Third International Seminar on Global Health (3rd ISGH) Technology Transformation in Healthcare for a Better Life ISGH 3 | Vol 3. No. 1 | Oktober 2019 | ISSN : 2715-1948 ISOLATION AND IDENTIFICATION OF CORAL REEF BACTERIA POTENTIAL CANDIDATES FOR PRODUCING BIOACTIVE COMPOUND Maelita Ramdani Moeis1*, Rohmah Nasada Tuita1, Firdha Rachmawati2 [email protected] 1Institut Teknologi Bandung 2Department of Medical Laboratory Technology, School of Health Sciences Jenderal Achmad Yani Cimahi, Indonesia ABSTRACT Background: There is an urgent need to develop new drugs to control serious pathogen. Terrestrial origin of bioactive compound and secondary metabolite has been diminished to be studied. Therefore, more exploration of bioactive compound and secondary metabolite from marine environment becomes important. Acropora digitifera is a coral that is widespread in Indo-Pacific. Many microorganisms live associated with coral reef doing their role. Many active compound have been isolated and characterized from marine microorganisms, which potentially to be used for therapeutic purposes. Therefore, this research was conducted as an initial stage of research to obtain bacteria that have the potential to produce bioactive compounds and secondary metabolites. Objectives: Isolation and identification bacteria from coral reef Acropora digitifera Methods: DNA sample Acropora digitifera was amplified using bacterial marker gene, 16S rRNA. 16S rRNA gene was ligated to cloning vector pGEM-T Easy and transformated to E. coli DH5a. The inserted gene in recombinant plasmid was confirmed by PCR. The gene was sequenced by Macrogen, Korea. The sequence was analysed using BLAST (Basic Local Alignment Search Tool) (http://www.blast.ncbi.nlm.nih.gov/Blast.cgi) and phylogenetic tree by MEGA6 with parameter (distance-based) Neighbor Joining (NJ) bootstrap 1000. Results:Twelve isolates were isolated. Seven isolates were Pseudomonas. Three isolates identified as Endozoicomonas euniceicola and one isolate identified as Endozoicomonas elysicola. Endozoicomonas was known has antimicrobial activity. One isolate identified as Brevibacterium permense. Many of Brevibacterium strain was known as bacteriocins producers. Conclusions:Several genus and species bacteria can be isolated from coral reef Acropora digitifera. There are Endozoicomonas euniceicola, Endozoicomonas elysicola, Brevibacterium permense and several genus of Pseudomonas Keywords: Acropora digitifera, Bio-active compound, Marine bacteria, Metagenome, Secondary metabolite. INTRODUCTION microbial infections (Rohwer, Seguritan, Azam, & Indonesia has a coral reef ecosystem covering Knowlton, 2002). an area of 50,875 km2 which is composed by Bioactive compound and secondary metabolite various coral species. As many as 18% of coral from terrestrial environment has been diminished. species in the world can be found in Indonesia Therefore, marine environment must to be explored (Roza, 2017). The association between for a novel bioactive compound (Bajpai, 2016). The microorganisms and corals is called coral holobiont. condition between marine environment and Coral consists of symbiosis between coral animals, terrestrial are very different. Therefore, bacteria endosymbiotic algae or zooxanthellae and the might produces different bioactive compound diversity of bacterial communities. Like (Nithyanand & Pandian, 2009). microbiomas in humans, the bacterial community Acropora digitifera coral is widespread in on corals has an important role for corals. The role Indo-Pacific. Amed area is a diving tourism area of microorganisms in corals is to carry out the cycle located in Karang Asem Bali. Amed is divided into of carbon, sulfur, nitrogen and other nutrients for 3 locations, namely Jemeluk, Lipah and Japanese coral hosts. Bacteria also play a role in producing Shipwreck. In this area there are various types of secondary metabolites as protection against corals such as Acropora, Ganiostrea, Pavites and School of Health Sciences Jenderal Achmad Yani Page 362 Jenderal Sudirman Canal Road – Cimahi 40533 Phone: +62-22-6631622 - 6631624 Isolation and Identification of Coral Reef Bacteria Potential Candidates for Producing Bioactive Compound Montipora. The author uses DNA samples of DNA will grow white on the media while the other Acropora digitifera that sampled from Jemeluk. is blue. This research was conducted as pre-liminary research to identify bacteria from Acropora Confirmation of transformed DNA digitifera that potentially to be explored more on Plasmid was isolated from transforman cell by bioactive compounds and secondary metabolite. alkalyne-lysis (minipreparation) method (Sambrook, 2003). Isolated plasmid was amplified METHODS by PCR using 16S rRNA universal primer. The 16S rRNA genes amplification