Development of a Nile tilapia (Oreochromis niloticus) gut microbiota-derived bacterial consortium with antibacterial activity against fish pathogens

As the world's population is rising, the demand for food is increasing. >59.7 million people worldwide rely directly on the fishing industry; 67.6% are fishery-based and 32.4% are aquaculture-based (FAO, 2019). Fish is a healthy alternative to the consumption of red meat because it represents a key source of protein, essential amino acids, lipids, and minerals (Giri, Sukumaran, Sen, & Jena, 2014; Ullah et al., 2018). The increased demand for aquaculture fish calls for improvements in crop technology and management practices (Sankar, Philip, Philip, & Singh, 2017). Large-scale production at aquaculture facilities exposes fish to stressful environments, increasing the vulnerability to disease outbreaks and the potential for significant economic losses (Salah Mesalhy Aly, Yousef Abdel-Galil Ahmed, et al., 2008).

For instance, a variety of pathogens, such as Streptococcus agalactiae and Aeromonas hydrophila, cause different disease outbreaks in tilapia, one of the most economically significant farmed fish in the world, resulting in massive economic losses to the tilapia industry (Ding et al., 2017; Kuebutornye, Abarike, & Lu, 2019; Mu et al., 2021; Wang, Du, Jiang, He, & Li, 2019). For that reason, most of the article of multistrain probiotic administration are focus in a challenge of commercial fish against A. hydrophila (62% of 81 articles) and S. agalactiae (15% of 81 articles) (Melo-Bolívar, Ruiz Pardo, Hume, & Villamil Díaz, 2021). In particular, streptococcal infections, which are prevalent in numerous countries and regions, are associated with high mortality rates and catastrophic illnesses. Streptococcal infections in tilapia generate an annual economic loss more than $250 million (Zhang et al., 2020).

Antibiotics are widely used as a conventional strategy for disease control, increased production, and improved feed conversion efficiency (Anuar, Omar, Noordiyana, & Sharifah, 2017). However, significant antibiotic misuse has been unacceptable for aquaculture due to the adverse effects of antibiotics on the environment and the well-being of fish, carcinogenic effects, and the development of antibiotic resistance by pathogens (Ullah et al., 2018). Therefore, numerous biotechnology approaches have been studied to prevent associated damage while improving livestock health and production due to the adverse effects of antibiotics on livestock, the ecosystem, and possibly end-use consumers. These aquaculture biotechnologies may include nonspecific immunostimulants, vaccines, probiotics, prebiotics, symbiotics, medicinal plants, and other ways of managing fishery disease (Assefa & Abunna, 2018). Of these biotechnologies, probiotics present unique potential since they can offer combined benefits of several strategies.

Probiotics are “live microorganisms that, when administered in adequate amounts, confer a health benefit on the host” (Hill et al., 2014). Probiotic microorganisms can colonize and multiply in the host gastrointestinal tract (GIT) and benefit the host and its environment (Abu-Elala, Marzouk, & Moustafa, 2013). Probiotic use in aquaculture can enhance growth and disease tolerance, improve water quality parameters, increase host nutrition through digestive enzyme production, eliminate harmful bacteria, and improve survival and stress tolerance (Kanwal & Tayyeb, 2019). Commercial probiotics in the aquaculture industry are often isolated from terrestrial animals and, therefore, cannot adapt suitably to the aquatic environment, which leads to a loss of viability and lowers the capacity to modulate the intestinal microbiota and elicit the expected benefits. For these reasons, fish-isolated microorganisms are recommended to be evaluated for probiotic potential and improved efficacy (Del'Duca, Evangelista Cesar, Galuppo Diniz, & Abreu, 2013).

Also, feed supplementation is a better technique for ensuring the efficacy of probiotic bacteria in the GIT of fish, which avoids the need for the bacteria to come into contact with the surrounding environment (Bidhan et al., 2014). For that reason, most freshwater fish studies have incorporated probiotics into the diet (Melo-Bolívar et al., 2021). Technological challenges, such as bacterial inclusion in extruded feeds, must be considered before industrial-scale application because, as previously mentioned, the loss of probiotic viability has a detrimental impact on the effectiveness of many products (Merrifield et al., 2010; Stephania Aragón-Rojas, Yolanda Ruiz-Pardo, Javier Hernández-Álvarez, & Ximena Quintanilla-Carvajal, 2020). Extreme temperatures and pressures experienced during extrusion or pellet manufacture may reduce the viability of probiotic microorganisms (Burr, Gatlin, & Ricke, 2005). Therefore, applying the probiotic organism to the feed after extrusion is necessary to maintain probiotic viability because the probiotic organism would be subjected to intense heat and pressure if incorporated before extrusion (Gatlin & Peredo, 2012).

Strategies have yet to be adequately established for maintaining probiotic efficacy while preserving the viability of the organisms (Ganguly, Banerjee, Mandal, & Mohapatra, 2019). Some approaches recognize that probiotic microbe viability depends on specific storage requirements and that many probiotic bacteria experience reduced viability while in storage (Choudhury & Kamilya, 2018; Maji, Mohanty, Pradhan, & Maiti, 2017). For that reason, in the present work, we extend our previous work by defining the proportions of each bacterium in the mixture with the highest antibacterial activity against S. agalactiae and A. hydrophila, incorporating the potential probiotic mixture into the fish feed, and evaluating viability during storage over 28 days at 4 °C and 25 °C.

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