Efficient disease management in aquaculture is crucial for sustainable and successful production of aquatic animals (Abarike et al., 2018). The widespread and improper use of antibiotics has raised significant biological and ecological concerns, particularly regarding the emergence of antibiotic-resistant bacteria (Yang et al., 2019). Beneficial microbes, known as probiotics, are recommended as antibiotic substitutes because they promote health, effectiveness, environmental friendliness, and pathogen resistance (Vijayaram et al., 2024). Probiotic Bacillus sp. exhibit various characteristics, including the production of non-pathogenic substances, enhancement of water quality, heat tolerance, extended shelf life, and resilience under harsh conditions owing to their sporulation capability (Kuebutornye et al., 2019; Amoah et al., 2021). In aquaculture, Probiotic Bacillus sp. are isolated from diverse sources, such as decaying matter, water, commercial sources, fish and vertebrate gastrointestinal tracts, and soil. Recently, Bacillus sp. have shown beneficial effects in aquaculture, with multiple studies highlighting their positive effects on Nile tilapia cultivation (Kuebutornye et al., 2019; Atef et al., 2024; Shija et al., 2023). In this study, the probiotic potential of three Bacillus sp. (B. amyloliquefaciens AV5(OR647358), B. subtilis AV7 (LC781790), and B. velezensis AV50 (OR647359)) isolated from Nile tilapia intestines was investigated. The strains AV5, AV7, and AV50 were identified based on morphological traits and biochemical tests and confirmed by 16S rDNA gene sequencing. All isolates demonstrated the ability to utilize various carbon sources, including amino acids, such as arginine, inositol, adonitol, citrate, gelatin, lactose, starch, rhamnose, sorbitol, and glucose. These characteristics suggest their potential utility as probiotics and in the production of value-added products in the food industry, as documented in previous studies (Lee et al., 2017; Kavitha et al., 2018; Kuebutornye et al., 2020). In contrast to other probiotics, Bacillus strains produce heat-resistant spores (Kuebutornye et al.,2019; Amoah et al., 2021), and are resistant to low pH and high bile concentrations (Šimunović et al., 2022). They have also demonstrated the ability to thrive in the gastrointestinal tract of fish (Amoah et al., 2021; Šimunović et al., 2022). According to Garcia-Ruiz et al.(2014), there is a high survival rate of Bacillus sp. in the human gut with varying bile contents (0.3–0.5%). The ability to withstand harsh heat conditions, often used in animal feed production for pathogen elimination and flavor enhancement, is a crucial biotechnological trait of probiotics (Guo et al., 2016). In our study, all isolated strains (AV5, AV7, and AV50) demonstrated high sporulation efficiency, enabling them to survive at 0.5% bile concentration (Fig. 5), pH as low as 1 (Fig. 8), and post-heat treatment, surpassing the control group (Fig. 6). Furthermore, higher heat treatments (80°C to 100°C) activated the selected isolates, promoting faster growth. Bacillus velezensis (Ye et al., 2018), Bacillus subtilis (Guo et al., 2016), and Bacillus amyloliquefaciens (Reda et al., 2018) have been extensively studied for their sporulation ability. Probiotics possess a crucial ability to colonize and adhere to epithelial cells and mucosal surfaces, thereby ensuring resistance to alterations in intestinal contents and impeding the attachment of harmful bacteria throughout the intestine, ultimately minimizing inflammation (Shinde et al., 2019; Rohith & Halami, 2021; Li et al., 2020). Auto-aggregation and hydrophobicity are indirect measures of bacterial adhesion (Kuebutornye et al., 2020; Amoah et al., 2021). AV5 exhibited significantly higher hydrophobicity (97.5% and 97.1% with chloroform and xylene, respectively) than AV50 (92.8% and 95.8%) and AV7 (85.1% and 96.6%, respectively), indicating its superior attachment to hydrocarbons. AV7 showed a higher hydrophobicity than AV50 and AV5 with ethyl acetate (90.6%). Our study found significantly higher hydrophobicity results compared to those of Lee et al.(2017), suggesting enhanced electron donation and acceptance for better epithelial cell adhesion. In addition, auto-aggregation activity is directly proportional to cell adhesion in the digestive tract, which is a prerequisite for beneficial probiotic bacteria (Amoah et al., 2021). AV5, AV7, and AV50 exhibited significant auto-aggregation rates of 92.66%, 84.67%, and 89.33%, respectively, consistent with previous studies (Kuebutornye et al., 2020; Amoah et al., 2021).
Another vital aspect for identifying potential probiotic bacteria in vitro is the evaluation of hemolytic activity, compatibility, and biofilm formation (Kuebutornye et al., 2020; Amoah et al., 2021). Either γ-hemolysis or α-hemolysis is safe, whereas β-hemolysis is harmful (Deng et al., 2023). In the current study, all strains exhibited γ-hemolysis, indicating non-hemolytic behavior and enhancing the safety of host organisms, which aligns with the results of previous studies (Amoah et al., 2021; Lee et al., 2017; Kavitha et al., 2018). Compatibility assays determine whether strains can be used together as multispecies probiotics, which is crucial for maintaining product quality (Kuebutornye et al.,2020). Our study confirmed mutual compatibility among the AV5, AV7, and AV50 strains. Biofilm formation, which is advantageous in some contexts, requires careful evaluation owing to potential antimicrobial resistance implications (Kuebutornye et al., 2020). None of our isolates (AV5, AV7, and AV50) formed biofilms, which is consistent with previous findings (Kuebutornye et al., 2020; Kavitha et al., 2018). To be labeled as a probiotic, a bacterium must be non-harmful to the host organism and incapable of transmitting antibiotic resistance genes (Elshaghabee et al., 2017; Ouwehand et al., 2016). Biosafety assessments demonstrated that all isolates were suitable for Nile tilapia, and the results were consistent with those of previous studies (Amouh et al., 2021; Kuebutornye et al 2020). Additionally, antibiotic susceptibility testing indicated high susceptibility of all isolates to 17 of the 20 antibiotics tested, highlighting their potential safety and efficacy in aquaculture settings (Guo et al., 2016; Kuebutornye et al., 2020). The AV7 strain was resistant to ampicillin, vancomycin, furazolidone, and clindamycin and demonstrated moderate susceptibility to medecamycin and sulfamethoxazole. In contrast, the AV5 strain exhibited only moderate susceptibility to furazolidone. Notably, AV7 exhibited resistance to these antibiotics possibly because of the presence of natural resistance genes (Larsen et al., 2014). The isolated strains AV5, AV7, and AV50 also demonstrated significant antagonistic effects against common fish pathogens, such as V. alginolyticus, S. iniae, V. harveyi, and S. agalactiae, suggesting their potential for disease prevention and enhancing aquaculture sustainability, which aligns with previous results (Bluford et al., 2017; Shoemaker et al., 2001; Bowater et al., 2012). The results suggest that the three strains AV5, AV7, and AV50 possess the ability to combat fish diseases, thereby promoting the sustainability of the aquaculture sector.