Identification of bacteria found in holothuria tubulosa and detection of their antibacterial activity

Abstract

Sea cucumbers have been shown to produce antibacterial agents. This research tested the hypothesis that the antibacterial agents were produced by bacteria indigenous to the sea cucumber, as part of a symbiotic relationship. Research was done using 2 specimens of Holothuria tubulosa and a total of 23 bacterial strains were isolated from 5 zones of the sea cucumber and also from the sea-water as a control. All strains were grown using brain-heart agar and broth prepared with sterile sea-water. From the 23 strains, seven strains were selected for further analysis. These 7 were identified as J1:2 = K. pneumoniae, J2:2 = B. subtillis, J2:3 = B. licheniformis, J2:6 = B. licheniformis, J2:7 = K. pneumoniae, J2:10 = B. subtillis and J1:12 = K. pneumoniae, based on PCR 16S rRNA gene specific forward and reverse primers, and subsequent sequencing of the approximately 700bp products. Selection was based on antibacterial well diffusion testing results, with the 7 selected strains having higher and more consistent antibacterial activity in comparison to the other isolated strains from H. tubulosa. In turbidometric analysis, all 7 strains’ extracts delayed the exponential growth phase of test pathogens K. pneumoniae and S. typhimurium compared to the negative control (negative control = approx. 120 minutes, test samples = approx. 200 minutes). Size exclusion chromatography using Sephadex G-25 (1-5kDa separation) was carried out on all 7 strains. A hundred 1ml fractions were collected for each strain and absorbance readings were taken at 600nm. Two major peaks were seen in the chromatograms for all 7 strains, the first between fractions 20 to 40 (5kDa range) and the second at fractions 50 to 70 (1kDa range). SDS-PAGE analysis on fractions 27-33 of bacterial strain J2:2 (B. subtilis strain) showed 3 peptide bands, X (MW = 20413), Y (MW = 18742) and Z (MW = 5792). The results from this research showed that is possible that antibacterial agents found in sea cucumbers are made by microflora indigenous to the sea cucumber.