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 invasive species³. This shift in the prevalence and distribution of Candida species may be influenced by local healthcare practices, patient demographics, the utilization of antifungal agents, and the prevalence of risk factors for Candida infections.

Petite mutants in Candida, lacking functional mitochondria, reveal how this organelle affects drug response, virulence, and stress resistance^4,5. C. glabrata readily forms these colonies, gaining sensitivity to polyenes while losing it to azoles⁶. Azole sensitivity and respiratory function are intricately linked, with diminished respiration leading to resistance via mitochondrial DNA loss⁷. Petite colonies of C. glabrata with azole resistance have been isolated from human stool samples from a bone marrow transplant recipient undergoing fluconazole treatment⁸ and from blood culture bottles of patients with bloodstream infections⁹. Their potential implications in drug resistance, virulence, and stress response highlight their clinical significance. Additionally, their distinct properties make them valuable tools for investigating fundamental questions in mitochondrial biology⁵. As research into petite mutants continues, their applications in both clinical and basic research are likely to expand.

This study discovered that photodynamic therapy (PDT) can induce petite colonies in C. glabrata, expanding the range of methods beyond the traditional techniques of exposing C. glabrata to ethidium bromide or fluconazole.

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 quantification compared to C. albicans, which forms hyphae at 37 °C¹⁰.

1. To prepare a log-phase culture of C. glabrata, pick a single colony from an agar plate and transfer it to a glass test tube with 3 mL of sterile yeast peptone dextrose (YPD) medium (see Table of Materials). Incubate for 14-16 h at 25 °C with shaking (155 rpm, 45° angle to increase air transmission to the medium).
2. After incubation, dilute the culture with fresh YPD to an OD₆₀₀ of 0.1 using sterile technique. Incubate at 25 °C for 6 h with shaking at 155 rpm. Verify the log phase of C. glabrata by measuring the OD₆₀₀. Aim for 0.65-1.00, which corresponds to 1 × 10⁷-1.5 × 10⁷ cells/mL.

