Isolasi dan Identifikasi Molekuler Bakteri Proteolitik dari Saluran Pencernaan Ikan Nila (Oreochromis niloticus) yang Dibudidayakan di Kabupaten Banyumas

Mohammad Nurhafid; Hamdan Syakuri; Oedjijono Oedjijono; Emyliana Listiowati; Anandita Ekasanti; Dewi Nugrayani; Hendro Pramono

doi:10.22146/jfs.64072

Isolasi dan Identifikasi Molekuler Bakteri Proteolitik dari Saluran Pencernaan Ikan Nila (Oreochromis niloticus) yang Dibudidayakan di Kabupaten Banyumas

https://doi.org/10.22146/jfs.64072

Mohammad Nurhafid⁽¹⁾, Hamdan Syakuri^(2*), Oedjijono Oedjijono⁽³⁾, Emyliana Listiowati⁽⁴⁾, Anandita Ekasanti⁽⁵⁾, Dewi Nugrayani⁽⁶⁾, Hendro Pramono⁽⁷⁾

(1) Program Magister Biologi, Fakultas Biologi, Universitas Jenderal Soedirman, Purwokerto, Jawa Tengah, Indonesia
(2) Program Studi Akuakultur, Fakultas Perikanan dan Ilmu Kelautan, Universitas Jenderal Soedirman, Purwokerto, Jawa Tengah, Indonesia
(3) Laboratorium Mikrobiologi, Fakultas Biologi, Universitas Jenderal Soedirman, Purwokerto, Jawa Tengah, Indonesia
(4) 3Program Studi Akuakultur, Fakultas Perikanan dan Ilmu Kelautan, Universitas Jenderal Soedirman, Purwokerto, Jawa Tengah, Indonesia
(5) Program Studi Akuakultur, Fakultas Perikanan dan Ilmu Kelautan, Universitas Jenderal Soedirman, Purwokerto, Jawa Tengah, Indonesia
(6) Program Studi Akuakultur, Fakultas Perikanan dan Ilmu Kelautan, Universitas Jenderal Soedirman, Purwokerto, Jawa Tengah, Indonesia
(7) Laboratorium Mikrobiologi, Fakultas Biologi, Universitas Jenderal Soedirman, Purwokerto, Jawa Tengah, Indonesia
(*) Corresponding Author

Abstract

Keberadaan bakteri proteolitik pada komoditas akuakultur penting untuk dipelajari, salah satunya terkait dengan praktek budidaya ikan skala kecil di daerah pedesaan. Penelitian ini bertujuan untuk mengetahui keberadaan dan melakukan identifikasi secara molekuler bakteri proteolitik yang diisolasi dari saluran pencernaan ikan nila (Oreochromis niloticus). Sampel ikan nila diambil dari tiga unit kegiatan akuakultur yang menggunakan pakan berbeda di Kabupaten Banyumas yaitu dari Desa Pandak (dengan probiotik, pakan pellet), Desa Beji (tanpa probiotik, pakan tumbuhan) dan Desa Tambaksogra (dengan probiotik, kombinasi pakan pellet dan tumbuhan). Jumlah bakteri, proporsi bakteri proteolitik, dan indeks aktifitas proteolitik diamati dari usus bagian anterior, middle, dan posterior. Sampel isolat bakteri proteolitik dikelompokkan berdasarkan hasil analisis restriksi 16S rDNA menggunakan software PhyElp. Bakteri dari setiap kelompok diidentifikasi berdasarkan sekuen gen 16S rDNA dengan menggunakan analisis BLAST dan analisis filogenetik. Jumlah bakteri di saluran pencernaan ikan nila dari tiga tempat relatif sama dan cenderung meningkat ke arah posterior. Hasil penelitian menunjukkan ikan nila dari Desa Pandak memiliki proporsi bakteri proteolitik yang lebih tinggi dibandingkan sampel ikan dari Desa Beji dan Tambaksogra. Nilai aktivitas bakteri proteolitik saluran pencernaan ikan nila dari Desa Pandak relatif lebih tinggi dibandingkan dari dua desa lainnya. Bakteri proteolitik dari saluran pencernaan ikan nila dapat dikelompokkan menjadi 15 kelompok berdasarkan polimorfisme hasil digesti fragment gen 16S rDNA. Sampel dari 15 kelompok tersebut memiliki sekuen 16S rDNA yang mirip dengan Pseudomonas aeruginosa (4 isolat), Plesiomonas shigelloides, Escherichia coli, Aeromonas veronii, Klebsiella variicola, Enterobacter ludwigii, Enterobacter hormaechei (2 isolat), Enterobacter cloacae, Bacillus subtilis, Bacillus amyloliquefaciens dan Bacillus sp.

