Identification and Characterization of four Bacillus species from the intestine of hybrid grouper (Epinephelus fuscoguttatus♀ × E. lanceolatus♂), their antagonistic role on common pathogenic bacteria, and effects on intestinal health.

As an alternative to the criticized antibiotics, probiotics have been adopted for their eco-friendly nature and ability to enhance host growth and immunity. Nevertheless, reports suggest ineffectiveness in commercially available probiotics since most are from non-fish sources; thus, this study was envisaged to isolate and characterize new Bacillus spp. from the gut of hybrid grouper (Epinephelus fuscoguttatus♀×Epinephelus lanceolatus♂) as potential probiotics. The isolation and characterization were performed based on their morphological and biochemical properties and 16S rRNA sequencing homology analysis, and a subsequent 4-week in vivo biosafety feeding trial was conducted to ascertain the effects on isolates' non-pathogenicity, growth, and intestinal mucosal microvilli via scanning electron microscopy (SEM) analysis. Four Bacillus spp. strains, namely, B. velezensis strain PGSAK01 (accession number OQ726606), B. stercoris strain PGSAK05 (accession number OQ726607), B. velezensis strain PGSAK17 (accession number OQ726601), and B. subtilis strain PGSAK19 (accession number OQ726605), were identified and characterized in the current study. The strains showed promising probiotic properties such as showing higher adhesion capability, higher thermotolerance, displaying higher survivability to 0.5% bile, lower pH tolerance, γ-haemolytic activity, and multispecies characteristics. Among the 24 antibiotics tested, isolates were susceptible to 21, whereas the PGSAK01 strain showed resistance to furazolidone antibiotics. None of the isolates showed possession of i) virulence factor genes encoding enterotoxigenic (hblA, hblC, hblD, nheA, nheB, and entFM) and emetic (cereulide synthetase gene, ces) genes, and ii) streptomycin resistance gene (vat c), ampicillin-resistant genes (mecA and bla), and vancomycin-resistant gene (van B). Nevertheless, the PGSAK01 and PGSAK17 strains showed possession of tek K, cat, and ant(4')-Ia (adenylyltransferase) (except the PGSAK01) resistant genes. All isolates displayed better antimicrobial effects against pathogenic bacteria Streptococcus agalactiae, S. iniae, Vibrio harveyi, and V. alginolyticus. The vivo biosafety trial involved hybrid grouper fish being grouped into five (average weight 32±0.94 g), namely, the group fed the basal diet void of isolate's supplementation (control), and the remaining four groups fed the basal diet with 1×108 CFU/g diet of individual strain PGSAK01, PGSAK05, PGSAK17, and PGSAK19 supplementation. At the end of the study, a significantly higher WGR, K (except the PGSAK01 group), VSI; lysozyme (except PGSAK01 group), superoxide dismutase, total antioxidant activity, alkaline phosphatase enzyme activities; highly dense intestinal mucosal villi (based on the scanning electron microscopy analysis); and significantly lower malondialdehyde levels were witnessed in the isolated treated groups compared to the control, supporting the results obtained in the auto-aggregation and cell-surface hydrophobicity test. This work's results have provided thought-provoking targets; thus, studies involving extensive genome sequencing and functional annotation analysis will be explored to offer unfathomable insights into their mechanisms of action and potential health benefits, further establishing the four Bacillus strains PGSAK01, PGSAK05, PGSAK17, and PGSAK19 potential role in probiotic fields and functional foods.