Antimicrobial Activity and Identification of Fungus Associated Stylissa Flabelliformis Sponge Collected from Menjangan Island West Bali National Park, Indonesia

Indonesian J. Pharm. Vol. 29 No. 2 : 66 – 73 ISSN-p : 2338-9427 DOI: 10.14499/indonesianjpharm29iss2pp66 Research Article Antimicrobial activity and Identification of fungus associated Stylissa flabelliformis sponge collected from Menjangan Island West Bali National Park, Indonesia Erna Prawita Setyowati1*, Sylvia Utami Tunjung Pratiwi1, Purwantiningsih1, Putu Oka Samirana2 1Faculty of Pharmacy, ABSTRACT Universitas Gadjah Mada The Fungus is a very important microorganism as a producer (UGM). Jl. Sekip Utara of bioactive secondary metabolites. Active substances of microbial 55281, Yogyakarta, origin have been sought through the process of screening methods Indonesia to obtain antimicrobial compounds. The purpose of this study was 2 Dept of Pharmacy, Faculty to isolate fungi associated with sponge taken from Menjangan of Math and Natural Island National Park West Bali (Indonesia) and identify fungi that Sciences, Udayana have antimicrobial activity. Isolation of fungus from sponge was University, Bali, Indonesia carried out by spread plate method using Saboroud Saline Agar medium. Each fungi will be tested to Staphylococcus aureus ATCC Submitted: 08-02-2018 29213, Escherichia coli ATCC 25922 and Candida albicans ATCC Revised: 10231. Identification of fungi is based on the observation of Accepted: macroscopic, microscopic and also using 16rRNA/ITS phylogeny tree. The results showed that S. flabelliformis sponge had 10 *Corresponding author fungal isolates. Most of them have antimicrobial activity. The name Erna Prawita Setyowati associated with a sponge fungus is These 10 fungus are Email: Aspergillus flavus strain UPMZ02, Aspergillus fumigatus strain [email protected] CD1621, Trichoderma reesei strain JCM 2267, Aspergillus nomius strain KUB105, Aspergillus sp. strain TLWK-09, Aspergillus flavus strain MC-10-L, Penicillium sp. strain RMA-2, Aspergillus sp. strain TLWK-09, Aspergillus fumigatus and Trichoderma reesei strain TV221 Keyword: Sponge-associated fungi, Stylissa flabelliformis, Staphylococcus aureus, Escherichia coli, Candida albicans INTRODUCTION Marine sponges often contain diverse Sponges are the subject of interesting and abundant microbial communities, including antibiotic development studies because the bacteria, archaea, microalgae, and fungi. In sponges form associations with various some cases, these microbial associates comprise microbes and are rich in bioactive compounds. as much as 40% of the sponge volume and can Sponges are the largest source of new bioactive contribute significantly to host metabolism compounds than other marine products (Taylor, et al., 2007). Correspondingly, the (Mehbub, et al., 2014). Bioactive compounds studies of natural products from sponge- obtained from sponges are known to have associated microorganisms, particularly bacteria various activities such as cytotoxic, and fungi, are also plenty. Secondary antimicrobial, anti-inflammatory, antiparasite metabolites produced by fungi associated with and others (Proksch, et al., 2002; Setyowati et al, sponges are much larger when compared with 2016). The bioactive compound on the sponge secondary metabolites produced by bacteria acts as a self-protection mechanism against the that associate with sponges. In more detail, environment. It was surprising to find that Ascomycota dominates the proportion of many secondary metabolites reported from fungal producer by division (Kim, et al., 2013). marine invertebrate are produced by their The main focus on secondary metabolites microorganism symbionts (Thakur and Muller, produced by fungi is due to their diverse 2004). biological activities having both toxic and 66 Volume 29 Issue 2 (2018) Erna Prawita Setyowati curative effects in maintaining human and chloramphenicol, and cultivated at 25°C. veterinary health. More intense studies on Emerging fungal mycelia were isolated and marine fungi have been done in the past three taken into the culture. decades. These continuous studies revealed that marine fungi are rich sources of bioactive Antimicrobial activity of fungi natural products (Ebada and Prosch, 2010; Bills The concentration of microbes used was and Gloer, 2016). 