Keywords

Aktivitas proteolitik; bakteri; 16S rDNA; Oreochromis niloticus; saluran pencernaa;

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References

Adeoye, A.A., R. Yomla, A. Jaramillo-Torres, A. Rodiles, D.L. Merrifield & S.J. Davies. 2016. Combined effects of exogenous enzymes and probiotics on nile tilapia (Oreochromis niloticus) growth, intestinal morphology and microbiome. Aquaculture. 463: 61-70. doi:https://doi.org/10.1016/j.aquaculture.2016.05.028

Algammal, A.M., M. Mabrok, E. Sivaramasamy, F.M. Youssef, M.H. Atwa, A.W. El-kholy, H.F. Hetta & W.N. Hozzein. 2020. Emerging MDR-Pseudomonas aeruginosa in fish commonly harbor oprL and toxA virulence genes and blaTEM, blaCTX-M, and tetA antibiotic-resistance genes. Sci. Rep. 10: 15961. doi:10.1038/s41598-020-72264-4

Ali, N.G., T.E.S. Ali, I.M. Aboyadak & M.A. Elbakry. 2021. Controlling Pseudomonas aeruginosa infection in Oreochromis niloticus spawners by Cefotaxime Sodium. Aquaculture. 544: 737107. doi:https://doi.org/10.1016/j.aquaculture.2021.737107

Altschul, S.F., W. Gish, W. Miller, E.W. Myers & D.J. Lipman. 1990. Basic Local Alignment Search Tool. J. Mol. Biol. 215: 403-410. doi:10.1016/S0022-2836(05)80360-2

Aly, S.M., W.G. Nouh & M.M. Salem-Bekhit. 2012. Bacteriological and histopathological studies on enterobacteriacea in nile tilapia Oreochromis Niloticus. J. Pharm. Biomed. Sci. 2: 94-104.

Armada, C.D & R.M.C. Simora. 2016. Isolation and identification of protease-producing Pseudomonas sp. PD14 in the gut of rabbitfish Siganus guttatus (Bloch 1787). Asian Fish. Sci. 29. 82-95.

Askarian, F., Z. Zhou, R.E. Olsen, S. Sperstad & E. Ringø. 2012. Culturable autochthonous gut bacteria in atlantic salmon (Salmo salar L.) fed diets with or without chitin. Characterization by 16S rRNA gene sequencing, ability to produce enzymes and in vitro growth inhibition of four fish pathogens. Aquaculture. 326-329: 1-8. doi:https://doi.org/10.1016/j.aquaculture.2011.10.016

Badan Pusat Statistik. 2017. Produksi Ikan di Kabupaten Banyumas (Ekor), 2014-2016 [WWW Document]. URL https://banyumaskab.bps.go.id/indicator/56/90/1/pr (accessed 11.21.21).

Bairagi, A., K.S. Ghosh, S.K. Sen & A.K. Ray. 2002. Enzyme producing bacterial flora isolated from fish digestive tracts. Aquac. Int. 10: 109-121. doi:10.1023/A:1021355406412

Baldissera, M.D., C.F. Souza, S.N. Descovi, C.M. Verdi, C.C. Zeppenfeld, L. de Lima Silva, A.L. Gindri, M.A. Cunha, R.C.V. Santos, B. Baldisserotto & A.S. da Silva. 2019. Effects of dietary grape pomace flour on the purinergic signaling and inflammatory response of grass carp experimentally infected with Pseudomonas aeruginosa. Aquaculture. 503: 217-224. doi:https://doi.org/10.1016/j.aquaculture.2019.01.015