amplification of 16S rRNA gene was confirmed by DNA sample of Acropora digitifera was running the amplification product in 1% agarose obtained from the research of Pitasari, 2015 gel. Full-length sequencing of the rRNA gene (Pitasari, 2015). The 16S rRNA genes were (about 1500 bp) for all the coral-associated bacterial amplified by PCR with the universal primer 27F isolates was carried out in Macrogen (Seoul, (5’AGAGTTTGATCCTGGCTC-3’) and 16492R Korea). (5’GGTTACCTTGTTACGACTT-3’) (Oligo Macrogen) and GoTaq Green Master Mix Nucleotide sequence analysis (Promega). PCR was performed with the initial The full-length sequences obtained were DNA denaturation step at 95℃ for 3 min, followed matched with previously published sequences by 30 cycles of DNA denaturation at 95℃ for 30 available in NCBI using BLAST. Multiple sequence sec, primer annealing at 45℃ for 30 sec, and primer analysis was carried out using CLUSTALX ver 2.3. extention at 72℃ for 2 min. The PCR products were MEGA6 with Neighbor Joining method were used analyzed by electrophoresis in 1% agarose gel. to construct the phylogenetic tree. To validate the reproducibility of the branching pattern, 1000 Cloning of 16S rRNA genes bootstrap analysis was performed PCR products were purified by GeneAid Gel/PCR DNA Fragments Extraction Kit RESULT (GeneAidTM) according to manufacturer's The results of the transformation showed that instructions. The purified PCR products was cloned there were 67 bacterial colonies growing on agar in pGEM-T Easy (Promega) vector. Ligation was media. There were 34 transformed colonies that are carried out by centrifugation for 10 sec and white. The white colonies were grown on LB broth incubation at 4℃ for 16 hours. media. Twenty six isolates grew while 8 isolates did not grow. Plasmid isolation was carried out on 26 Transformation of ligated 16S rRNA genes isolates. The 16S rRNA gene amplification results Ligated DNA was transformed to competent showed that only 12 isolates were successfully cell E. coli DHα by heat-shock method. The cold inserted by the full-lenght 16S rRNA gene (1500 competent cell was transferred into a cold microtube bp). containing ligated DNA and incubated at 42℃ for Twelve isolates were identified by 16SrRNA 45-50 sec. Microtube was put back on the ice and gene sequencing (see table 1). Sequence analysis should not be shaken. LB media was added into revealed that 92% belonged to the microtube then incubated at 37℃, 150 rpm, for 1.5 Gammaproteobacteria and 8% to the hours. Transformed DNA was selected by growing Actinobacteria. All the bacteria has the identity the cells on the LB agar containing ampisilin, X- more than 90%. Some of the bacteria has the Gal, and IPTG. Cell colonies with transformed identity more than 97% which are closely related to the previous bacterial spesies. Third International Seminar on Global Health (3rd ISGH) Page 363 Vol 3 | No. 1 | October 2019 | Isolation and Identification of Coral Reef Bacteria Potential Candidates for Producing Bioactive Compound Table 1.BLAST Results of the Sequences Third International Seminar on Global Health (3rd ISGH) Page 364 Vol 3 | No. 1 | October 2019 | Isolation and Identification of Coral Reef Bacteria Potential Candidates for Producing Bioactive Compound Phylogenetic analysis of the bacterial isolates in methane cycle, carbon cycle and nitrogen cycle . were constructed based on the isolate that closely (Madigan, 2014) Pseudomonas and related to the other isolate. The result revealed that Endozoicomonas are genera belonging to the class four isolates (p1, p8, p12 and p13) were closely Gammaproteobacteria. Pseudomonas is able to use related to the genus Endozoicomonas, seven isolates organic compound as carbon and energy source. (p3, p7, p16, p22, p25, p31, p33) were closely This genera has an important role on energy cycle related to genus Pseudomonas and one isolate (p23) and bioremediation agent (Madigan, 2014). was closely related to Brevibacterium. Percentage Pseudomonas flourescence is known to produce an identity <95% indicated isolates of different antibiotic called mupirocin that has the potential to species. Likewise with the bootstrap value <70% fight MRSA (Methicillin-Resistant Staphylococcus indicating that the isolate was not closely related aureus) (El-Sayed et al., 2003). Pseudomonas (Hillis & Bull, 1993). p13 isolate is a close relative synxantha has an important role at nitrogen fixation with Endozoicomonas euniceicola while p1 and p8 (Wechter et al., 2002). Pseudomonas are not. p12 is closely related to Endozoicomonas extremaustralis was found at antartica and this elysicola. p7 and p25 are close relative with bacteria can produce polyhydroxybutyrate (López Pseudomonas flourescence while p22 and p33 are et al., 2009). Polyhydroxybutyrate