Isolation, Characterization and Molecular Identification of Putative Probiotic Bacteria From Red Tilapia Gut

The use of probiotics is a promising strategy to enhance fish health and sustainability in aquaculture as alternatives to antibiotics. This study applied a systematic multi-criteria screening approach to identify autochthonous probiotic candidates from the intestinal microbiota of healthy red tilapia (Oreochromis sp.). A total of 26 culturable bacterial isolates were recovered and evaluated for antimicrobial activity against major fish pathogens (Aeromonas hydrophila, Streptococcus agalactiae and Streptococcus iniae), safety, tolerance to simulated gastrointestinal conditions, adhesion-related properties, enzymatic activities and environmental robustness. Only a small subset of isolates exhibited broad-spectrum antimicrobial activity and satisfied subsequent safety and functional criteria. Based on integrated performance, three isolates were shortlisted for advanced evaluation, of which two were retained for molecular identification. The selected isolates were identified by 16S rRNA gene sequencing as Pseudomonas aeruginosa PA4 (PA4) and Bacillus sp. BA4P (BA4P). Both strains demonstrated tolerance to gastrointestinal stress, measurable adhesion-related hydrophobicity, diverse enzymatic activities and stability under varying temperature and salinity conditions. These findings provide a robust framework for screening host-associated probiotics and support the further evaluation of PA4 and BA4P as potential candidates for sustainable tilapia aquaculture.