Antimicrobial activity and Identification of fungus associated Stylissa flabelliformis sponge collected from Menjangan Island West Bali National Park , Indonesia

Email: erna_prawita@ugm.ac.id ABSTRACT The Fungus is a very important microorganism as a producer of bioactive secondary metabolites. Active substances of microbial origin have been sought through the process of screening methods to obtain antimicrobial compounds. The purpose of this study was to isolate fungi associated with sponge taken from Menjangan Island National Park West Bali (Indonesia) and identify fungi that have antimicrobial activity. Isolation of fungus from sponge was carried out by spread plate method using Saboroud Saline Agar medium. Each fungi will be tested to Staphylococcus aureus ATCC 29213, Escherichia coli ATCC 25922 and Candida albicans ATCC 10231. Identification of fungi is based on the observation of macroscopic, microscopic and also using 16rRNA/ITS phylogeny tree. The results showed that S. flabelliformis sponge had 10 fungal isolates. Most of them have antimicrobial activity. The name associated with a sponge fungus is These 10 fungus are Aspergillus flavus strain UPMZ02, Aspergillus fumigatus strain 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


INTRODUCTION
Sponges are the subject of interesting antibiotic development studies because the sponges form associations with various microbes and are rich in bioactive compounds.Sponges are the largest source of new bioactive compounds than other marine products (Mehbub, et al., 2014).Bioactive compounds obtained from sponges are known to have various activities such as cytotoxic, antimicrobial, anti-inflammatory, antiparasite and others (Proksch, et al., 2002;Setyowati et al, 2016).The bioactive compound on the sponge acts as a self-protection mechanism against the environment.It was surprising to find that many secondary metabolites reported from marine invertebrate are produced by their microorganism symbionts (Thakur and Muller, 2004).
Marine sponges often contain diverse and abundant microbial communities, including bacteria, archaea, microalgae, and fungi.In some cases, these microbial associates comprise as much as 40% of the sponge volume and can contribute significantly to host metabolism (Taylor, et al., 2007).Correspondingly, the studies of natural products from spongeassociated microorganisms, particularly bacteria and fungi, are also plenty.Secondary metabolites produced by fungi associated with sponges are much larger when compared with secondary metabolites produced by bacteria that associate with sponges.In more detail, Ascomycota dominates the proportion of fungal producer by division (Kim, et al., 2013).The main focus on secondary metabolites produced by fungi is due to their diverse biological activities having both toxic and curative effects in maintaining human and veterinary health.More intense studies on marine fungi have been done in the past three decades.These continuous studies revealed that marine fungi are rich sources of bioactive natural products (Ebada and Prosch, 2010;Bills and Gloer, 2016).
Previous study conducted by Setyowati et al. (2004) showed that Stylissa flabelliformis sponge taken from Menjangan Island's waters of the West Bali National Park could produce jaspamide compounds.This compound is highly potent against C. albicans and more active than Miconazole nitrate as a comparison, this compound also has cytotoxic power equivalent to vincristine (leukemia cancer drug) in myeloma cells.This compound is usually found from the genus Jaspis sp and has never been found from the species S. flabelliformis.Often metabolites with compound structures similar to those produced from sponges with different taxonomies (Mehbub et al., 2014).This suggests that the structural characteristics of a compound are strongly thought to originate from sponge microorganisms and are responsible for the production of various bioactive compounds (Koopmans, et al., 2009).Therefore it is estimated that some sponge metabolites are produced by symbiotic microorganisms (Thomas, et al., 2010).Antimicrobial assay against fungi associated with S. flabelliformis sponge and molecular identification of the sponge fungus was performed in this study.

Sampling and Isolation of the fungal association sponge.
Specimens of the marine sponge S. flabelliformis (Axenellida) were collected from Menjangan Island National Park West Bali (Indonesia) through Scuba diving.Since the stringent surface sterilization procedures have previously proven to be too rigorous for delicate sponge tissues, the sponges were either briefly sterilized for 30s in 70% v/v ethanol followed by three subsequent washing in sterile commercial sea water or merely washed three times in sterile seawater.The sponge tissue was then cut into approximately 1.5mm segments and planted on Sabouroud agar growth medium with sea water, 250ug/mL chloramphenicol, and cultivated at 25°C.Emerging fungal mycelia were isolated and taken into the culture.

