Evaluating the probiotic potential of Bacillus pumilus HFS2 isolated from the gut microbiota of Heteropneustes fossilis for aquaculture applications

Abstract

Background Probiotics are increasingly used in aquaculture to enhance fish survival, growth, and health, offering a sustainable approach to fish farming. Objective This study assessed the probiotic properties of Bacillus pumilus HFS2, isolated from the digestive tract of Heteropneustes fossilis. Method Among fourteen isolates, HFS2 was characterized using physiological, biochemical, and 16S rRNA analyses, and it's in vitro probiotic properties as well as in vivo safety were evaluated. Results The isolate was Gram-positive, rod-shaped, motile, and aerobic, showing positive results for methyl red, catalase and Voges–Proskauer tests, and a negative result for the indole test. It fermented dextrose, lactose, maltose, and mannitol with acid production but did not ferment sucrose. In vitro probiotic assessments showed that the strain tolerated a temperature range of 4–35 °C, survived at pH 2–5, and tolerated bile concentrations of 2.5–7.5%. The isolate demonstrated promising adhesion-related properties, including autoaggregation (66%), cell surface hydrophobicity (69% with xylene and 68% with toluene), and coaggregation with Lactococcus lactis (53%) and Staphylococcus arlettae (49%). It also exhibited inhibitory activity against fish pathogens such as Lactococcus garvieae, Aeromonas caviae, A. jandaei, A. veronii, and A. hydrophila. The isolate exhibited γ-hemolytic activity, strong biofilm-forming ability, and susceptibility to commonly used antibiotics, including ampicillin, vancomycin, erythromycin, tetracycline, chloramphenicol, and ciprofloxacin, while resistance observed only against cefotaxime. In vivo safety evaluation through intraperitoneal injection and immersion challenge at 108 CFU/mL indicated no pathogenic effects in H. fossilis. Conclusion These results suggest that B. pumilus HFS2 is a resilient and promising potential probiotic candidate for aquaculture applications.