Introduction
Intensive fish farming involves high population densities and stressful conditions, can lead to disease outbreaks, and may result in the use of antibiotics. However, the development of antibiotic-resistant strains in fish farms has rendered the use of antibiotics ineffective. This has also contributed to a negative perception of the aquaculture industry among consumers (Martinez et al., 2012). In this study we conducted two series of experiments to monitor the antimicrobial activity of microalgae species, isolated from lagoons in Western Greece against four fish pathogenic bacteria (Vibrio anguillarum, Aeromonas veronii, Vibrio alginolyticus, and Vibrio harveyi), using axenic cultures and extracellular assays in two experimental series.
Materials and methods
In this first experiment series, we tested the antimicrobial activity of the axenic microalgae strains: Amphidinium carterae, Asteromonas gracilis, Tetraselmis sp. (red var.), Tetraselmis sp. (palmella), Tetraselmis sp. (red var., Pappas), and Dunaliella salina, at different salinities. The microalgae were cultured under aeration, and their cell numbers were counted using a microscope at the start and end of the six-day experiment (Stanier et al., 1978). Experiments were conducted with and without light, using Chlorella minutissima as a reference microalgae (Makridis et al., 2006). In the second series of experiments, supernatants from Amphidinium carterae, Asteromonas gracilis, Tetraselmis sp. (red var. Pappas), Nephroselmis sp., Phormidium sp., Anabaena sp., and Cyanothece sp. were tested against the same fish pathogens (Katircioglu et al., 2006, Jlidi et al., 2022). The optical density was measured at 600 nm at 0, 2, 4, 6, 21, 23, 25, 48, 72, and 168 h after inoculation and calculated the inhibition efficiency using a formula according to Jlidi et al. (2022).
Results and Discussion
In the first series of experiments, all microalgae tested reduced the growth of bacteria compared with the control treatments. The largest differences were observed on the 4th day of the experiment for all microalgae tested, with the best results observed against the pathogen V. anguillarum.
In the case of V. anguillarum (Fig.1a,b), the control treatment had a microbial concentration of 33×106 CFU/mL, while in exposure to light, the microalgae A. gracilis, and Tetraselmis species (red var., palmella, red var. Pappas) resulted in a concentration range of 2×104-89×105 CFU/mL. Similarly, in exposure to light, the microalgae A. carterae and D. salina resulted in a concentration range of 66×104-16,6×105 CFU/mL, compared to the control treatment of 6×106 CFU/mL. In the absence of light, the microbial concentration range for A. gracilis, and Tetraselmis species (red var., palmella, red var. Pappas) was 3×104–77×105 CFU/mL, and the concentration range for A. carterae and D. salina was 78×104-18,8×105 CFU/mL.
Seven species of microalgae were then tested for extracellular antimicrobial activity, where our experiments with V. alginolyticus yielded promising results compared with the control treatment, particularly for the cyanobacteria strains. The inhibitory effect on Phormidium sp. and Anabaena sp. was observed during the period from 21 to 48 hours, and for Cyanothece sp. it was observed during the period from 25 to 48 hours (p<0.05). The samples containing Tetraselmis sp. (red var., Pappas) with V. alginolyticus showed statistically significant inhibition between 4 and 25 hours. A. gracilis showed inhibition at 72 hours, while Nephroselmis sp. had a peak inhibition at 48 hours.
Our results with V. anguillarum indicate that A. carterae demonstrated inhibitory activity between 48 and 72 hours while in the experiment with Tetraselmis sp. (red var., Pappas), the growth of the pathogen was inhibited during the period from 6 to 21 hours. The results of our experiments with V. harveyi revealed statistically significant inhibition of growth for the Anabaena sp. strain between 23 and 25 hours while in our experiments with the Tetraselmis sp. (red var., Pappas) strain, inhibition was observed between 21 and 23 hours. Nephroselmis sp. exhibited inhibition against V. harveyi between 4 and 6 hours.
Acknowledgements
This study was supported through the project “Isolation and cultivation of local microalgae species from lagoons with the vision of mass production of antimicrobial substances, fatty acids, pigments and antioxidants” funded by the General Secretariat for Research and Innovation in Greece and EU funds (ref. nr. 5048496).
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