Isolation and characterization of lactic acid bacteria isolated from blacktip shark (carcharhinus limbatus) with probiotic potential

Abstract

A complex and diverse range of microorganism communities live within the gastrointestinal (GI) tract of living organisms. Amongst the bacterial communities in the GI tract, lactic acid bacteria (LAB) is generally regarded as safe, and probiotic due to their inherent benefits in improving the overall host GI tract, limiting intestinal infections, combatting pathogenic bacteria and controlling some types of cancer. In this study, 80 isolates were obtained from brain, epidermal organ, stomach and the end of intestinal tract of Blacktip shark (Carcharhinus limbatus). Preliminary inhibition test using spot-on-lawn assay was conducted to eliminate the isolate with no antagonistic activity against selected indicator strains. Out of these 80, only 4 isolates showed antagonistic activities against selected indicator strains in which they were selected for further biochemical tests such as Gram-staining, catalase, oxidase, and motility, the were found to be Gram positive coccoid shape, lactose fermentation positive, and negative to both oxidase and catalase tests. The isolates FA1, FA2, FA3 and FA4 demonstrated broad range of activity spectrum by inhibiting the Gram-positive indicator strains (Staphylococcus aureus and Bacillus cereus) and the Gram-negative indicator strains (Escherichia coli, Pseudomonas aeruginosa, salmonella typhimurium, Vibrio alginolyticus and Vibrio parahaemolyticus); with the highest inhibition zone recorded at 20 ± 0.1 mm on V. parahaemolyticus using agar well diffusion method as a confirmatory test. The isolates were chosen for further characterization using 16S rRNA PCR amplification. Each sequence was respectively deposited in GenBank (NCBI) with an accession number: MN975529 for FA1, MN982712 for FA2, MN982711 for FA3 and MN982710 for FA4. These isolates were identified to be belonged to member of the Lactococcus lactis group with 98% homology. The cell free supernatant (CFS) of the isolates was harvested for further tests. Treatment of the CFS with pH and catalase ruled out any inhibition caused by organic acids or hydrogen peroxide (H2O2) and was considered as crude bacteriocin. Complete inactivation of the isolates CFS antimicrobial activity was observed after treatment with proteinase K, thus proving its proteinaceous nature. This is one of a very few reports on LAB strains isolated from the Blacktip shark (Carcharhinus limbatus) producing antimicrobial proteins or bacteriocin. These isolated strains could prove to be a very useful probiotic strain in aquaculture or animal feed as a mean to fight the ever-growing problem of drug resistant pathogenic bacteria as well as, promoting growth and the overall host health.