International Journal of Aquaculture, 2026, Vol.16, No.4, 234-242 http://www.aquapublisher.com/index.php/ija 238 Table 5 Acid tolerance of isolates Isolate pH 2.5 pH 3.0 pH 3.5 pH 4 pH 7 Bacillus spp. 1.8±0.2 2.2±0.4 2.6±0.2 2.8±0.3 3.4±0.2 Cocci bacteria 0.4±0.1 0.7±0.2 0.9±0.1 1.1±0.2 1.8±0.3 Statistical analysis showed significantly higher acid tolerance (p < 0.05) for Bacillus isolates across all pH levels. Table 6 Bile tolerance Isolate 0.2% 0.4% 0.6% 1.0% Bacillus spp. 2.8±0.3 2.2±0.1 1.9±0.2 1.6±0.1 Cocci bacteria 2.7±0.1 2.1±0.3 1.6±0.1 1.3±0.2 The superior tolerance exhibited by Bacillus spp. indicates their adaptation to intestinal bile conditions. 3.4 Antimicrobial activity As presented in table 7 and figure 2, the inhibitory activity of the tested probiotic isolates against Escherichia coli isolates showed clear differences between Cocci spp. and Bacillus spp., as demonstrated by their respective zones of inhibition. Bacillus spp exhibited the highest antagonistic effect with a mean inhibition zone of 34 mm, while Cocci spp. produced a comparatively smaller zone of 26 mm. The significantly larger inhibition zone produced by Bacillus spp. indicates a stronger antimicrobial potency against the target pathogen. Table 7 Zone of inhibition against pathogens Pathogen Cocci spp. (mm) Bacillus spp. (mm) Escherichia coli 26 34 Salmonella typhi 29 29 Staphylococcus aureus 39 39 Bacillus isolates demonstrated strong antagonistic activity, suggesting production of antimicrobial compounds. Figure 2 Diameter of inhibition zone of Bacillus clausii tested against A=Escherichia coli B=Salmonella typhi C=Staphylococcus aureus isolates. Mean radii with different superscripts indicate significant (α0.05) difference according to DMRT. 3.5 Molecular identification PCR amplification of the 16S rRNA gene produced single bands of approximately 1500 bp, confirming successful amplification. Sequencing analysis identified the isolate as Bacillus clausii. Phylogenetic analysis revealed a close evolutionary relationship between the identified strain and previously reported Bacillus clausii strains retrieved from the NCBI GenBank database (Figure 3). The identified strain showed close clustering with Bacillus clausii PRA25, Bacillus clausii AK31, and Bacillus clausii XJU2. Bootstrap values ranged from 86% to 98%, indicating strong support for the phylogenetic clustering.
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