Sains Malaysiana 43(7)(2014): 1013–1021

Microbial Population in the Coelomic Fluid of Stichopus chloronotus and

Holothuria (Mertensiothuria) leucospilota Collected from Malaysian Waters

(Populasi Mikrob dalam Cecair Selom Stichopus chloronotus dan Holothuria

(Mertensiothuria) leucospilota dari Perairan Malaysia)

AHMAD LUTFI LUKMAN¹, NOOR FAIZUL HADRY NORDIN² & KAMARUL RAHIM KAMARUDIN²*

¹Department of Biotechnology, Faculty of Agriculture and Biotechnology, Universiti Sultan Zainal Abidin (UniSZA), Kampus Kota, Jalan Sultan Mahmud, 20400 Kuala Terengganu, Terengganu Darul Iman, Malaysia

²Department of Biotechnology, Kulliyyah of Science, International Islamic University Malaysia (IIUM), Jalan Istana, Bandar Indera Mahkota, 25200 Kuantan, Pahang Darul Makmur, Malaysia

Received: 22 August 2011/Accepted: 16 September 2013

ABSTRACT

This preliminary study aimed to isolate and identify microbes that inhabit the coelomic fluid of two local species of sea cucumbers collected from Malaysian waters - a ‘gamat’ species i.e. Stichopus chloronotus Brandt, 1835 and the most abundant ‘timun laut’ species in Malaysia i.e. Holothuria (Mertensiothuria) leucospilota (Brandt 1835). Phylogenetic analyses of partial 16S rRNA mtDNA gene sequences suggested the presence of at least eight microbial genera i.e. five bacterial genera - Bacillus, Exiguobacterium, Pseudomonas, Stenotrophomonas and Vibrio - isolated from the coelomic fluid of H. leucospilota and three genera of gram-positive bacteria from the Micrococcaceae family - Kytococcus, Micrococcus and either Kocuria or Rothia - isolated from the coelomic fluid of S. chloronotus. We speculate that less diverse microbial population in S. chloronotus as compared to H. leucospilota could be due to a number of environmental factors e.g. penetration of light surrounding the habitats of both species, the feeding behaviour of H. leucospilota and the higher level of antimicrobial properties of coelomic fluid in S. chloronotus. In terms of antimicrobial-resistance capability test, an isolate from genus Pseudomonas that is suspected to be P. alcaligenes exhibited high resistance towards streptomycin. Another isolate from genus Stenotrophomonas that was suspected to be S. maltophilia showed moderate resistance towards streptomycin and lower resistance towards kanamycin. Both isolates were from the coelomic fluid of H. leucospilota. Tetracycline inhibited the growth of all bacterial isolates tested. Further studies with more specimens of S. choronotus and H. leucospilota from broader geographical locations and the use of complete mtDNA genes along with morphological approaches for species identification may facilitate to provide better insights into the microbial population in the coelomic fluid of both local sea cucumber species.

Keywords: Antimicrobial-resistance; coelomic fluid; microbial diversity; partial sequences of 16S ribosomal RNA mitochondrial gene; phylogenetic analyses; sea cucumbers

REFERENCES

Al-Jasser, A. 2006. Stenotrophomonas maltophiliaresistant to trimethoprim-sulfamethoxazole: An increasing problem. Annals of Clinical Microbiology and Antimicrobials 5: 23.

Black, J.G. 2002. Microbiology: Principles and Explorations. 5th ed. USA: John Wiley and Sons.

Brandt, J.F. 1835. Echinodermata ordo Holothurina. Pp. 42-62. In Prodromus Descriptionis Animalium Ab. H. Mertensio In Orbis Terranum Circumnavigatione Observatorum. Fasc. I: 75 pps. Petropoli.

Choo, P.S. 2008. Population status, fisheries and trade of sea cucumbers in Asia. In Sea Cucumbers. A Global Review of Fisheries and Trade, edited by Toral-Granda, V., Lovatelli, A. & Vasconcellos, M. FAO Fisheries and Aquaculture Technical Paper. No. 516.

Dayang, F.B., Jacinta, S., Chong, S.L. & Rohasmizah, I. 2004. Antimycotic activity of the extracts of Stichopus chloronotusbrandt in the treatment of experimental dermatophytosis. Jurnal Sains Kesihatan Malaysia 2(2): 97-102.

Farouk, A.E., Ghouse, F.A.H. & Ridzwan, B.H. 2007. New bacterial species isolated from Malaysian sea cucumbers with optimized secreted antibacterial activity. American Journal of Biochemistry and Biotechnology 3(2): 60-65.

Felsenstein, J. 2004. 'Phylip', Phylogeny Inference Package. version 3.6b (beta release). Seattle, WA: Department of Genome Sciences, University of Washington.

Fredalina, B.D., Ridzwan, B.H., Zainal, A.A., Kaswandi, M.A., Zaiton, H., Zali, I., Kittakoop, P. & Mat Jais, A.A. 1999. Fatty acid compositions in local sea cucumber, Stichopus chloronotus, for wound healing. General Pharmacology 33(4): 337-340.

Hawa, I., Zulaikah, M., Jamaludin, M., Zainal Abidin, A.A., Kaswandi, M.A. & Ridzwan, B.H. 1999. The potential of the coelomic fluid in sea cucumber as an antioxidant. Malaysian Journal of Nutrition 5(1&2): 55-59.

Isaacson, P., Jacobs, P.H., Mackenzie, A.M.R. & Mathews, A.W. 1976. Pseudotumour of the lung caused by infection with Bacillus sphaericus. Journal of Clinical Pathology 29: 806- 811.

