ANTIBACTERIAL AND ANTICANCER ACTIVITIES OF MARINE BACTERIAL EXTRACTS AND DETECTION OF GENES FOR BIOACTIVE COMPOUNDS SYNTHESIS
Objective: This study was aimed to determine the antimicrobial, toxicity, and anticancer activities of ethyl acetate extracts from sponge-associated bacteria and to detect the presence of PKS and NRPS genes.
Methods: Crude extracts of sponge-associated bacteria HAA-01, HAL-13, and HAL-74 were used as samples for toxicity test using Brine Shrimp Lethality Test (BSLT). The Minimum Inhibitory Concentration (MIC) of bacterial extracts were tested using agar diffusion method. Cytotoxicity test was carried out by 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT)method against HeLa cell line. To determine the presence of keto synthase (KS) domain of polyketide synthase (PKS) and adenylation (A) domain of nonribosomal peptide synthetase (NRPS) genes were carried out using Polymerase Chain Reaction (PCR). DNA of PCR products were purified and sequenced. The DNA sequences were analyzed by the Blast program, and phylogenetic tree analysis was constructed using MEGA5 program with neighbor-joining method.
Results: The research results indicated that the best value of MIC of bacterial extracts against five bacteria such as Escherichia coli, Enteropathogenic E. coli (EPEC), Pseudomonas aeruginosa, Staphylococcus aureus and Bacillus subtilis were 0.1 mg/ml. The half maximal lethal concentration (LC50) value of HAL-74, HAA-01 and HAL-13 bacterial metabolite extracts against the brine shrimp Artemia salina were 378 Î¼g/ml, 463.028 Î¼g/ml and 444.796 Î¼g/ml, respectively. The bacterial metabolite extract has cytotoxicity effect on HeLa cell line with the best half maximal inhibitory concentration (IC50) value was 132.877 Âµg/ml as performed by extract of HAL-74. Genetic analysis showed that HAL-74 and HAA-01 genomes contained genes that encoded the KS domain of PKS gene and A domain of NRPS gene, while HAL-13 genome encoded the KS domain of PKS gene, which played a role in the synthesis of bioactive compounds.
Conclusion: Bacterial metabolite extracts of HAA-01, HAL-13, and HAL-74 had antimicrobial, toxicity against A. salina, and anticancer against HeLa cell line activities. The best toxicity and anticancer activity were performed by HAL-74 crude extract with LC50 and IC50 were 378 mg/ml and 132.877 mg/ml, respectively. HAA-01 and HAL-74 genomes contained PKS and NRPS genes while HAL-13 genome contained PKS gene.
2. World Health Organization; 2014. Cancer Health Topic. Available from: http://www.who.int/cancer/en/index.html. [Last accessed on 15 Aug 2014].
3. Taylor MW, Radax R, Steger D, Wagner M. Spons associated microorganisms: evolution, ecology, and biotechnological potential. Microbiol Mol Biol Rev 2007;71:295-347.
4. Imhoff JF, Stohr R. Sponge-associated bacteria: general overview and special aspects of bacteria associated with Halichondria panicea. Prog Mol Subcell Biol 2003;37:35-57.
5. Hoffmann F, Larsen O, Thiel V, Rapp HT, Pape T, Michelish W, et al. An anaerobic world in sponges. J Geomicrobiol 2005;22:1-10.
6. Proksch P, Edrada RA, Ebel R. Drugs from sea current status and microbiological implications. Appl Microbiol Biotechnol 2002;59:125-34.
7. Tokasaya P. Sponge-associated bacteria producing antimicrobial compounds and their genetic diversity analysis [thesis]. Bogor (ID): Institut Pertanian Bogor; 2010.
8. Banoet Y. Aktivitas senyawa bioaktif antimikrob dari bakteri yang berasosiasi dengan spons Haliclona sp. dan telaah genetiknya [thesis]. Bogor (ID): Institut Pertanian Bogor; 2011.
9. Muller WEG, Grebenjuk VA, Pennec G, Schroder H, Brummer F, Hentschel U, et al. Sustainable production of bioactive compounds by sponges-cell culture and gene cluster approach: a review. Mar Biotechnol 2004;6:105-17.
10. Sudirman LI. Partial purification of antimicrobal compounds isolated from mycelia of tropical Lentinus cladopus LC4. Hayati J Biosci 2010;17:63-7.
11. Meyer BN, Ferrigni NR, Putnam JE, Jacobsen LB, Nichols DE, McLaughlin JL. Brine shrimp: a convenient general bioassay for active constituent. J Med Plants Res 1982;45:31-4.
12. Thakur AN, Thakur NL, Indap MM, Pandit RA, Datar VV, Muller WEG. The antiangiogenic, antimicrobial, and cytotoxic potential of sponge-associated bacteria. Mar Biotechnol 2005;7:245-52.
13. Sambrook W, Russel DW. Molecular cloning: a laboratory manual. 3rd Ed. New York: Gold Spring Harbor Laboratory; 2001. p. 362.
14. Schirmer A, Gadkari R, Reeves CD, Ibrahim F, DeLong EF, Hutchinson CR. Metagenomic analysis reveals diverse polyketide synthase gene cluster in microorganisms associated with the marine sponge Discodermia dissoluta. Appl Environ Microbiol 2005;71:4840-9.
15. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. MEGA5 molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 2011;28:2731-9.
16. Wahi AK, Singh A, Singh AK. Determination of minimum inhibitory concentration (MIC) of some novel triazole. Int J Res Pharm Chem 2011;4:1108-14.
17. Holetz FB, Pessini GL, Sanches NR, Cortez DAG, Nakamura CV, Filho BPD. Screening of some plants used in the Brazilian folk medicine for the treatment of infectious diseases. Mem Inst Oswaldo Cruz 2002;97:35-47.
18. Schwarzer D, Finking R, Marahiel MA. Nonribosomal peptides: from genes to products. Nat Prod Rep 2003;20:275-87.
19. Kim TK, Fuerst JA. The diversity of polyketide synthase genes from bacteria associated with the marine sponge Pseudoceratina clavata: culture-dependent and culture-independent approaches. Environ Microbiol 2006;8:1460-70.
20. Zhao J, Yang N, Zeng R. Phylogenetic analysis of type I polyketide synthase and nonribosomal peptide synthetase genes in antarctic sediment. Extremophiles 2008;12:97-105.
21. Shen B, Du L, Sanchez C, Edwards DJ, Chen M, Murrell JM. The biosynthetic gene cluster for the anticancer drug bleomycin from Streptomyces verticillus ATCC15003 as a model for hybrid peptide-polyketide natural product biosynthesis. J Ind Microbiol Biotechnol 2001;27:378-85.
22. Muscholl-Silberhorn A, Thiel V, Imhoff JF. Abundance and bioactivity of cultured sponge-associated bacteria from the mediterranean sea. Microb Ecol 2008;55:94-106.
23. Nagai K, Kamigiri K, Arao N, Suzumura K, Kawano Y, Yamaoka M, et al. YM-266183 and YM-266184, novel thiopeptide antibiotics produced by Bacillus cereus isolated from a marine sponge. J Antibiot 2003;56:123-8.