PERTANIKA JOURNAL OF SOCIAL SCIENCES AND HUMANITIES

Probiotics have been increasingly considered an alternative to antibiotics in combating disease outbreaks. Combined probiotics have been studied to possibly harbor synergistic effects that could provide better protection for aquatic species. Three potential probiotics, which had shown in vitro antagonism towards Aeromonas hydrophila in this study, were Bacillus amyloliquefaciens (L9, isolated from the blue swimming crab), Lysinibacillus fusiformis (A2, isolated from a microalga), and Enterococcus hirae (LAB3, isolated from the Asian seabass) were combined into a probiotic mixture. The probiotic mixture produced significantly higher biofilm (P < 0.05) (2.441 ± 0.346) than A. hydrophila (0.578 ± 0.124) during 24-h and showed a continuous increase in production at 48-h and 72-h time intervals, respectively. Furthermore, no hemolytic action was observed when the probiotic mixture was streaked on sheep blood agar (5%), whereas A. hydrophila presented α-hemolysis. The lowest concentration of the probiotic mixture (107 CFU mL-1) significantly inhibited (P < 0.05) the growth of A. hydrophila at 106 CFU mL-1 after 24 h of incubation, where bacterial count in the treatment was 6.595 ± 0.218 CFU mL-1, which was significantly lower (P < 0.05) than the control (7.247 ± 0.061 CFU) mL-1. Significant reduction (P < 0.05) in Aeromonas count from 7.532 ± 0.026 CFU mL-1 to 6.883 ± 0.015 CFU mL-1 was observed at 12 hours of co-incubation. Hence, this research suggests that the probiotic mixture of L9, A2, and LAB3 potentially confers protection against A. hydrophila infection due to their characteristics meeting the criteria of probiotics.

• Akanmu, O. A. (2018). Probiotics, an alternative measure to chemotherapy in fish production. In S. Enany (Ed.), Probiotics: Current knowledge and future prospects. Intech Open. https://doi.org/10.5772/intechopen.72923

• Alonso, S., Carmen Castro, M., Berdasco, M., de la Banda, I. G., Moreno-Ventas, X., & de Rojas, A. H. (2019). Isolation and partial characterization of lactic acid bacteria from the gut microbiota of marine fishes for potential application as probiotics in aquaculture. Probiotics and Antimicrobial Proteins, 11(2), 569-579. https://doi.org/10.1007/s12602-018-9439-2

• AlYahya, S. A., Ameen, F., Al-Niaeem, K. S., Al-Sa’adi, B. A., Hadi, S., & Mostafa, A. A. (2018). Histopathological studies of experimental Aeromonas hydrophila infection in blue tilapia, Oreochromis aureus. Saudi Journal of Biological Sciences, 25(1), 182–185. https://doi.org/10.1016/j.sjbs.2017.10.019

• Andrews, J. M. (2001). Determination of minimum inhibitory concentrations. Journal of Antimicrobial Chemotherapy, 48(Supplement 1), 5-16. https://doi.org/10.1093/jac/48.suppl_1.5

• Azrin, N. A. R., Yuzine, E., Ina-Salwany, M. Y., & Karim, M. (2019). The efficacy of potential probiont Bacillus amyloliquefaciens strain L11 in protecting Artemia nauplii and blue crab juveniles against Vibrio harveyi infection. Journal of Pure and Applied Microbiology, 13(2), 923-932. https://dx.doi.org/10.22207/JPAM.13.2.29

• Bhandary, T., Riyaz, A. L., & Paari, K. A. (2021). Probiotic properties of Bacillus subtilis isolated from dried anchovies (Stolephorus indicus) and evaluating its antimicrobial, antibiofilm and growth-enhancing potential in Danio rerio. Journal of Animal Health and Production, 9(3), 205-212. https://doi.org/10.17582/journal.jahp/2021/9.3.205.212

• Bruhn, J. B., Gram, L., & Belas, R. (2007). Production of antibacterial compounds and biofilm formation by Roseobacter species are influenced by culture conditions. Applied and Environmental Microbiology, 73(2), 442–450. https://doi.org/10.1128/AEM.02238-06