Banerjee, G., A. Nandi, S.K. Dan, P. Ghosh & A.K. Ray. 2017. Mode of association, enzyme-producing ability and identification of autochthonous bacteria in the gastrointestinal tract of two indian air-breathing fish, murrel (Channa punctatus) and stinging catfish (Heteropneustes fossilis). Proc. Zool. Soc. 70: 132-140. doi:10.1007/s12595-016-0167-x

Behera, B.K., A.K. Bera, P. Paria, A. Das, P.K. Parida, S. Kumari, S. Bhowmick & B.K. Das. 2018. Identification and pathogenicity of Plesiomonas shigelloides in silver carp. Aquaculture. 493: 314-318. doi:https://doi.org/10.1016/j.aquaculture.2018.04.063

Bereded, N.K., M. Curto, K.J. Domig, G.B. Abebe, S.W. Fanta, H. Waidbacher & H. Meimberg. 2020. Metabarcoding analyses of gut microbiota of nile tilapia (Oreochromis niloticus) from Lake Awassa and Lake Chamo, Ethiopia. Microorganisms. doi:10.3390/microorganisms8071040

Castresana, J. 2000. Selection of Conserved Blocks from Multiple Alignments for Their Use in Phylogenetic Analysis. Mol. Biol. Evol. 17: 540-552. doi:10.1093/oxfordjournals.molbev.a026334

Chevenet, F., C. Brun, A.L. Bañuls, B. Jacq & R. Christen. 2006. TreeDyn: Towards dynamic graphics and annotations for analyses of trees. B.M.C. Bioinformatics 7: 1-9. doi:10.1186/1471-2105-7-439

Das, P., S. Mandal, A. Khan, S.K. Manna & K. Ghosh. 2014. Distribution of extracellular enzyme-producing bacteria in the digestive tracts of four brackish water fish species. Turkish J. Zool. 38. 79-88. doi:10.3906/zoo-1205-3

Davin-Regli, A., J.P. Lavigne & J.M. Pagès. 2021. Enterobacter spp.: Update on taxonomy, clinical aspects, and emerging antimicrobial resistance. Clin. Microbiol. Rev. 32: e00002-19. doi:10.1128/CMR.00002-19

Diwan, A.D., S.N. Harke, G. Gopalkrishna & A.N. Panche. 2021. Aquaculture industry prospective from gut microbiome of fish and shellfish: An overview. J. Anim. Physiol. Anim. Nutr. (Berl). n/a. doi:https://doi.org/10.1111/jpn.13619

F.A.O. 2020. The State of World Fisheries and Aquaculture 2020. Sustainability in action. Rome. doi:https://doi.org/10.4060/ca9229en

Ganguly, S & A. Prasad. 2012. Microflora in fish digestive tract plays significant role in digestion and metabolism. Rev. Fish Biol. Fish. 22. 11-16. doi:10.1007/s11160-011-9214-x

Gao, X., H. Zhang, Q. Jiang, N. Chen, X. Li, X. Liu, H. Yang, W. Wei & X. Zhang. 2019. Enterobacter cloacae associated with mass mortality in zoea of giant freshwater prawns Macrobrachium rosenbergii and control with specific chicken egg yolk immunoglobulins (IgY). Aquaculture. 501: 331-337. doi:https://doi.org/10.1016/j.aquaculture.2018.11.050

Gao, X., Y. Zhou, X. Zhu, H. Tang, X. Li, Q. Jiang, W. Wei & X. Zhang. 2021. Enterobacter cloacae: A probable etiological agent associated with slow growth in the giant freshwater prawn Macrobrachium rosenbergii. Aquaculture. 530: 735826. doi:https://doi.org/10.1016/j.aquaculture.2020.735826

Gascuel, O. 1997. BIONJ: An Improved Version of the N.J. Algorithm Based on A Simple Model of Sequence Data. Mol. Biol. Evol. 14, 685–695. doi:10.1093/oxfordjournals.molbev.a025808

Giri, S.S., J.W. Jun, S. Yun, H.J. Kim, S.G. Kim, S.W. Kim, K.J. Woo, S.J. Han, W.T. Oh, J. Kwon, V. Sukumaran & S.C. Park. 2020. Effects of dietary heat-killed Pseudomonas aeruginosa strain VSG2 on immune functions, antioxidant efficacy, and disease resistance in Cyprinus carpio. Aquaculture. 514: 734489. doi:https://doi.org/10.1016/j.aquaculture.2019.734489