Antimicrobial activity of fungi
The concentration of microbes used was 10 7 Colony Forming Unit.The microbial suspension was inoculated into 10mL nutrientagar medium (E.coli, S. aureus) and Saboroud Dextrose Agar (C.albicans), then poured into a petri dish.
Sterilized disc paper, sprayed as much as 10μL of fungi methanol extract dissolved with a concentration of 10 mg/mL.As a positive control, 1mg/mL of tetracycline was used for antibacterial test and used 1mg/mL of griseofulvin for an antifungal test, and methanol as a solvent control.Paper discs were placed on top of pre-prepared solid media.Petri was incubated overnight at 35ºC-37°C.Observed zone barriers formed around the paper disc (Niyomkam, et al., 2010).

Identification of fungi
Identification of fungi associated sponge was carried out by observing fungi macroscopically, microscopically, and molecularly based on the genetic analysis of partial locus Internal Transcribed Spacer (ITS) ribosomal DNA fungi.
DNA isolation was initiated by growing fungal isolates in Potato Dextrose Broth liquid medium (PDB) and incubated for 72h.The biomass of the fungal mycelia is further harvested for the DNA extraction process.DNA fungi extraction was performed using PHYTOpure nucleon reagents (Amersham LIFE Science).PCR Amplification on ITS using primer ITS4.Purification of PCR product was performed by PEG precipitation method and continued with cycle sequencing.The results of the sequencing cycle are reversed with ethanol purification method.Analysis of sequence sequencing of nitrogen-based using an automated DNA sequencer (ABI PRISM 3130 Genetic analyzer) (Applied Biosystems).The sequenced raw data was then streamed and dissembled using the Bioedit program.Sequencing data was already assembled at BLAST with genome data registered at DDBJ/DNA Data Bank of Japan or NCBI/National Centre for Biotechnology Volume 29 Issue 2 (2018) Information to determine which taxon/species has the greatest homology/similarity and is molecularly related (Atashpaza, et al., 2010;Santosa, 2011).

RESULTS AND DISCUSSION
S. flabelliformis sponges (Figure 1) are red with a thin shape and soft, porous surface with irregular protrusions on the whole body, inside yellowish-white body.Sponge classification is as follows: Kingdom Animalia, Division Porifera, Class Demospongiae, Subclass Ceractinomorpha, Order Halicondrida, Family Axenellida, Genus Stylissa, Species S. flabelliformis (Hooper and Soest, 2002).
The screening was done to obtain the active microbial (fungi) potential.Isolation of fungus from S. flabelliformis sponge observed at least 10 fungi as (Figure 2).All of the 10 fungi were then being tested against the 3 kinds of microbes such as Staphylococcus aureus ATCC 29213, Escherichia coli ATCC 25922 and Candida albicans ATCC 10231.These microbes used are representative of gram-positive bacteria, gramnegative and yeast.
The antimicrobial activity of fungi against several bacteria showed that most of the collected fungi were possible to inhibit the growth of microorganisms tested.Fungi No. 3, 5, 6, 7, 9 and 10 were inhibiting S. aureus, C. albicans and E. coli.The fungus has broad-spectrum properties because it can inhibit the growth of almost all tested bacteria and yeast in vitro.
Table I also shows that 7 of the 10 fungi can inhibit Staphylococcus aureus.It is known that S. aureus causing various infections, such as acne, ulcers, pneumonia, endocarditis, and implantation of any part of the body (Jawetz et al., 1996).The prevalent treatment to deal with bacterial infections is to use antibiotics such as methicillin, amoxicillin, penicillin, oxacillin, and others.Nonetheless, what seems to be happening is the occurrence of S. aureus resistance to these antibiotics.One of the most commonly reported cases of resistance is Methicillin Resistant S. aureus (MRSA), a methicillin-resistant S. aureus bacterium.The mechanism of methicillin resistance does not depend on the formation of beta-lactamase.However, it is more due to changes in Protein  Binding Penicillins (PBPs), a penicillin-receiving protein that occurs as a result of chromosome mutations (Hugo and Russel, 2011).There are 5 fungi that can inhibit the growth of three microbes.In addition, sponge-derived strain collections that comprise isolates that tested negative for antimicrobial activity at first may have done so, because the compound of interest is not produced under standard laboratory conditions (Indraningrat, et al, 2016).From the results of the data above can be seen that the 10 fungi, 7 fungi belong to the genus Aspergillus, 2 fungi belong to the genus Trichoderma and 1 fungi belong to the genus Penicillium.These three genera are included in the Ascomycota division.Although the genus is the same (Aspergillus) not all fungi have antimicrobial effects on the microbes tested.Most fungi of the Ascomycota division having an antimicrobial effect are Aspergillus, as expressed in the Indraningrat et al paper (Indraningrat et al, 2016).Sponge-associated Ascomycota found to produce antimicrobials can be further classified into 12 genera.Of these 12 fungal genera Aspergillus (30%) and Penicillium (23%) are currently the two most prominent groups of sponge-associated fungi reported as antimicrobial producers.