2. Induction of petite colonies by ethidium bromide, fluconazole and photodynamic therapy

1. Ethidium bromide petite colony induction
   1. Adjust a yeast suspension to an OD₆₀₀ of 0.1 (5 × 10⁶ cells/mL) with YPD to a final volume of 3 mL, then add 30 µL of ethidium bromide stock (10 mg/mL) (see Table of Materials) for a final concentration of 100 µg/mL.
   2. Incubate the yeast suspension overnight (16-18 h) at 25 °C, 155 rpm, 45° angle. Adjust to an OD₆₀₀ of 0.65 (1 × 10⁷ cells/mL). Perform a 10-fold dilution series in a 96-well plate by adding 20 µL of the adjusted yeast suspension to 180 µL of PBS per well. This creates six dilutions ranging from 10^-1 to 10^-5.
   3. Select 4 dilution factors (e.g., 10⁰, 10¹, 10², and 10³) and plate 3 drops (20 µL) each on 4 quadrants of YPD agar plates (triplicate).
   4. Incubate plates overnight at 37 °C. Select quadrants with 5-80 colonies from the previously chosen dilutions for triphenyltetrazolium chloride (TTC) staining¹¹ (see step 3). Scan plates at 1200 dpi using a 48-bit full-color optical scanner.
2. Fluconazole petite colony induction
   NOTE: To expedite the process, use T3 cells (a mixture of normal and petite cells obtained after 3 RB-PDT treatments; see step 2.3) for fluconazole-induced petite colony formation. This is because standard treatment typically results in slower formation.
   1. Prepare and adjust a yeast cell suspension as described in step 2.1.1.
   2. Incubate overnight (16-18 h) at 25 °C. Adjust the OD₆₀₀ to 0.1 again.
   3. Transfer 1 mL of the adjusted suspension to a sterile 5 mL tube.
   4. Dip a sterile cotton swab (15 cm long, with a 0.9 cm x 2.6 cm tip, see Table of Materials) in the yeast suspension, ensuring contact with the tube bottom and twisting to remove excess liquid from the tube wall.
   5. Prepare the plates in the hood using Mueller-Hinton agar (see Table of Materials).
   6. Swab the cotton back and forth on the agar, rotate 60° twice, then swab the perimeter for even coverage.
   7. Sterilize forceps over a flame for 1-2 s, allowing them to cool briefly, then use them to pick up the blank disks.
   8. Divide the plate into three equal sectors using a marker. Place one blank disk in the center of each sector.
   9. Add 12.5 µL fluconazole stock (2 mg/mL, see Table of Materials) to each disk (25 µg/disk), mix thoroughly to avoid bubbles, and incubate at 37 °C for 20-24 h.
   10. Perform the TTC staining (see step 3). Scan plates at 1200 dpi using a 48-bit full-color optical scanner.
3. PDT petite colony induction
   NOTE: Different fungi may produce different numbers of petite colonies after PDT. Some strains may not produce any at all.
   1. Prepare the aPDT system.
      NOTE: The setup procedure for the aPDT system device follows the method reported by Hung et al¹². Briefly, the aPDT system consists of a green LED array with a peak at 520 nm (see Table of Materials), shining light from the bottom with good alignment with each well of a 96-well plate.
   2. Adjust the yeast cell suspension in the YPD medium to an OD₆₀₀ of 0.65 (about 1 × 10⁷ cells/mL).
   3. Mix 1 mL of the yeast suspension with 111 µL of 2% rose bengal (RB, Figure 1) (see Table of Materials) in a round-capped tube to give a final RB concentration of 0.2%. Incubate the mixture at 25 °C for 15 min, rotating it at a 45° angle at 155 rpm.
   4. Transfer the mixture to a 1.5 mL microcentrifuge tube and centrifuge at 16,100 x g for 2.5 min (at room temperature).
   5. Discard the supernatant and gently scrape the tube five times on the hood floor to resuspend the pellet.
   6. Wash the suspension with 1x PBS four times to remove all the RB. Each wash is followed by resuspension of the pellet with brief vortexing or pipetting for thorough resuspension.
      1. After the final wash, add 1000 µL of PBS. The solution is light pink in color. Transfer 200 µL of the washed RB-loaded yeast suspension to each of three wells in a 96-well plate (triplicate).
   7. Position the 96-well plate on the photodynamic light system with LED bulbs shining green light from below. Turn off the room lights to ensure uniform illumination and prevent interference. Activate the LED light system to deliver the PDT dose of 4.38 J/cm² over 2 min; ensure the system is properly aligned with the wells.
   8. After irradiation, dilute the yeast suspension in a 96-well plate. Add 20 µL of yeast suspension to a well containing 180 µL PBS, creating a 10-fold dilution. Repeat for the remaining dilutions to achieve a range of 10^-1 to 10^-5.
   9. Choose four dilution factors (e.g., 10^-2 to 10^-5) based on the yeast cell concentration adjustment.
   10. For each dilution factor, plate 3 drops (20 µL each) per quadrant on YPD agar plates.
   11. After the yeast suspension is completely absorbed, around 10 min by the agar plates, invert and incubate overnight at 37 °C.
   12. Define T0 as the control parental C. glabrata without PDT treatment. Prepare T0 fungi in the log growth phase as described above and expose them to 4.38 J/cm² green light in the presence of 0.2% RB. This PDT condition consistently inhibits 3 to 3.5 logs of fungal growth.
       1. Denote the survived fungi as T1 and expose them to the same PDT dose again after they are expanded in vitro to a log growth phase. The survived fungi after the second PDT are denoted as T2 and so forth.
   13. On the next day, choose quadrants with colony counts between 5 and 80. Count the number of colonies in each quadrant and calculate the titer with the following formula: Colony-forming unit (CFU)/mL = Numbers of colonies (average of triplicate) × Dilution factor × 50.
   14. Perform the TTC staining (see step 3). Scan the plate as described earlier (step 2.2).

3. Mitochondrial function analysis (TCC staining test)

NOTE: TTC is a redox indicator and an electron acceptor. It turns red when white compounds are broken by electrons¹¹. Note that not all cells necessarily form visible colonies within 24 h after culture on an agar plate. Colonies with functional mitochondria turn red, while those with non-functional mitochondria remain white. This allows differentiation between colonies with different mitochondrial functionality.

1. After treatment, dilute Candida suspensions on YPD agar plates according to the desired cell density to obtain distinct colonies for easy visualization and staining.
2. After 24 h of growth at 37 °C, visible colonies are formed from a single cell. Pipette 20 µL of 20% TTC directly onto the center of each colony.
3. After complete absorption, around 10 min of TTC by the colonies, incubate at 37 °C for 30-40 min.

4. Growth kinetics of normal and petite C. glabrata

NOTE: Three strains of yeast were compared: C. glabrata C2-1000907 T0 (clinical isolate without PDT treatment), T3n (C. glabrata C2-1000907 after 3 consecutive RB-PDT exhibiting colonies with an average diameter of 1.5 ± 0.8 mm similar to the parental cells), and T3p (petite colonies of C. glabrata C2-1000907 after 3 consecutive RB-PDT).

1. Adjust the yeast cell suspension in YPD medium to an OD₆₀₀ of 0.1 (5 × 10⁶ cells/mL) in a 3 mL tube.
2. Automatically measure the OD₆₀₀ of the statical culture every 20 min using the multi-mode microplate reader (see Table of Materials) for 24 h.

Results

The data is presented as the mean with ± standard error and was obtained from three independent experiments, with at least triplicates in each group. Experimental data, including colony counts, OD₆₀₀ measurements, and TTC staining results, were graphed and statistically analyzed using graphing and statistical software (see Table of Materials). One-way ANOVA or t-test was used to analyze the data, and a p-value <0.05 was considered significant. Scanning was performed ...

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