Giri, S.S., S.S, Sen & V. Sukumaran. 2012. Effects of dietary supplementation of potential probiotic Pseudomonas aeruginosa VSG-2 on the innate immunity and disease resistance of tropical freshwater fish, Labeo rohita. Fish Shellfish Immunol. 32: 1135-1140. doi:https://doi.org/10.1016/j.fsi.2012.03.019

Guo, T-Y., W-Zhao, J-Y. He, S-Y. Liao, J-J. Xie, S-W. Xie, K. Masagounder, Y-J. Liu, L-X. Tian & J. Niu. 2020. Dietary dl-Methionyl-dl-Methionine supplementation increased growth performance, antioxidant ability, the content of essential amino acids and improved the diversity of intestinal microbiota in nile tilapia (Oreochromis niloticus). Br. J. Nutr. 123: 72-83. DOI: 10.1017/S0007114519002289

Kar, N., R.N. Roy, S.K. Sen & K. Ghosh. 2008. Isolation and characterization of extracellular enzyme-producing bacilli in the digestive tracts of rohu, Labeo rohita (Hamilton) and Murrel, Channa punctatus (Bloch). Asian Fish. Sci. 21: 421-434.

Kuebutornye, F.K.A., Z. Wang, Y. Lu, E.D. Abarike, M.E. Sakyi, Y. Li, C.X. Xie & V. Hlordzi. 2020. Effects of three host-associated Bacillus species on mucosal immunity and gut health of nile tilapia, Oreochromis niloticus and its resistance against Aeromonas hydrophila infection. Fish Shellfish Immunol. 97. 83-95. doi:https://doi.org/10.1016/j.fsi.2019.12.046

Kurniasih, T., A.M. Lusiastuti, Z.I. Azwar & I. Melati. 2014. Isolasi dan seleksi bakteri saluran pencernaan ikan lele sebagai upaya mendapatkan kandidat probiotik untuk efisiensi pakan ikan. J. Ris. Akuakultur. 9: 99. doi:10.15578/jra.9.1.2014.99-109

Lagacé, L., M. Pitre, M. Jacqeus & D. Roy. 2004. Identification of the bacterial community of maple sap by using amplified ribosomal DNA (rDNA) restriction analysis and rDNA sequencing. Appl. Environ. Microbiol. 70. 2052-2060. doi:10.1128/AEM.70.4.2052-2060.2004

Li, J., J. Ni, J. Li, C. Wang, X. Li, S. Wu, T. Zhang, Y. Yu & Q. Yan. 2014. Comparative study on gastrointestinal microbiota of eight fish species with different feeding habits. J. Appl. Microbiol. 117: 1750-1760. doi:10.1111/jam.12663

Maatallah, M., M. Vading, M.H. Kabir, A. Bakhrouf, M. Kalin, P. Nauclér, S. Brisse & C.G. Giske. 2014. Klebsiella variicola is a frequent cause of bloodstream infection in the Stockholm area, and associated with higher mortality compared to K. pneumoniae. PLoS One. 9: e113539.

Marchesi, J.R., T. Sato, A.J. Weightman, T.A. Martin, J.C. Fry, S.J. Hiom & W.G. Wade. 1998. Design and evaluation of useful bacterium-specific PCR primers that amplify genes coding for bacterial 16S rRNA. Appl. Environ. Microbiol. 64: 795-799. doi:10.1128/aem.64.2.795-799.1998

Martins, A.F.M., T.L. Pinheiro, A. Imperatori, S.M. Freire, L. Sá-Freire, B.M. Moreira & R.R. Bonelli. 2019. Plesiomonas shigelloides: A notable carrier of acquired antimicrobial resistance in small aquaculture farms. Aquaculture. 500: 514-520. doi:https://doi.org/10.1016/j.aquaculture.2018.10.040

Michael, T.M & M.M. John. 2006. Brock Biology of Microorganisms, Clinical Microbiology and Parasitology (For DMLT Students). doi:10.5005/jp/books/12721_4