• Cavalcante, R. B., Telli, G. S., Tachibana, L., de Carla Dias, D., Oshiro, E., Natori, M. M., da Silva, M. M., & Ranzani-Paiva, M. J. (2020). Probiotics, prebiotics and synbiotics for Nile tilapia: Growth performance and protection against Aeromonas hydrophila infection. Aquaculture Reports, 17, 100343. https://doi.org/10.1016/j.aqrep.2020.100343

• Chean, M. Y. B., Puvanasundram, P., Yaminudin, J., & Karim, M. (2021). Evaluation of antagonism activity and control of Vibrio alginolyticus in Artemia culture using mixed probiotic. Pertanika Journal of Tropical Agricultural Science, 44(1), 117-137. https://doi.org/10.47836/pjtas.44.1.07

• Duarte, C. M., Agusti, S., Barbier, E., Britten, G. L., Castilla, J. C., Gattuso, J., Fulweiler, R. W., Hughes, T. P., Knowlton, N., Lovelock, C. E., Lotze, H. K., Predragovic, M., Poloczanska, E., Roberts, C., & Worm, B. (2020). Rebuilding marine life. Nature, 580, 39–51. https://doi.org/10.1038/s41586-020-2146-7

• El-Saadony, M. T., Alagawany, M., Patra, A. K., Kar, I., Tiwari, R., Dawood, M., Dhama, K., & Abdel-Latif, H. (2021). The functionality of probiotics in aquaculture: An overview. Fish and Shellfish Immunology, 117, 36–52. https://doi.org/10.1016/j.fsi.2021.07.007

• Elsabagh, M., Mohamed, R., Moustafa, E. M., Hamza, A., Farrag, F., Decamp, O., Dawood, M. A. O., & Eltholth, M. (2018). Assessing the impact of Bacillus strains mixture probiotic on water quality, growth performance, blood profile and intestinal morphology of Nile tilapia, Oreochromis niloticus. Aquaculture Nutrition, 24(6), 1613-1622. https://doi.org/10.1111/anu.12797

• Fazio, F. (2019). Fish hematology analysis as an important tool of aquaculture: A review. Aquaculture, 500, 237-242. https://doi.org/10.1016/j.aquaculture.2018.10.030

• Flemming, H. C., Wingender, J., Szewzyk, U., Steinberg, P., Rice, S. A., & Kjelleberg, S. (2016). Biofilms: An emergent form of bacterial life. Nature Reviews Microbiology, 14(9), 563–575. https://doi.org/10.1038/nrmicro.2016.94

• Food and Agriculture Organization of the United Nations. (2020). The state of world fisheries and aquaculture: Sustainability in action. FAO. https://doi.org/10.4060/ca9229en

• Foulquié Moreno, M. R., Callewaert, R., Devreese, B., Van Beeumen, J., & De Vuyst, L. (2003). Isolation and biochemical characterisation of enterocins produced by enterococci from different sources. Journal of Applied Microbiology, 94(2), 214–229. https://doi.org/10.1046/j.1365-2672.2003.01823.x

• Gewaily, M. S., Shukry, M., Abdel-Kader, M. F., Alkafafy, M., Farrag, F. A., Moustafa, E. M., Van Doan, H., Abd-Elghany, M. F., Abdelhamid, A. F., Eltanahy, A., & Dawood, M. A. O. (2021). Dietary Lactobacillus plantarum relieves Nile Tilapia (Oreochromis niloticus) juvenile from oxidative stress, immunosuppression, and inflammation induced by Deltamethrin and Aeromonas hydrophila. Frontiers in Marine Science, 8, 621588. https://doi.org/10.3389/fmars.2021.621558

• Gobi, N., Vaseeharan, B., Chen, J. C., Rekha, R., Vijayakumar, S., Anjugam, M., & Iswarya, A. (2018). Dietary supplementation of probiotic Bacillus licheniformis Dahb1 improves growth performance, mucus and serum immune parameters, antioxidant enzyme activity as well as resistance against Aeromonas hydrophila in tilapia Oreochromis mossambicus. Fish and Shellfish Immunology, 74, 501-508. https://doi.org/10.1016/j.fsi.2017.12.066

