Colony morphology and molecular identification of Vibrio spp. on green mussels (Perna viridis) in Yogyakarta, Indonesia tourism beach areas
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Abstract. Hikmawati F, Susilowati A, Setyaningsih R. 2019. Colony morphology and molecular identification of Vibrio spp. on green mussels (Perna viridis) in Yogyakarta, Indonesia tourism beach areas. Biodiversitas 20: 2891-2899. Green mussels (Perna viridis) have filter feeder properties that allow pathogenic bacteria from the water environment to accumulate in relatively high levels. About 20% of foodborne diseases are caused by large quantities of seafood contaminated with bacteria. The purpose of this study is to determine the morphological characteristics, pathogenicity, identity, and the kinship of Vibrio species on green mussels in Yogyakarta coastal tourism areas. Vibrio spp. were grown on selective differential TCBS media. In this media, the suspected Vibrio spp. would produce yellow or green colonies. The ability of hemolysis of Vibrio was blood agar media, the species was molecularly identified using 16S rRNA gene sequence, and the phylogenetic relationship of the Vibrio spp., was analyzed using MEGA X Neighbor-Joining program. Based on morphological analysis, we obtained 23 bacterial isolates suspected to be Vibrio spp. Two Isolates (L1K2 6 and L2K2 13) were positive for ?-hemolysis activity and 4 isolates (L1K1 3, L2K1 8, L2K2 16, and L3K2 22) were positive for ?-hemolysis activity. The molecular analysis involved 18 Vibrio species, and 4 of them represented the Vibrio genus and 14 species represented 97-99% similarity species in accordance with the 16S rRNA sequence in database, namely: Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio cholerae, Vibrio neocaledonicus, Vibrio mimicus, Vibrio azureus, Vibrio diabolicus, Vibrio tapetis, Vibrio natriegens, and Vibrio owensii. The most dominant number of Vibrio isolates was V. alginolyticus, while the lowest was V. owensii. The highest number of Vibrio species in green mussels was found in Goa Cemara beach while the lowest was in Kwaru beach. Vibrio spp bacteria found in green clams in coastal tourism areas in Yogyakarta have close phylogenetic relationships with other Vibrio in seafood in Indonesian coastal waters.
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References
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Ilmiah, Sukenda, Widanarni dan Enang H. 2012. Isolation and characterization of Pathogenic Vibrio on Tiger grouper Epinephelus fuscoguttatus. Indonesian Aquaculture Journal. Bogor Vol. 11 (1), 28-37.
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Kharirie. 2013. Diagnosa Vibrio cholerae dengan Metode Kultur dan Polimerase Chain Reaction (PCR) pada Sampel Sumber Air Minum. Indonesian Medisiana Biotech Journal. Biomedical Center and Basic Technology for Balitbangkes Health, Indonesian Ministry of Health. Vol 2.2.2013:51-58.
Kurniawan RA. 2016. Identification and Molecular Characterization of Vibrio Bacteria in Seawater Fish in the Batam and Mataram Regions. Veterinary Science Thesis. Yogyakarta's Gadjah Mada University Sain Verener Study Program.
Liu PC, and KK Lee. 1999. Cysteine Protease is a Major Exotoxin of Pathogenic Luminous Vibrio harveyi in The Tiger Prawn, Penaeus monodon. Letters in Applied Microbiology, 28(6):428- 430.
Madigan, MT, JM.Martinko dan J. Parker. 2000. Biology of Microorganisms. 9th edition. Prentice Hall International, Inc. New Jersey.
Marchesi, JR, Sato T. Wightman AJ, Martin TA, Fry JC, Hiom SJ, Wade WG. 1998. Design and evaluation of useful bacterium-specific PCR primers that amplify genes coding for bacterial 16S rRNA. Appl Environ. Microbiol 64:795-9.
Montieri S, Suffredini, Elisabetta, Cicozza M, dan Croci Luciana. 2010. Phylogenic and Evolutionary Analysis of Vibrio parahaemolyticus and Vibrio alginolyticus isolated based on toxR Gene Sequence. Journal New Microbiological. 33:359-372.
Murdinah. 2009. Handling and Differentiation of Processed Green Shell Products. Researcher at the Research Center for Marine and Fisheries Biotechnology and Product Processing. SQUALEN Vol. 4. No. 2.
Nei M, and Kumar S. (2000). Molecular Evolution and Phylogenetics. Oxford University Press, New York.
