Light-Activated Treatment of Bacterial Biofilms Using a Photosensitizer Precursor

Abstract

Source: Zhao, K., et al. An In Vitro Model to Study the Effect of 5-Aminolevulinic Acid-mediated Photodynamic Therapy on Staphylococcus aureus Biofilm. J. Vis. Exp. (2018)

The video demonstrates a photodynamic therapy protocol to assess antibacterial effects on Gram-positive bacterial biofilms. Biofilms are divided into four groups: two receive a photosensitizer precursor, and two receive a buffer. All are incubated in the dark, followed by red light exposure to one precursor-treated group and one buffer-only group. Reactive oxygen species are generated only in the group receiving both precursor and light, leading to bacterial cell death. Viability is compared across all groups to evaluate treatment efficacy.

Protocol

1. Biofilm Formation

1. Biofilm formation in 96-well microplates
   1. Retrieve the Staphylococcus aureus strain USA300 and 3 biofilm-forming clinical strains (C1-C3) stored at -80 °C.
      NOTE: The ability of the clinical strains to form biofilms was determined by the microtiter plate assay.
   2. Inoculate the bacterium in 5 mL tryptone soya broth (TSB) medium, and cultivate in an incubator with shaking at 37 °C overnight to the stationary phase.
   3. Centrifuge the overnight bacterial culture at 4,000 × g for 10 min at 25 °C and discard the supernatant. Resuspend the pellets in phosphate-buffered saline (PBS) to a final concentration of 2.0 × 10⁹ Colony-forming units (CFU)/mL.
      NOTE: The concentration of the bacteria was estimated by measuring optical density and further determined by plate count, revealing that 1 OD₆₀₀ of suspension contained 1.5 × 10⁸ CFU/mL.
   4. Dilute the bacterial suspension to 1:200 (1.0 × 10⁷ CFU/mL) in TSB medium containing 0.5% glucose. Inoculate 200 µL of bacterial suspension into each well of a cell-culture-treated polystyrene 96-well microplate.
   5. Incubate the microplate statically at 37 °C for 24 h in a well-oxygenated environment.
      NOTE: The incubation time for mature biofilm formation may vary for different bacterial strains; this should be determined before the Photodynamic Therapy (PDT) experiment.
   6. Discard the media in the wells and wash the microplate wells gently with PBS three times and then discard the supernatant.
      NOTE: The step should be carried out very gently to avoid disturbing the formed biofilm.

2. Light Irradiation

1. Store 5-Aminolevulinic Acid (5-ALA) in a 4 °C refrigerator. Before the experiment, dilute 5-ALA with PBS to 10 mM.
   NOTE: 5-ALA solution should be freshly prepared before the experiment.
2. In the experimental group, add 200 µL of 10 mM ALA to each well of the microplate or 2 mL to the culture dish. Cover the plate/dish with aluminum foil, and incubate for 1 h. Then, irradiate the plate/dish with a light-emitting diode (LED) with a light intensity of 100 mW/cm² for 1 h to achieve a light dose of 360 J/cm² at a major wavelength of 633 ± 10 nm.
   NOTE: In order to let the light energy be effectively and equally delivered to the biofilm in all of the wells/dishes, fix the distance from the peak of the light source to the well/dish at 6.0 cm, and limit the experimental region to the central irradiation area (10 cm × 8 cm). To ensure that the results are reproducible, the experiments should be performed at the same room temperature.In the LED irradiation step, to avoid direct exposure of the plate to other light sources, such as sunlight, room lighting, or lamplight, the LED was turned on before moving the plate/dish to the irradiation area, and the light was bright enough to finish the operation.
3. Set up the control groups (three control groups were set up in our experiment).
   1. For the first control group (ALA-LED-), add 200 µL of PBS to each well of the microplate or 2 mL to the culture dish. Cover the plate/dish with aluminum foil, and incubate it for 2 h.
   2. For the second control group (ALA+LED-), add 200 µL of 10 mM ALA to each well of the microplate or 2 mL to the culture dish. Cover the plate/dish with aluminum foil, and incubate it for 2 h.
   3. For the third control group (ALA-LED+), add 200 µL of PBS to each well of the microplate or 2 mL to the culture dish. Cover the plate/dish with aluminum foil, and incubate it for 1 h. Then, expose the plate to the LED with 360 J/cm² light irradiation at a major wavelength of 633 ± 10 nm.
      NOTE: In the LED irradiation step, avoid direct exposure of the plate to other light sources, such as sunlight, room lighting, or lamplight.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Tryptone Soya Broth (TSB)	OXOID	CM0129B
SYTO9	Thermo Fisher Scientific	L7012	The LIVE/DEAD BacLight Bacterial Viability Kits
Propidium iodide (PI)	Thermo Fisher Scientific	L7012	The LIVE/DEAD BacLight Bacterial Viability Kits
5-aminolevulinic acid (ALA)	Fudan Zhangjiang Bio-Pharm	3.1
Staphylococcus aureus strain USA300			USA 300 references
Staphylococcus aureus clinical strains (C1-C3)			All clinical strains were isolated from patients with chronic rhinosinusitis
96-well microplate	Corning Inc	3599	Clear Flat Bottom Polystyrene TC-Treated Microplates, Individually Wrapped, with Lid, Sterile
Eppendorf microcentrifuge 5417	Eppendorf	Z365998 | SIGMA
Incubator	Thermo Fisher Scientific	SHKE4000	MaxQ 4000 Benchtop Orbital Shakers
Light emitting diode (LED)	Wuhan Yage Optic and Electronic Technique CO	LED-IB