• Gopi, N., Iswarya, A., Vijayakumar, S., Jayanthi, S., Nor, S. A. M., Velusamy, P., & Vaseeharan, B. (2022). Protective effects of dietary supplementation of probiotic Bacillus licheniformis Dahb1 against ammonia induced immunotoxicity and oxidative stress in Oreochromis mossambicus. Comparative Biochemistry and Physiology Part C: Toxicology and Pharmacology, 259, 109379. https://doi.org/10.1016/j.cbpc.2022.109379

• Gudmundsdottir, B. K., & Bjornsdottir, B. (2017). Aeromonas salmonicida and A. hydrophila. In P. T. K. Woo & R. C. Cipriano (Eds.), Fish viruses and bacteria: Pathobiology and protection (pp. 173-189). CABI. https://doi.org/10.1079/9781780647784.0000

• Hai, N. (2015). The use of probiotics in aquaculture. Journal of Applied Microbiology, 119(4), 917-935. https://doi.org/10.1111/jam.12886

• Janda, J. M., & Abbott, S. L. (2010). The genus Aeromonas: Taxonomy, pathogenicity, and infection. Clinical Microbiology Reviews, 23(1), 35-73. https://doi.org/10.1128%2FCMR.00039-09

• Jinendiran, S., Archana, R., Sathishkumar, R., Kannan, R., Selvakumar, G., & Sivakumar, N. (2021). Dietary administration of probiotic Aeromonas veronii V03 on the modulation of innate immunity, expression of immune-related genes and disease resistance against Aeromonas hydrophila infection in common carp (Cyprinus carpio). Probiotics and Antimicrobial Proteins, 13, 1709-1722. https://doi.org/10.1007/s12602-021-09784-6

• Kaktcham, P. M., Temgoua, J. B., Zambou, F. N., Diaz-Ruiz, G., Wacher, C., & Pérez-Chabela, M. D. L. (2018). In vitro evaluation of the probiotic and safety properties of bacteriocinogenic and non-bacteriocinogenic lactic acid bacteria from the intestines of Nile tilapia and common carp for their use as probiotics in aquaculture. Probiotics and Antimicrobial Proteins, 10(1), 98-109. https://doi.org/10.1007/s12602-017-9312-8

• Kesarcodi-Watson, A., Kaspar, H., Lategan, M. J., & Gibson, L. (2008). Probiotics in aquaculture: The need, principles and mechanisms of action and screening processes. Aquaculture, 274(1), 1-14. https://doi.org/10.1016/j.aquaculture.2007.11.019

• Kuebutornye, F. K., Abarike, E. D., Lu, Y., Hlordzi, V., Sakyi, M. E., Afriyie, G., Wang, Z., Li, Y., & Xie, C. X. (2020). Mechanisms and the role of probiotic Bacillus in mitigating fish pathogens in aquaculture. Fish Physiology and Biochemistry, 46(3), 819-841. https://doi.org/10.1007/s10695-019-00754-y

• Lin, H. L., Shiu, Y. L., Chiu, C. S., Huang, S. L., & Liu, C. H. (2017). Screening probiotic candidates for a mixture of probiotics to enhance the growth performance, immunity, and disease resistance of Asian seabass, Lates calcarifer (Bloch), against Aeromonas hydrophila. Fish and Shellfish Immunology, 60, 474-482. https://doi.org/10.1016/j.fsi.2016.11.026

• Masduki, F., Zakaria, T., Min, C. C., & Karim, M. (2020). Evaluation of Enterococcus hirae LAB3 as potential probiont against Vibrio harveyi in Artemia nauplii and Asian seabass larvae (Lates calcarifer) cultures. Journal of Environmental Biology, 41, 1153-1159. https://doi.org/10.22438/jeb/41/5(SI)/MS_06

• Moraes, F. R., & Martins, M. L. (2004). Favourable conditions and principal teleostean diseases in intensive fish farming. In J. E. P. Cyrino, E. C. Urbinati, D. M. Fracalossi, N. Castagnolli (Eds.), Especial topics in tropical intensive freshwater fish farming (pp. 343-383). TecArt.