Nitimulyo KH, Alim I, Triyanto, Indah I, Muhammad M. 2005. Isolation, Identification and Characterization of Vibrio spp. Pathogens that cause Vibriosis in grouper shells at the Brackish Water Aquaculture Center in Situbondo. Journal of Fisheries. Vol 12 (2): 80-94.
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Parjitno. 1995. Primadona Penyakit Udang Windu di Tambak. Makalah Penelitian Nasional Keterampilan dan Bina Usaha Mandiri bidang Budidaya Air Payau dan tawar. Malang: Mahasiswa Pemuda Pedesaan Brawijaya. Malang. 17 hal.
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Saitou N, and Nei M. (1987). The neighbour-joining method: A new method for reconstructing phylogenetic trees. Molecular Biology and Evolution 4:406-425.
Sethi L. 2014. Pathogenicity, genetic aspects and characterization of Vibrio species Isolated from Marine Environment. Thesis. Department of Life Science National Institue of Technology Rourkela, Odisha.
Supardi I, and Sukamto. 1999. Microbiology in Food Processing and Safety. Adikarya IKAPI Foundation & The Food Foundation. Alumni Publisher. Bandung, pp. 1-290.
Suryono CA. 2013. Filtration of Green Mussels (Perna viridis) Against Micro Algae in Heavy Metal Contaminated Media. Marine Science Journal. Faculty of Fisheries and Marine Science. Diponegoro University. Semarang. Vol 2 (5): 1-4. Pp. 41-47.
Tamura K, dan Nei M. (1993). Perkiraan jumlah substitusi nukleotida di wilayah kontrol DNA mitokondria pada manusia dan simpanse. Biology and Molecular Evolution. 10: 512-526.
Tortora G, Funke B, Case C. 2001. Microbiology an introduction. Edition 7. United States of America: Addison Wesley Longman, Inc. p.407.
Wati SA. 2014. Synthesis and Characterization of Hydroxyapatite from Shellfish Feathers Waste (Andara antiquate). Essay. Faculty of Math and Science. The University of North Sumatra.
Wayan IYW. 2015. The existence of Vibrio cholera pathogenic bacteria in some fishery products sold in traditional markets in Denpasar City. Biology Thesis. Biology Study Program at Udayana University Denpasar.
Yital M, Peter HF, Hammes F, and Egli T. 2007. Growth of Vibrio cholerae Ol Ogawa Eltor in freshwater. Microbiol. 153 : 1993-2001
Zulkifli Y, NB, Alithenn R, Son SK, Yeap MB, Lesley, dan Raha AR. 2009. Identification of Vibrio parahaemolyticus isolates by PCR targeted to the toxR gene and detection of virulence genes. International Food Research Journal 16: 289-296.
National Agency of Drug and Food Control. 2009. Regulation of the Head of the Indonesian Food and Drug Supervisory Agency Number. HK. 00.06.1.52.4011. About Determining the Maximum Limit of Microbial Contaminants and Food Chemistry. Jakarta.
Buxton R. 2013. Blood agar plates and hemolysis protocols. American Society for Microbiology.
Campanelli C M. 2012. American Geriatrics Society Updated Beers Criteria for Potentially Inappropriate Medication Use in Older Adults. Journal of the American Geriatrics Society, 60: 616–631.
Daniels R, Nicoll L H. 2012. Contemporary medical surgical nursing second editions, Delmar: Cengage Learning, Canada, 1591-1614.
Davies BR, Fanning GR, Madden JM, Steigerwalt G, Gradford HB, Smith HL Jr, Brenner DF. 1981. Characterisation of biochemically atypical Vibrio cholera trains and designation of a new pathogenic species, Vibrio mimicus. J. Clin. Microbiol. 14:631-639.
De Paola, A, C.A. Kaysner, and B. John. 1998. Environmental Investigation of Vibrio parahaemolitycus in oyster after Outbreaks in Washington, Texas and New York.
Eshmat ME, Mahasri G, Rahardja BS. 2014. Analysis of Lead (pb) and Cadmium (Cd) Heavy Metals in Green Mussels (Perna viridis L.) in the Ngemboh Waters of Gresik Regency, East Java. Journal of Fisheries. Faculty of Fisheries and marine affairs. Airlangga University. Surabaya.
Fajriani B, Budiharjo A, and Pujiyanto S. 2018. Isolation and Identification of Molecular Antagonist Bacteria Against Vibrio parahaemolyticus Pathogen in Litopenaeus vannamei Shrimp from Mangrove Probiotic and Sediment Products in Rembang. Journal of Biology. Vol 7 (1) 52-53.