107 Colony Forming Unit. The microbial Previous study conducted by Setyowati et suspension was inoculated into 10mL nutrient- al. (2004) showed that Stylissa flabelliformis agar medium (E. coli, S. aureus) and Saboroud sponge taken from Menjangan Island's waters Dextrose Agar (C. albicans), then poured into a of the West Bali National Park could produce petri dish. jaspamide compounds. This compound is Sterilized disc paper, sprayed as much as highly potent against C. albicans and more active 10μL of fungi methanol extract dissolved than Miconazole nitrate as a comparison, this with a concentration of 10 mg/mL. As a compound also has cytotoxic power equivalent positive control, 1mg/mL of tetracycline was to vincristine (leukemia cancer drug) in used for antibacterial test and used 1mg/mL of myeloma cells. This compound is usually found griseofulvin for an antifungal test, and from the genus Jaspis sp and has never been methanol as a solvent control. Paper found from the species S. flabelliformis. Often discs were placed on top of pre-prepared metabolites with compound structures similar solid media. Petri was incubated overnight at to those produced from sponges with different 35ºC-37°C. Observed zone barriers formed taxonomies (Mehbub et al., 2014). This suggests around the paper disc (Niyomkam, et al., 2010). that the structural characteristics of a compound are strongly thought to originate Identification of fungi Identification of fungi associated from sponge microorganisms and are sponge was carried out by observing responsible for the production of various fungi macroscopically, microscopically, and bioactive compounds (Koopmans, et al., 2009). molecularly based on the genetic analysis of Therefore it is estimated that some sponge partial locus Internal Transcribed Spacer (ITS) metabolites are produced by symbiotic ribosomal DNA fungi. microorganisms (Thomas, et al., 2010). DNA isolation was initiated by growing Antimicrobial assay against fungi associated fungal isolates in Potato Dextrose Broth liquid with S. flabelliformis sponge and molecular medium (PDB) and incubated for 72h. The identification of the sponge fungus was biomass of the fungal mycelia is further performed in this study. harvested for the DNA extraction process. MATERIAL AND METHODS DNA fungi extraction was performed using Sampling and Isolation of the fungal PHYTOpure nucleon reagents (Amersham association sponge. LIFE Science). PCR Amplification on ITS Specimens of the marine sponge S. using primer ITS4. Purification of PCR product flabelliformis (Axenellida) were collected from was performed by PEG precipitation method Menjangan Island National Park West Bali and continued with cycle sequencing. The (Indonesia) through Scuba diving. Since the results of the sequencing cycle are reversed stringent surface sterilization procedures have with ethanol purification method. Analysis of previously proven to be too rigorous for sequence sequencing of nitrogen-based using delicate sponge tissues, the sponges were either an automated DNA sequencer (ABI PRISM briefly sterilized for 30s in 70% v/v ethanol 3130 Genetic analyzer) (Applied Biosystems). followed by three subsequent washing in sterile The sequenced raw data was then streamed and commercial sea water or merely washed three dissembled using the Bioedit program. times in sterile seawater. The sponge tissue was Sequencing data was already assembled at then cut into approximately 1.5mm segments BLAST with genome data registered at and planted on Sabouroud agar growth DDBJ/DNA Data Bank of Japan or medium with sea water, 250ug/mL NCBI/National Centre for Biotechnology Volume 29 Issue 2 (2018) 67 Antimicrobial activity and Identification of fungus Figure 1. Stylissa flabelliformis sponge. Figure 2. Isolate of fungus from Stylissa flabelliformis Information to determine which taxon/species The antimicrobial activity of fungi against has the greatest homology/similarity and is several bacteria showed that most of the collected molecularly related (Atashpaza, et al., 2010; fungi were possible to inhibit the growth of Santosa, 2011). microorganisms tested. Fungi No. 3, 5, 6, 7, 9 and 10 were inhibiting S. aureus, C. albicans and RESULTS AND DISCUSSION E. coli. The fungus has broad-spectrum S. flabelliformis sponges (Figure 1) are red properties because it can inhibit the growth of with a thin shape and soft, porous surface with almost all tested bacteria and yeast in vitro. irregular protrusions on the whole body, inside Table I also shows that 7 of the 10 fungi yellowish-white body. Sponge classification is can inhibit Staphylococcus aureus. It is known that as follows: Kingdom Animalia, Division S. aureus causing various infections, such as Porifera, Class Demospongiae, Subclass acne, ulcers, pneumonia, endocarditis, and Ceractinomorpha, Order Halicondrida, Family implantation of any part of the body (Jawetz et Axenellida, Genus Stylissa, Species S. al., 1996). The prevalent treatment to deal with flabelliformis (Hooper and Soest, 2002). bacterial infections is to use antibiotics such as The screening was done to obtain the methicillin, amoxicillin, penicillin, oxacillin, and active microbial (fungi) potential. Isolation of others. Nonetheless, what seems to be fungus from S. flabelliformis sponge observed at happening is the occurrence of S. aureus least 10 fungi as (Figure 2). All of the 10 fungi

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