First Report on Probiotic Potential Characterization and Clustering Using Unsupervised Algorithm of Lactic Acid Bacteria Isolated from Air Breathing Fish

This study represents the first effort in Northeast India to isolate lactic acid bacteria from the gut of Channa punctata and to assess their probiotic attributes, as well as clustering them based on their probiotic potential. Following isolation, a comprehensive analysis was conducted, including morphological differentiation, catalase activity, IMViC tests, acid and bile tolerance, autoaggregation and coaggregation, hydrophobicity, hemolytic activity, and biosafety assays, to evaluate their probiotic potential. The most potent isolates were identified through 16S rRNA sequencing and tested for pathogen antagonism, antibiotic susceptibility, growth performance, and coexistence between the isolated probiotic strains, as well as the antagonism of the consortia against pathogens. For cluster analysis, heat maps and principal component analysis were performed. Two isolates, exhibiting the most promising probiotic characteristics among the screened isolates, were identified via Sanger’s dideoxy sequencing of the 16S rRNA gene as Leuconostoc pseudomesenteroides strain BICP3 and Streptococcus equinus strain BICP2. These two strains effectively inhibited pathogens Aeromonas hydrophila and Aeromonas jandaei and exhibited sensitivity to all the antibiotics tested, except for streptomycin. Both strains were found to be compatible and demonstrated higher in vitro inhibition against pathogens. This investigation successfully screened the probiotic potential of lactic acid bacteria colonizing the gut of Channa punctata and isolated two safe, potential probiotic strains for use in the aquaculture industry.