Figure 4 .
Figure 4. Phylogeny tree of fungi Sal 1 KY698416.1 Aspergillus flavus strain UPMZ02 18S ribosomal RNA gene partial sequence internal transcribed spacer 1 5.8S ribosomal RNA gene and internal transcribed spacer 2 complete sequence and 28S re; KU561919.1 Aspergillus flavus strain aC4 internal transcribed spacer 1 partial sequence 5.8S ribosomal RNA gene and internal transcribed spacer 2 complete sequence and 28S ribosomal RNA gene partial e; KT828546.1 Aspergillus flavus strain KAU internal transcribed spacer 1 partial sequence 5.8S ribosomal RNA gene and internal transcribed spacer 2 complete sequence and 28S ribosomal RNA gene partial e; KR296888.1 Aspergillus flavus strain PUXX-FS06 18S ribosomal RNA gene partial sequence internal transcribed spacer 1 5.8S ribosomal RNA gene and internal transcribed spacer 2 complete sequence and 28e; KU561917.1 Aspergillus flavus strain sM4 internal transcribed spacer 1 partial sequence 5.8S ribosomal RNA gene and internal transcribed spacer 2 complete sequence and 28S ribosomal RNA gene partial (Figure 4).Sal 2: Based on BLAST results on the NCBI database on the ribosomal RNA 18S sequence Gene sample number 1702.00468 obtained a homology of 99% with Aspergillus fumigatus strain CD1621 18S ribosomal gene, partial sequence; Internal transcribed spacer 1, 5.8S ribosomal RNA gene, and internal transcribed spacer 2, complete sequence; And 28S ribosomal RNA gene, partial sequence

Sal 6 :
Based on BLAST results on the NCBI database against the ribosomal RNA 18S sequence Gene sample number 1702.00472 obtained homology of 100% with Aspergillus flavus strain MC-10-L internal transcribed spacer 1, partial sequence; 5.8S ribosomal RNA gene and internal transcribed spacer 2, complete sequence; And 28S ribosomal RNA gene, partial sequence (Figure7).

Sal 7 :
Figure 10.Phylogeny tree of fungi Sal 7 KP296145.1Penicillium griseofulvum isolate 018 internal transcribed spacer 1 partial sequence 5.8S ribosomal RNA gene; KC506183.1 Fungal sp.AM2013 strain 16 Mp internal transcribed spacer 1 partial sequence 5.8S ribosomal RNA gene KF914643.1Penicillium sp.4M3 internal transcribed spacer 1 partial sequence 5.8S ribosomal RNA gene; KY883661.1 Penicillium sp.strain RMA-2 internal transcribed spacer 1 partial sequence 5.8S ribosomal RNA gene; KY439022.1 Penicillium citrinum strain 1S.12 internal transcribed spacer 1 partial sequence 5.8S ribosomal RNA gene (Figure 10).Sal 8: Based on BLAST results on the NCBI database on the ribosomal RNA 18S sequence Gene sample number 1702.00474 Figure and fungus identification data for Sal 8 are the same as figure and data in Sal 5 (Figure 8), so Sal 8 is not further elaborated Sal 9: Based on BLAST results on the NCBI database on the ribosomal RNA 18S sequence Gene sample number 1702.00475 obtained a homology of 100% with Aspergillus fumigatus small ribosomal subunit RNA gene, partial sequence; Internal transcribed spacer 1, 5.8S ribosomal RNA gene, and internal transcribed spacer 2, complete sequence; And large ribosomal subunit of RNA gene, partial sequence.

Sal 10 :
Based on BLAST results on the NCBI database on the ribosomal RNA 18S sequence Gene Sal 10 obtained a homology of 99% with Trichoderma reesei strain TV221 internal transcribed spacer 1, partial sequence; 5.8S ribosomal RNA gene and internal transcribed spacer 2, complete sequence; And 28S ribosomal RNA gene, partial sequence.