Kamarudin, K.R., Rehan, A.M., Hashim, R., Usup, G., Ahmad, H.F., Anua, M.H. & Idris, M.Y. 2011. Molecular phylogeny of Holothuria (Mertensiothuria) leucospilota (Brandt 1835) as inferred from cytochrome C oxidase I mitochondrial DNA gene sequences. Sains Malaysiana 40(2): 125-133.

Kamarudin, K.R., Rehan, A.M., Hussin, R. & Usup, G. 2010. An update on diversity of sea cucumber (Echinodermata: Holothuroidea) in Malaysia. Malayan Nature Journal 62(3): 315-334.

Kaswandi, M.A., Azraul-Mumtazah, R., Sahalan, A.Z., Normalawati, S., Aziz, A.H.A. & Abu Bakar, M.A. 2007. Activity of crude extracts mixture from sea cucumbers Stichopus badionotusand Stichopus chloronotuson fungal growth. Journal of Microscopy Society of Thailand 24: 151- 154.

Khartini, A.W., Dayang Fredalina, B., Kaswandi, A., Hing, H.L., Oon, K.W. & Sit, N.W. 2003. Antimicrobial and cytotoxic activities of Holothuria sp. and B. marmorata vitiensis. Malaysian Journal of Biochemistry and Molecular Biology 8: 49-53.

Kuske, C.R., Banton, K.L., Adorada, D.L., Stark, P.C., Hill, K.K. & Jackson, P.J. 1998. Small-scale DNA sample preparation method for field PCR detection of microbial cells and spores in soil. Applied and Environmental Microbiology 64(7): 2463-2472.

McGowan, J. 2006. Resistance in nonfermenting gram-negative bacteria: Multidrug resistance to the maximum. American Journal of Medicine 119(6 Suppl 1): S29-S36, discussion S62-S70.

Osama, Y.A., Ridzwan, H., Muhammad, T., Jamaludin, M.D., Masa-Aki, I. & Zali, B.I. 2009. In vitro antioxidant and antiproliferative activities of three Malaysian sea cucumber species. European Journal of Scientific Research 37(3): 376-387.

Page, R.D.M. 1996. Treeview: An application to display phylogenetic trees on personal computers. Computer Applications in the Biosciences 12: 357-358.

Ridzwan, B.H. 1993. Sumber Makanan Persisiran Laut Sabah. Kuala Lumpur: Dewan Bahasa dan Pustaka.

Ridzwan, B.H. 2007. Sea Cucumber: The Malaysian Heritage. Malaysia: Research Management Centre, International Islamic University Malaysia (IIUM).

Ridzwan, B.H., Kaswandi, M.A., Azman, Y. & Fuad, M. 1995. Screening for anti-bacterial agents in three species of sea cucumber from coastal areas of Sabah. General Pharmacology 26(7): 1539-1543.

Ridzwan, B.H., Leong, T.C. & Idid, S.Z. 2003. The antinociceptive effects of water extracts from sea cucumber Holothuria leucospilotabrandt, Bohadschia marmorata vitiensisjaeger and coelomic fluid from Stichopus hermanii. Pakistan Journal of Biological Sciences 6(24): 2068-2072.

Ridzwan, B.H., Zarina, M.Z., Kaswandi, M.A., Nadirah, M. & Shamsuddin, A.F. 2001. The antinociceptive effects of extracts from Stichopus chloronotusbrandt. Pakistan Journal of Biological Sciences 4(3): 244-246.

Sim, Y.K., Tan, S.H., Zulfigar, Y., Wong, W.S., Chai, J., Ng, B.W. & Luar, L. 2008. The diversity and distribution of Holothuroidea in Pulau Aur, Johor. In Research and Information Series of Malaysian Coast 2. Pusat Penyelidikan Ekosistem Marin (EKOMAR), UKM. pp. 13-22.

Stackebrandt, E., Rainey, F.A. & Ward-Rainey, N.L. 1997. Proposal for a new hierarchic classification system, Actinobacteria classis nov. International Journal of Systematic Bacteriology 47: 479-491.

Stackebrandt, E., Koch, C., Gvozdiak, O. & Schumann, P. 1995. Taxonomic dissection of the genus Micrococcus: Kocuria gen. nov., Nesterenkonia gen. nov., Kytococcus gen. nov., Dermacoccus gen. nov., and Micrococcus Cohn 1872 gen. emend. International Journal of Systematic Bacteriology 45: 682-692.

Tan, W.T., Fong, Y.S. & Ridzwan, B.H. 2005. The effect of coelomic fluid Stichopus hermaniion isolated perfused rat hearts and the involvement of prostaglandin in its mechanism of action. Pakistan Journal of Biological Sciences 8(1): 78-84.

Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F. & Higgins, D.G. 1997. The clustalX windows interface: Flexible strategies for multiple sequence alignment aided by the quality analysis tools. Nucleic Acids Research 24: 4876-4882.

Zulfigar, Y., Sim, Y.K. & Tan, S.H. 2007. The distribution of sea cucumbers in Pulau Aur, Johore, Malaysia. In Publications of the Seto Marine Biological Laboratory, Special Publication Series Volume 8 (from Selected Paper of NaGISA World Congress 2006). Japan: Seto Marine Biological Laboratory, Field Science Education and Research Center, Kyoto University. pp 73-86.

Zulfigar, Y., Sim, Y.K., Tan, S.H. & Shirayama, Y. 2008. Field Guide to the Echinoderms (Sea Cucumber and Sea Stars) of Malaysia. The Nippon Foundation, Japan: Kyoto University Press. p. 103.

*Corresponding author; email: physique481@yahoo.com