|Year : 2015 | Volume
| Issue : 1 | Page : 33-36
Absence of anti-Staphylococcus aureus (MRSA) activity of secondary metabolite actinomycetes associated sponges from Pulau Panjang, Indonesia
Yatnita Parama Cita1, Farid Kamal Muzaki2, Nurlita Abdulgani2, Syamsudin Abdillah3
1 Department of Microbiology, Faculty of Medicine, University of Indonesia, Jakarta, Indonesia
2 Laboratorium of Ecology and Zoology, Department of Biology, Faculty of Mathematic and Sciences, Sepuluh Nopember Institute of Technology, Surabaya, East Java, Indonesia
3 Department of Pharmacology, Faculty of Pharmacy, Pancasila University, Jagakarsa, Jakarta-Selatan, Indonesia
|Date of Web Publication||20-May-2015|
Department of Pharmacology, Faculty of Pharmacy, Pancasila University, Jagakarsa, Jakarta Selatan, Jakarta
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Objective: To study the anti-Staphylococcus aureus MRSA of the secondary metabolite actinomycetes associated sponges from Pulau Panjang, Central Java, Indonesia. Materials and Methods: The sponge samples were taken by scuba diving in Pulau Panjang, and the sponge was identified using external morphology as well as identification of spicules from the fresh preparation. Isolation of actinomycetes associated with sponge was conducted by means of direct plating. The symbion bacteria were planted (scratched in a circular way) on agar medium Zobell 2216E, medium actinomycetes using the existing method, namely, S.aureus MRSA and E. coli MRSA. Results: The results of identification from 6 sponges were Phakettia euctimenna, Gelliodes fibolatus, Lamellodysidea herbacea, Clathria reindwardtii and Callyspongia sp, while antibacterial assay for the secondary metabolite actinomycetes associated sponges showed negative results. Conclusion: Secondary metabolite actinomycetes associated sponges from Pulau Panjang were not found to have antibacterial activity against S. aureus MRSA.
Keywords: Anti-methicillin resistant Staphylococcus aureus, Pulau Panjang, secondary metabolite actinomycetes-associated sponges
|How to cite this article:|
Cita YP, Muzaki FK, Abdulgani N, Abdillah S. Absence of anti-Staphylococcus aureus (MRSA) activity of secondary metabolite actinomycetes associated sponges from Pulau Panjang, Indonesia. J Pharm Negative Results 2015;6:33-6
|How to cite this URL:|
Cita YP, Muzaki FK, Abdulgani N, Abdillah S. Absence of anti-Staphylococcus aureus (MRSA) activity of secondary metabolite actinomycetes associated sponges from Pulau Panjang, Indonesia. J Pharm Negative Results [serial online] 2015 [cited 2020 Jan 24];6:33-6. Available from: http://www.pnrjournal.com/text.asp?2015/6/1/33/157388
| Introduction|| |
Available resources for pharmacy can be found in the coral reef ecosystem/one of the components that form the coral reef ecosystem is sponge.  Sponge is a potential marine biota that is potential to produce bioactive secondary metabolites. This is evident from around 6000 bioactive substances (lead compound) isolated from marine biota within the last three decades. About 40% of them are derived from sponges. 
Metabolite activity of sponge is beneficial to prevent predator attack  and serve as anti-microbe.  The sponge metabolites include alkaloid, flavonoid, steroid-triterpenoid and saponin. The metabolites can be used as sources of nutrients for the living creature in the area.  Research on the potential of secondary metabolite of the sponges from Indonesia has begun three decades ago, when Corley et al.  isolated laulimalide and isolaulimalide from the sponge Hyatella sp, which was found to be cytotoxic. Antioxidant compound was successfully isolated from the sponge Callyspongia sp. from Kepulauan Seribu;  it was also reported that marine sponge Axinella carteri Dendy from Pulau Babi, South Sumatera, is potential as larvacide and antibacterial compounds isolated and identified from the extract of sponge Petrosia nigrans from Pulau Babi, South Sumatera. 
Pulau Panjang is a small island located 2 miles to the west of Jepara beach line. Almost all of the water around the island is populated with shallow coral reefs [Figure 1].  A study on the inventory of coral reefs around the northern beach line of Pulau Panjang found 19 genera and the ecosystem was in moderate condition.  The study was conducted to screen the antibacterial and antioxidant activities of some sponges found in Pulau Panjang, Jepara, Central Java.
|Figure 1: Pulau Panjang is situated at the northern part of Jepara, near Kartini and Bandengan beaches|
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| Materials and Methods|| |
The samples of bacteria in symbiosis potential sponges were taken from Pulau Panjang, Jepara, Central Java up to a depth of 2-10 m using scuba diving method in June 2012. After taken up from the sea, the sponges were photographed and some tissues of the sponge were taken, and then put into the medium % marine broth for bacterial isolation (direct plating method). The remaining sponges were introduced into antibiotic ampicilline containing medium for bacterial isolation using enrichment method. The sponges were identified by recording the specific characteristics (color, texture, shape, size, and depth of collection). The next steps of systematic and sponge identification followed the guideline in a textbook by Hooper  as well as a textbook by Mapstone. 
