Total Bacteria Isolated from 3 Sponges

Results

Isolation of sponge-associated bacteria from 3 different sponges resulted 80 bacterial isolates which are morphologically different. Nine bacterial isolates were obtained from sponge Hyrtios sp., 20 bacteria isolated from Smenospongia sp., and 51 bacteria isolated from Verungola sp.

Sponge-associated bacteria with anti-Vibrio spp properties

Twelve (15%) out of 80 bacterial isolates were able to inhibit Vibrio’s growth indicated by clear zone formation around the bacterial colony (Table 1). One bacterial strain (P2.24) could inhibit three Vibrio species, including V. harveyi, V. parahaemolyticus, and V. vulnificus. In addition, 10 bacterial strain specifically only inhibit V. harveyi, and 1 strain (P3.310) could inhibit both V. harveyi and V. parahaemolyticus. Twelve bacterial isolates showed a diverse clear zone diameter, ranging from 0.1 mm to 5 mm. The best anti-Vibrio spp. activity showed by P2.24 strain due to its ability to inhibit three Vibrio species with different clear zone diameter. To confirm its anti-Vibrio spp. activity, the concentrated culture, supernatants and metabolite extracts of the strain P2.24 were tested in antagonism assay.

Hemolytic Reaction of the potential bacteria

Twelve potential strains producing anti-Vibrio spp. compounds were hemolytic negative. They can not lysis blood cells as indicated by no clear zone formed around the colony after 24 h of incubation.

Three (P2.24, P3.310, D4.13) out of 4 potential strains (based on their ability in inhibiting Vibrio spp growth in wide spectra) has been identified to have both NRPS and PKS genes, and one strain (P2.211) do not have both genes, as shown by Table 2. Adenilase (A) domain of NRPS and ketosynthase (KS) domain of PKS gene from these bacteria have been amplified by PCR method resulting ±1000 bp and ±700 bp DNA fragment, respectively (Fig. 1). Alignment using BlastN showed all NRPS and PKS genes were similar with NRPS and PKS genes of Bacillus spp in various strains. In addition, amplification of 16S rRNA gene of four strains resulted ±1300 bp fragment. All three strains were highly homolog with Bacillus spp (Table 2).

The genetic relationship for NRPS-PKS gene of the potential strains was compared with their relative gene in GenBank NCBI database and some references by constructing the phylogenetic tree. The phylogenetic tree of A domain from NRPS gene and KS domain of PKS gene was shown in Figure 2 and Figure 3, respectively. The evolutionary relationship based on 16S rRNA of the potential strains with their close related strains was performed in Figure 4.

Antibacterial activity of culture, supernatant, and extract of the strain P2.24

Bacterial culture used in this assay contains approximately 7.8 x 107 cell/mL medium or 1.6 x 107 cell/ 20 µL. Culture, supernatant as well as the extract of the strain P2.24 consistently exhibited anti-Vibrio spp. activities as indicated by clear zone formation around paper disk (Fig.5). The strongest inhibitory effect was shown by the concentrated culture of the strain P2.24 against V. parahaemolyticus. Clear zone also was exhibited by ampicillin as positive control. There is no clear zone obtained from DMSO as negative control.