Aldrich, MO, USA	E1510
Fluconazole, 2 mg/mL	Pfizer, NY, USA	BC18790248
GraphPad Prism	GraphPad Software	Version 7.0
Green light emitting diode (LED) strip	Nanyi electronics Co.,Ltd, Tainan, Taiwan	5050	Excitation wave: 500~550 nm
Low Temperature. shake Incubators	Yihder, Taipei, Taiwan	LM-570D (R)
Mouth care cotton swabs	Good Verita Enterprise, Taipei, Taiwan	161357
Muller Hinton II agar	BD biosciences, California, USA	211438
Multimode microplate reader	Molecular Devices	SpectraMax i3x
OD600 spectrophotometer	Biochrom, London, UK	Ultrospec 10
Rose Bengal	Sigma-Aldrich, USA	330000	stock concentration 40 mg/mL = 4%, prepare in PBS, stored at 4 °C
Sterilized glass tube	Sunmei, Tainan, Taiwan	AK45048-16100
Yeast Extract Peptone Dextrose Medium	HIMEDIA, India	M1363