• Munir, M. B., Hashim, R., Mohd Nor, S. A., & Marsh, T. L. (2018). Effect of dietary prebiotics and probiotics on snakehead (Channa striata) health: Haematology and disease resistance parameters against Aeromonas hydrophila. Fish and Shellfish Immunology, 75, 99-108. https://doi.org/10.1016/j.fsi.2018.02.005

• Nayak, S. K., & Mukherjee, S. C. (2011). Screening of gastrointestinal bacteria of Indian major carps for a candidate probiotic species for aquaculture practices. Aquaculture Research, 42(7), 1034-1041. https://doi.org/10.1111/j.1365-2109.2010.02686.x

• Naylor, R. L., Hardy, R. W., Buschmann, A. H., Bush, S. R., Cao, L., Klinger, D. H., Little, D. C., Lubchenco, J., Shumway, S. E., & Troell, M. (2021). A 20-year retrospective review of global aquaculture. Nature, 591, 551–563. https://doi.org/10.1038/s41586-021-03308-6

• Ng, W. K., Kim, Y. C., Romano, N., Koh, C. B., & Yang, S. Y. (2014). Effects of dietary probiotics on the growth and feeding efficiency of red hybrid tilapia, Oreochromis sp., and subsequent resistance to Streptococcus agalactiae. Journal of Applied Aquaculture, 26(1), 22-31. https://doi.org/10.1080/10454438.2013.874961

• Puvanasundram, P., Chong, C. M., Sabri, S., Yusoff, M. S., & Karim, M. (2021). Multi-strain probiotics: Functions, effectiveness and formulations for aquaculture applications. Aquaculture Reports, 21, 100905. https://doi.org/10.1016/j.aqrep.2021.100905

• Qi, X., Xue, M., Cui, H., Yang, K., Song, K., Zha, J., Wang, G., & Ling, F. (2020). Antimicrobial activity of Pseudomonas monteilii JK-1 isolated from fish gut and its major metabolite, 1-hydroxyphenazine, against Aeromonas hydrophila. Aquaculture, 526, 735366. https://doi.org/10.1016/j.aquaculture.2020.735366

• Rengpipat, S., Rueangruklikhit, T., & Piyatiratitivorakul, S. (2008). Evaluations of lactic acid bacteria as probiotics for juvenile seabass Lates calcarifer. Aquaculture Research, 39(2), 134-143. https://doi.org/10.1111/j.1365-2109.2007.01864.x

• Restrepo, L., Domínguez-Borbor, C., Bajaña, L., Betancourt, I., Rodríguez, J., Bayot, B., & Reyes, A. (2021). Microbial community characterization of shrimp survivors to AHPND challenge test treated with an effective shrimp probiotic (Vibrio diabolicus). Microbiome, 9, 88. https://doi.org/10.1186/s40168-021-01043-8

• Román, L., Real, F., Sorroza, L., Padilla, D., Acosta, B., Grasso, V., & Acosta, F. (2012). The in vitro effect of probiotic Vagococcus fluvialis on the innate immune parameters of Sparus aurata and Dicentrarchus labrax. Fish and Shellfish Immunology, 33(5), 1071–1075. https://doi.org/10.1016/j.fsi.2012.06.028

• Rosland, N. A., Ikhsan, N., Min, C. C., Yusoff, F. M., & Karim, M. (2021). Influence of symbiotic probiont strains on the growth of Amphora and Chlorella and its potential protections against Vibrio spp. in Artemia. Current Microbiology, 78(11), 3901-3912. https://doi.org/10.1007/s00284-021-02642-2

• Soltani, M., Ghosh, K., Hoseinifar, S. H., Kumar, V., Lymbery, A. J., Roy, S., & Ringø, E. (2019). Genus Bacillus, promising probiotics in aquaculture: aquatic animal origin, bio-active components, bioremediation and efficacy in fish and shellfish. Reviews in Fisheries Science and Aquaculture, 27(3), 331-379. https://doi.org/10.1080/23308249.2019.1597010

