Photodynamic antimicrobial chemotherapy for pathogenic vibrio control in prawn hatcheries

In the last two decades, the aquaculture industry has grown significantly, providing fish, molluscs and crustaceans to the global market. Microbial pathogens are the principal cause of massive financial losses in many fish and crustacean farms. Disinfection using ozone, chlorine or UV irradiation are expensive and leave toxic byproducts. Biosecurity threats through the introduction of wild brood-stock and live feed is a major concern for the aquaculture industry. Microalgae and/or Artemia nauplii (brine shrimp), have been identified as potential vectors for microbial pathogens that can result in high mortalities, particularly in hatcheries. Bacteria can proliferate rapidly in intensive aquaculture productions due to the high density of target animals, live feed organisms and build-up of biological waste that generate ideal conditions for many potential pathogens. The current inability to bio-secure farmed aquaculture animals through appropriate disinfection strategies of live feed without altering the quality of the final product is an area requiring urgent research, as it is the main barrier to cost-effective product development; the main hurdle to competitive expansions into national and international markets. Recently, photodynamic antimicrobial chemotherapy (PACT) has emerged as a promising water sterilisation technique. PACT uses the activation of photosensitisers by light to generate highly reactive oxygen species (ROS), which indiscriminately oxidises cell wall and cell membrane components (i.e. lipids, proteins, carbohydrates). After entry of the photosensitiser into cells, produced singlet oxygen also targets intracellular components such as organelles, membrane compartments, and nucleic acids (i.e. DNA and RNA). Due to the indiscriminate action of ¹O₂, resistance cannot develop, unlike in the case of antibiotics. The choice of photosensitiser is crucial, and compounds that photobleach with time are preferred for aquaculture purposes to avoid build up in water or the farmed animal. Porphyrins are a group of natural or synthetic compounds that fit the photobleaching requirement, but to date no research has specifically investigated porphyrin-based sterilisation efficiency in prawn hatcheries. In addition, the porphyrin-based antimicrobial efficiency for the ones used in this research has not been tested against luminescent virulent strains of Vibrio harveyi-related species, the main causative agents for luminescent vibriosis, which causes large losses for finfish, bivalves and prawn industries. Each chapter of this thesis addresses specific objectives that, in conjunction with the other chapters, contribute to the principal aim of the research: investigate the potential of using PACT to control Vibrio bacteria in aquaculture. Chapter 2 details objectives relating to method development to select a suitable Vibrio harveyi-related strain to be used in the following chapters. While Chapter 1 reviews PACT history and its application with special interest to aquaculture, the method development chapter (chapter 2), pathogenicity towards prawns after injection and luminescence intensity of two selected Vibrio strains were evaluated. Challenge experiments were performed with Penaeus monodon and Koch's postulates were fulfilled. Precise identification was obtained using molecular techniques including multiplex PCR, housekeeping gene analysis and construction of a phylogenetic tree. The strongly luminescent Vibrio sp ISO7, was demonstrated to be highly virulent towards P. monodon, killing 100% of the injected animals and molecular techniques revealed that this species belongs to the V. campbellii group. In contrast, V. owensii 47666-1, previously described as a luminescent prawn larvae pathogen, caused only 25% mortality in the first challenge experiment. Pathogenicity was regained in bacteria re-isolated from sick prawns, with 100% mortality of P. monodon obtained in a second challenge experiment, however the strain was found to be not luminescent. Hence, V. campbellii ISO7 was selected as the model bacterium based on its high virulence after injection and strong luminescence signal, making it suitable for the development of a fast luminescence-based assay to determine the efficiency of different porphyrin treatment protocols. In chapter 3, the objectives were 1) to compare the photostability of the tetracationic 5,10,15,20-tetrakis (1-methyl-4-pyridinio) porphyrin tetra (p-toluenesulfonate) [TMPyP] and the tetra-anionic 4,4',4",4"-(porphine-5,10,15,20-tetrayl) tetrakis (benzenesulfonic acid) tetrasodium salt hydrate [TPPS₄] porphyrins in seawater to confirm their suitability for aquaculture purposes and 2) to investigate the capacity of TMPyP and TPPS₄ to inactivate the model bacterium Vibrio campbellii ISO7. For the first objective, the photostability of the cationic TMPyP and the anionic TPPS₄ porphyrins were investigated by recording their full light spectra between 350 and 750 nm after irradiating seawater containing different porphyrin concentrations (1, 5, 10 and 20 μM) of porphyrins with high power 150 W LED lights. Results obtained using the maximum peak of absorbance for each porphyrin, showed porphyrin degradation (photobleaching) for both the cationic and the anionic porphyrin within 24 h of irradiation. In addition, the dark control (samples not irradiated) did not photobleach, confirming that porphyrin degradation was due to exposure to light. The results confirmed that both porphyrins were good candidates for aquaculture applications based on their photobleaching properties showing relatively fast degradation(within 24-h at concentration of 1 μM and more than ¼ of absorbance reduction at the concentration of 20 μM). Antimicrobial activity of the porphyrins was investigated using dose-response and time-course experiments. Twenty μM of the cationic porphyrin achieved 100% lethality in the model bacterium (start concentration of V. campbellii ISO7 ~10⁷ CFU·mL⁻¹) after five hours, which was validated using biological activity (luminescence), growth experiments (CFU, absorbance) and 7-day regrowth experiments. Consistent with previous reports, the anionic photosensitizer did not achieve inactivation of the model bacterium and was therefore not investigated further. As demonstrated, water sterilization was achieved after between five and twelve hours depending on the concentration of the cationic porphyrin; however, the effectiveness of sterilization in mixed culture with live feed organisms still needed to be confirmed. In chapters 4 and 5, the potential of using the TMPyP porphyrin to reduce bacteria loads of microalgae cultures (free of the model bacterium) and Artemia cysts was investigated including an evaluation of possible toxic effects caused by singlet oxygen generated during PACT towards the live feed organisms themselves. In chapter 4, the viability of microalgae cells was first evaluated using flow cytometry based on chlorophyll fluorescence and the live-dead stain Propidium Iodide (PI) during a six-hour dose-response treatment with the porphyrin. The treatment time was chosen based on bacterial disinfection results shown in chapter 3. Sensitivity to ¹O₂ was speciesspecific and related to cell wall characteristics. Of the five different microalgae used, only Nannochloropsis oculata was highly resilient to the six-hour treatment with up to 50 μM of the cationic porphyrin. The results were unexpected, as photosynthetic microorganisms possess detoxification systems for ¹O₂ and other reactive oxygen species (ROS). However, ¹O₂ produced externally to the cell may be able to photooxygenate and destabilize cell membrane components, leading to cell death. The thick cell wall of N. oculata most likely protected the cell membrane from fast photooxidation. The highly resilient microalga, N. oculata was therefore used in mixed culture with the model pathogen and treated with 20 μM final concentration of the TMPyP porphyrin. Complete inactivation of the model bacterium was successfully achieved, as verified by absence of luminescent CFUs on agar plates and a species-specific molecular technique that can detect the model organisms with high sensitivity (Multiplex PCR combined with Most Probable Number enrichment). In chapter 5, possible toxicity of the cationic porphyrin and ¹O₂ against two different types of Artemia cysts (magnetic and unmodified) was tested in dose-response experiments using percentage of cyst hatching as the measured variable. Surprisingly, magnetic cysts showed improved hatching under sub-optimal hatching conditions in the presence of porphyrin-generated ROS and the porphyrin TMPyP alone (i.e. independent to the production of ¹O₂) relative to the controls. In contrast, dose-response experiments with unmodified cysts showed a positive hatching response only to TMPyP-generated ¹O₂ but not TMPyP itself. Further investigations showed that when magnetic cysts were mixed with the model pathogen, complete pathogen inactivation was achieved after six hours of incubation with 20 μM of TMPyP in the light. In conclusion, my research demonstrated that PACT is suitable as an additional (or alternative) sterilization method in prawn hatcheries and potentially for aquaculture water treatment in general.