Photodynamic antimicrobial chemotherapy in aquaculture: photoinactivation studies of Vibrio fischeri

Abstract

Background: Photodynamic antimicrobial chemotherapy (PACT) combines light, a light-absorbing molecule that initiates a photochemical or photophysical reaction, and oxygen. The combined action of these three components originates reactive oxygen species that lead to microorganisms' destruction. The aim was to evaluate the efficiency of PACT on Vibrio fischeri: 1) with buffer solution, varying temperature, pH, salinity and oxygen concentration values; 2) with aquaculture water, to reproduce photoinactivation (PI) conditions in situ.

Methodology/principal findings: To monitor the PI kinetics, the bioluminescence of V. fischeri was measured during the experiments. A tricationic meso-substituted porphyrin (Tri-Py(+)-Me-PF) was used as photosensitizer (5 µM in the studies with buffer solution and 10-50 µM in the studies with aquaculture water); artificial white light (4 mW cm(-2)) and solar irradiation (40 mW cm(-2)) were used as light sources; and the bacterial concentration used for all experiments was ≈10(7) CFU mL(-1) (corresponding to a bioluminescence level of 10(5) relative light units--RLU). The variations in pH (6.5-8.5), temperature (10-25°C), salinity (20-40 g L(-1)) and oxygen concentration did not significantly affect the PI of V. fischeri, once in all tested conditions the bioluminescent signal decreased to the detection limit of the method (≈7 log reduction). The assays using aquaculture water showed that the efficiency of the process is affected by the suspended matter. Total PI of V. fischeri in aquaculture water was achieved under solar light in the presence of 20 µM of Tri-Py(+)-Me-PF.

Conclusions/significance: If PACT is to be used in environmental applications, the matrix containing target microbial communities should be previously characterized in order to establish an efficient protocol having into account the photosensitizer concentration, the light source and the total light dose delivered. The possibility of using solar light in PACT to treat aquaculture water makes this technology cost-effective and attractive.

Figure 1. Structure of the 5,10,15-tris(1-methylpyridinium-4-yl)-20-(pentafluorophenyl)porphyrin tri-iodide.

Figure 2. Relationship between the bioluminescence signal and viable counts of an overnight culture of V. fischeri (≈10⁹ CFU mL⁻¹) serially diluted in PBS with 3% of NaCl.

Bioluminescence is expressed in relative light units (RLU) and viable counts in CFU mL⁻¹. Bacterial suspension in the absence of PS (unfilled triangle), R² = 0,9957; bacterial suspension with 5 µM of Tri-Py⁺-Me-PF incubated 4 h in the dark (black square), R² = 0,9495.Values represent the mean of two independent experiments; error bars indicate the standard deviation.

Figure 3. Photoinactivation of V. fischeri with 5 µM of Tri-Py⁺-Me-PF at different pH values of the suspension medium, under 4 mW cm⁻² irradiation.

Values represent the mean of two independent experiments; error bars indicate the standard deviation.

Figure 4. Photoinactivation of V. fischeri with 5 µM of Tri-Py⁺-Me-PF at different temperatures of the suspension medium, under 4 mW cm⁻² irradiation.

Values represent the mean of two independent experiments; error bars indicate the standard deviation.

Figure 5. Photoinactivation of V. fischeri with 5 µM of Tri-Py⁺-Me-PF at different concentrations of NaCl (in g L⁻¹) in the suspension medium, under 4 mW cm⁻² irradiation.

Values represent the mean of two independent experiments; error bars indicate the standard deviation.

Figure 6. Photoinactivation of V. fischeri with Tri-Py⁺-Me-PF at 10, 20 and 50 µM, in an aquaculture water sample collected in May 2010, under 4 mW cm⁻² irradiation.

A: non-filtered portion; B: portion filtered by 0.7 µm membrane; C: portion filtered by 0.2 µm membrane. Values represent the mean of two replicates of the same sample; error bars indicate the standard deviation.

Figure 7. Photoinactivation of V. fischeri with Tri-Py⁺-Me-PF at 10, 20 and 50 µM, in an aquaculture water sample collected in June 2010, under 4 mW cm⁻² irradiation.

A: Assays with non-filtered water portion; B: Assays with water filtered by 0.7 µm membrane; C: Assays with water filtered by 0.2 µm membrane. Values represent the mean of two replicates of the same sample; error bars indicate the standard deviation.

Figure 8. Photoinactivation of V. fischeri with 20 µM of Tri-Py⁺-Me-PF, in an aquaculture sample collected in October 2010 using different light sources.

A: Assays with artificial white light (4 mW cm^{− 2}). 64.8 J cm^{− 2} was the total light dose applied after 270 min of irradiation; B: Assays with solar light (40 mW cm^{− 2}). 64.8 J cm^{− 2} was the total light dose applied after 27 min of irradiation. Values represent the mean of two replicates of the same sample; error bars indicate the standard deviation.

Figure 9. Absorption spectrum of the tricationic meso-subtituted porphyrin and emission spectra of the two light sources used in the photoinactivation studies.