• Stein, T. (2005). Bacillus subtilis antibiotics: structures, syntheses and specific functions. Molecular Microbiology, 56(4), 845-857. https://doi.org/10.1111/j.1365-2958.2005.04587.x

• Toscano, M., De Vecchi, E., Gabrieli, A., Zuccotti, G. V., & Drago, L. (2014). Probiotic characteristics and in vitro compatibility of a combination of Bifidobacterium breve M-16 V, Bifidobacterium longum subsp. infantis M-63 and Bifidobacterium longum subsp. longum BB536. Annals of Microbiology, 65(2), 1079-1086. https://doi.org/10.1007/s13213-014-0953-5

• Tran, N. T., Yang, W., Nguyen, X. T., Zhang, M., Ma, H., Zheng, H., Zhang, Y., Chang, K., & Li, S. (2022). Application of heat-killed probiotics in aquaculture. Aquaculture, 548(Part 2), 737700. https://doi.org/10.1016/j.aquaculture.2021.737700

• Van Doan, H., Soltani, M., & Ringø, E. (2021). In vitro antagonistic effect and in vivo protective efficacy of Gram-positive probiotics versus Gram-negative bacterial pathogens in finfish and shellfish. Aquaculture, 540, 736581. https://doi.org/10.1016/j.aquaculture.2021.736581

• Vandenbergh, P. A. (1993). Lactic acid bacteria, their metabolic products and interference with microbial growth. FEMS Microbiology Reviews, 12(1-3), 221-237. https://doi.org/10.1111/j.1574-6976.1993.tb00020.x

• Vaseeharan, B., and Ramasamy, P. (2003). Control of pathogenic Vibrio spp. by Bacillus subtilis BT23, a possible probiotic treatment for black tiger shrimp Penaeus monodon. Letters in Applied Microbiology, 36(2), 83-87. https://doi.org/10.1046/j.1472-765x.2003.01255.x

• Yasmin, I., Saeed, M., Khan, W. A., Khaliq, A., Chughtai, M. F. J., Iqbal, R., Tehseen, S., Naz, S., Liaqat, A., Mehmood, T., & Ahsan, S. (2020). In vitro probiotic potential and safety evaluation (hemolytic, cytotoxic activity) of Bifidobacterium strains isolated from raw camel milk. Microorganisms, 8(3), 354. https://doi.org/10.3390/microorganisms8030354

• Yi, C. C., Liu, C. H., Chuang, K. P., Chang, Y. T., & Hu, S. Y. (2019). A potential probiotic Chromobacterium aquaticum with bacteriocin-like activity enhances the expression of indicator genes associated with nutrient metabolism, growth performance and innate immunity against pathogen infections in zebrafish (Danio rerio). Fish and Shellfish Immunology, 93, 124-134. https://doi.org/10.1016/j.fsi.2019.07.042

• Yilmaz, S., Yilmaz, E., Dawood, M. A. O., Ringø, E., Ahmadifar, E., Abdel-Latif, H. M. R. (2022). Probiotics, prebiotics, and synbiotics used to control vibriosis in fish: A review. Aquaculture, 547, 737514. https://doi.org/10.1016/j.aquaculture.2021.737514

• Zabidi, A., Rosland, N. A., Yaminudin, J., & Karim, M. (2021). In vitro assessment of bacterial strains associated with microalgae as potential probiotics. Pertanika Journal of Tropical Agricultural Science, 44(1), 205-220. https://doi.org/10.47836/pjtas.44.1.12

• Zabidi, A., Yusoff, F. M., Amin, S. M., Yaminudin, N. J. M., Puvanasundram, P., & Karim, M. M. A. (2021). Effects of probiotics on growth, survival, water quality and disease resistance of red hybrid Tilapia (Oreochromis spp.) fingerlings in a biofloc system. Animals, 11(12), 3514. https://doi.org/10.3390/ani11123514