The sponges were taken from Pulau Panjang beach, Kediri, Central Java by means of direct collection. The samples were determined in the Zoological Laboratory, Department of Biology, FMIPA ITS.
Isolation and bacterial purification
The surface of sponge sample was sprayed with sterile sea water, 5 ml of seawater for 1 cm of sponge. This way, only bacterial with high affinity would be sampled. Mesohil was taken with the size of 1 × 1 cm, eroded and diluted in sterile saltwater. Isolation of bacteria from the outer surface was conducted using sterile swab.  The swab was rubbed on the outer surface of the sponge in one-way direction. Sterile swab, which had been rubbed on the sample surface, was put into a tube, which contained 5 m: of sterile saltwater, and made to a series of solution, namely 10 0 , 10 1 , 10 2 ,10 3 ,10 4 ,10 5. From the solution series of 10 -2 , 10 -3 , 10 -4 , and 10 -5 , 100 μL of the sample was taken and spread into the sterile Petri dish, which contained medium mixture of actino and Zobell 2216E. Media that had been filled with samples were then incubated under a temperature of 26°C for 7 days. After incubation, bacterial colonies that grew were closely observed for shape, color, and surface. Each colony had different shape and color. Scratch method was used to isolate and purify isolates of each bacterium.  Bacterial isolation was conducted using scratch method. The purified bacterial colonies were isolated using Ose needle, based on the difference in color, texture, and shape of colony on the medium Zobell 2216E in the Petri dish. The purified isolates of bacteria were then stored in sloping agar media.
Symbion bacteria was planted (scratched in a circular way) on agar medium Zobell 2216E using scratch method and then incubated for 2 × 24 hours under a temperature of 37 0 C. The liquid culture of pathogenic bacteria/test bacteria (S.aureus MRSA) with a density of 10 9 cell/ml was introduced into soft agar medium Zobell 2216E (Volume of liquid culture 1% of the soft agar Zobell 2216E volume). The soft agar medium Zobell 2216E, which had been inoculated with bacteria, was poured onto the surface of agar media Zobell 2216E, which had been planted with symbion bacteria. Inoculation took 4 days under room temperature, and inhibitory zone was observed and recorded accordingly.
| Results|| |
Besides identification using outer morphology, the spicules were also identified from the fresh preparation [Figure 1]. Guide to sponge collection and identification. Queensland. Guide to sponge collection and identification. Queensland Museum South Brisbane Australia: The results of identification are the following.
| Discussion|| |
The sponge samples used as mother biota consisted of 5 specimens of Phakettia euctimenna, Gelliodes fibolatus, Lamellodysidea herbacea, Clathria reindwardtii and Callyspongia sp. The bacteria isolated with direct plating method generally have relatively higher number of colonies. Even some samples were more than 10 5 , compared to it they were with direct plating method. It was because in direct plating method, the culture solution was directly planted on marine broth medium, which is a common medium for marine bacteria.
The capability of symbion bacteria of the sponge isolate to fight against the pathogens could be observed by conducting anti-bacterial assay. Their capability to fight against pathogen bacteria was characterized with a clear zone that forms around the bacterial scratching. Anti-bacterial assay was conducted on the bacterial isolate of 5 sponges, which were taken against MRSA and resistant E. Coli. The anti-bacterial assay showed that the bacterial isolated did not have antibacterial activity. [Figure 2] and [Figure 3] shows the result of anti-bacterial assay for the sponge bacterial isolated against S. aureus MRSA.
|Figure 2: The spreading of bacteria colony associated with sponge Sp.P5 on marine agar medium|
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|Figure 3: The results of anti-bacterial assay of secondary metabolite actinomycetes associated sponge (Sp.5)|
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This proves that there was no growth of pathogenic bacteria. Another research on the extract of Callyspongia sp showed anti-bacterial activity of S. aureus MRSA, but this research used S aureus MRSA. Methicillin-resistant Staphylococcus aureus (MRSA) is a specific type of Staphylococcus aureus bacterium, which endures methiciline. Staphylococcus aureus and non-resistant methiciline Staphylococcus aureus is known as methicillin-susceptible Staphylococcus aureus (MSSA). Some types of staph have been immune against antibiotics methiciline, which had been used to treat infected staph. Consequently, infection caused by MRSA is harder to treat, because many antibiotics cannot kill this type of bacteria.
| Conclusion|| |
Secondary metabolite compounds of actinomycetes associated sponge from Pulau Panjang do not have anti-bacterial activity against S. aureus MRSA
| Acknowledgment|| |
Thanks to Handung Naryadi, M.Sc of the Integrated Laboratory of Diponegoro University, for helping in this research.
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[Figure 1], [Figure 2], [Figure 3]