Feliatra F, Titania TN, Sila S, Yuslina O. 2011. Screening Of Indonesian Original Bacteria Vibrio sp. As A Cause Of Shrimp Diseases Based On 16S Ribosomal DNA- Technique. Jurnal ilmu dan Teknologi Kelautan, Vol. 3, No. 2, Halaman. 85-99.
Felsenstein J. (1985). Confidence limits on phylogenies: An approach using the bootstrap. Evolution 39:783-791.
Ferdian BF, Sarjito, Slamet BP. 2014. Identifikasi Agensia Penyebab Vibriosis Pada Penggemukan Kepiting Bakau (Scylla serrata) Di Pemalang. Jurnal Of Aquaqulture Management and Technology Volume 3, Nomor 2. Halaman. 126-134.
Fitriatin, E, Manan A. 2015. Examination of Viral Nervous Nevrosis (VNN) in Fish with the Polymerase Chain Reaction (PCR) Method. Fisheries and Marine Scientific Journal. Vol 7 (1): 2088-5842.
Fransisca G D M, Marina TTR. 2016. Phatogenic Vibrio Species Isolated from Estuarine Environment (Ceara, Brazil)- Antimicrobial Resistance and Virulance Potential Profiles. Journal Annals of The Brazilian Academy of Science. 89 (2); 1175-1188.
Fukui T, Shiraki K, Hamada D, Hara K, Miyata T, Fujiwara S, Mayanagi K, Yanagihara K, Lida K, Fukusaki. 2005. Thermostable direct hemolysin of Vibrio parahaemolyticus is a Bacterial Reversible Amyloid Toxin. Biochemistry, 44 (29), 9825-9832.
Hagstrom A, Pinhassi JF, and Zweiefel UL. 2000. Biogeographical Diversity Among Marine Bacterioplankton. Journal Microbial Technology Aquatic, 21: 231-244.
Hanny W. Mewengkang. 2010. Identifikasi Vibrio sp. Pada Gonad Ikan Cakalang (Katsuwonus pelamis L). Journal of Fisheries and Marine Affairs. Vol VI (1): 18-21.
Hasrida M, Indra R, Yusran U. 2016. Genomic DNA Concentration and Purity Measurement of Anopheles barbirostris. Disease Vector Journal. P2B2 Donggala Research and Development Center, Health Research and Development Agency, Ministry of Health of the Republic of Indonesia, Central Sulawesi.
Ilmiah, Sukenda, Widanarni dan Enang H. 2012. Isolation and characterization of Pathogenic Vibrio on Tiger grouper Epinephelus fuscoguttatus. Indonesian Aquaculture Journal. Bogor Vol. 11 (1), 28-37.
Jawetz, Melnick, Adelberg. 2007. Medical Microbiology. Surabaya. Salemba Medika.
Jesus L, Romalde, Ana L, Dieguez, Aide Lasa, Sabela Balboa. 2013. New Vibrio Species Associated to Molluscan Microbiota: a review. Journal Frontiers in Mirobiology. Universidad de Santiago de Compostela. 1664-302X.
Kumar S, Stecher G, Li M, Knyaz C, and Tamura K. (2018). MEGA X: Analisis Genetika Evolusi Molekul di seluruh platform komputasi. Biology and Molecular Evolution 35: 1547-1549.
Kharirie. 2013. Diagnosa Vibrio cholerae dengan Metode Kultur dan Polimerase Chain Reaction (PCR) pada Sampel Sumber Air Minum. Indonesian Medisiana Biotech Journal. Biomedical Center and Basic Technology for Balitbangkes Health, Indonesian Ministry of Health. Vol 2.2.2013:51-58.
Kurniawan RA. 2016. Identification and Molecular Characterization of Vibrio Bacteria in Seawater Fish in the Batam and Mataram Regions. Veterinary Science Thesis. Yogyakarta's Gadjah Mada University Sain Verener Study Program.
Liu PC, and KK Lee. 1999. Cysteine Protease is a Major Exotoxin of Pathogenic Luminous Vibrio harveyi in The Tiger Prawn, Penaeus monodon. Letters in Applied Microbiology, 28(6):428- 430.
Madigan, MT, JM.Martinko dan J. Parker. 2000. Biology of Microorganisms. 9th edition. Prentice Hall International, Inc. New Jersey.
Marchesi, JR, Sato T. Wightman AJ, Martin TA, Fry JC, Hiom SJ, Wade WG. 1998. Design and evaluation of useful bacterium-specific PCR primers that amplify genes coding for bacterial 16S rRNA. Appl Environ. Microbiol 64:795-9.
Montieri S, Suffredini, Elisabetta, Cicozza M, dan Croci Luciana. 2010. Phylogenic and Evolutionary Analysis of Vibrio parahaemolyticus and Vibrio alginolyticus isolated based on toxR Gene Sequence. Journal New Microbiological. 33:359-372.
Murdinah. 2009. Handling and Differentiation of Processed Green Shell Products. Researcher at the Research Center for Marine and Fisheries Biotechnology and Product Processing. SQUALEN Vol. 4. No. 2.
Nei M, and Kumar S. (2000). Molecular Evolution and Phylogenetics. Oxford University Press, New York.
Nitimulyo KH, Alim I, Triyanto, Indah I, Muhammad M. 2005. Isolation, Identification and Characterization of Vibrio spp. Pathogens that cause Vibriosis in grouper shells at the Brackish Water Aquaculture Center in Situbondo. Journal of Fisheries. Vol 12 (2): 80-94.
Nor M, Najwa A, Muhd DD, Mat AKAAW, Effendy M. 2015. Detection of Virulence Genes in Vibrio alginolyticus Isolated from Green Mussel Perna viridis. Journal Teknologi. Institute of Marine Biotechnology, Universitas Malaysia Terengganu, Malaysia.
Oliver JD, dan Kepper JB. 2001. Vibrio Sp.esies.In: M.P Doyle, L.R Beuchat and TJ Montville, eds. Food Microbiology: Fundamentals and Frontiers. 2nd Washington: ASM Press 263-300.
Parjitno. 1995. Primadona Penyakit Udang Windu di Tambak. Makalah Penelitian Nasional Keterampilan dan Bina Usaha Mandiri bidang Budidaya Air Payau dan tawar. Malang: Mahasiswa Pemuda Pedesaan Brawijaya. Malang. 17 hal.
Richie JP. 2005. Analisis Bakteri Vibrio Pada Udang Windu (Penaeus monodon) Tambak di Bengkalis Propinsi Riau. Skripsi, Faperikan UNRI. Pekanbaru (tidak diterbitkan).
Saitou N, and Nei M. (1987). The neighbour-joining method: A new method for reconstructing phylogenetic trees. Molecular Biology and Evolution 4:406-425.
Sethi L. 2014. Pathogenicity, genetic aspects and characterization of Vibrio species Isolated from Marine Environment. Thesis. Department of Life Science National Institue of Technology Rourkela, Odisha.
Supardi I, and Sukamto. 1999. Microbiology in Food Processing and Safety. Adikarya IKAPI Foundation & The Food Foundation. Alumni Publisher. Bandung, pp. 1-290.
Suryono CA. 2013. Filtration of Green Mussels (Perna viridis) Against Micro Algae in Heavy Metal Contaminated Media. Marine Science Journal. Faculty of Fisheries and Marine Science. Diponegoro University. Semarang. Vol 2 (5): 1-4. Pp. 41-47.
Tamura K, dan Nei M. (1993). Perkiraan jumlah substitusi nukleotida di wilayah kontrol DNA mitokondria pada manusia dan simpanse. Biology and Molecular Evolution. 10: 512-526.
Tortora G, Funke B, Case C. 2001. Microbiology an introduction. Edition 7. United States of America: Addison Wesley Longman, Inc. p.407.
Wati SA. 2014. Synthesis and Characterization of Hydroxyapatite from Shellfish Feathers Waste (Andara antiquate). Essay. Faculty of Math and Science. The University of North Sumatra.
Wayan IYW. 2015. The existence of Vibrio cholera pathogenic bacteria in some fishery products sold in traditional markets in Denpasar City. Biology Thesis. Biology Study Program at Udayana University Denpasar.
Yital M, Peter HF, Hammes F, and Egli T. 2007. Growth of Vibrio cholerae Ol Ogawa Eltor in freshwater. Microbiol. 153 : 1993-2001
Zulkifli Y, NB, Alithenn R, Son SK, Yeap MB, Lesley, dan Raha AR. 2009. Identification of Vibrio parahaemolyticus isolates by PCR targeted to the toxR gene and detection of virulence genes. International Food Research Journal 16: 289-296.
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