Mondal, S., T. Roy, S.K. Sen & A.K. Ray. 2008. Distribution of enzyme-producing bacteria in the digestive tracts of some freshwater fish. Acta Ichthyol. Piscat. 38. 1-8. doi:10.3750/AIP2008.38.1.01

Mukherjee, A., A. Rodiles, D.L. Merrifield, G. Chandra & K. Ghosh. 2020. Exploring intestinal microbiome composition in three Indian major carps under polyculture system: A high-throughput sequencing-based approach. Aquaculture. 524: 735206. doi:https://doi.org/10.1016/j.aquaculture.2020.735206

Pavel, A.B & C.I. Vasile. 2012. PyElph - a software tool for gel images analysis and phylogenetics. B.M.C. Bioinformatics. 13: 9. doi:10.1186/1471-2105-13-9

Ray, A.K., K. Ghosh & E. Ringø. 2012. Enzyme-producing bacteria isolated from fish Gut: A review. Aquac. Nutr. 18: 465-492. doi:https://doi.org/10.1111/j.1365-2095.2012.00943.x

Ray, A.K., T. Roy, S. Mondal & E. Ringø. 2010. Identification of gut-associated amylase, cellulase and protease-producing bacteria in three species of indian major carps. Aquac. Res. 41. 1462-1469. doi:https://doi.org/10.1111/j.1365-2109.2009.02437.x

Ringø, E., Z. Zhou, J.L.G. Vecino, S. Wadsworth, J. Romero, A. Krogdahl, R.W. Olsen, A. Dimitroglou, A. Foey, S. Davies, M. Owen, H.L. Lauzon, L.L. Martinsen, P. De Schryver, P. Bossier, S. Sperstad & D.L. Merrifield. 2016. Effect of dietary components on the gut microbiota of aquatic animals. A never-ending story? Aquac. Nutr. 22: 219-282. doi:10.1111/anu.12346

Romero, J., E. Ringø & D.L. Merrifield. 2014. The gut microbiota of fish. Aquac. Nutr., Wiley Online Books. doi:https://doi.org/10.1002/9781118897263.ch4

Standen, B.T., A. Rodiles, D.L. Peggs, S.J. Davies, G.A. Santos & D.L. Merrifield. 2015. Modulation of the intestinal microbiota and morphology of tilapia, Oreochromis niloticus, following the application of a multi-species probiotic. Appl. Microbiol. Biotechnol. 99: 8403-8417. doi:10.1007/s00253-015-6702-2

Su, S., B.P. Munganga, F. Du, J. Yu, J. Li, F. Yu, M. Wang, X. He, X. Li, R. Bouzoualegh, P. Xu & Y. Tang. 2020. Relationship between the fatty acid profiles and gut bacterial communities of the Chinese mitten crab (Eriocheir sinensis) from ecologically different habitats. Front. Microbiol.

Tachibana, L., G.S. Telli, D. De-C. Dias, G.S. Gonçalves, M.C. Guimarães, C.M. Ishikawa, R.B. Cavalcante, M.M. Natori, M.F. Fernandez-Alarcon, S. Tapia-Paniagua, M.A. Moriñigo, F.J. Moyano, E.R.L. de Araújo & M.J.T. Ranzani-Paiva. 2021. Bacillus subtilis and Bacillus licheniformis in diets for nile tilapia (Oreochromis niloticus): effects on Growth Performance, Gut Microbiota Modulation and Innate Immunology. Aquac. Res. 52. 1630-1642. doi:https://doi.org/10.1111/are.15016

Talukdar, S., E. Ringø & K. Ghosh. 2016. Extracellular tannase-producing bacteria detected in the digestive tracts of freshwater fishes (Actinopterygii: Cyprinidae and Cichlidae). Acta Ichthyol. Piscat. 46. 201-210.

Talwar, C., S. Nagar, R. Lal & R.K. Negi. 2018. Fish gut microbiome: current approaches and future perspectives. Indian J. Microbiol. 58: 397-414. doi:10.1007/s12088-018-0760-y

Tóth, E.M., A.K. Borsodi, T. Felföldi, B. Vajna, R. Sipos & K. Márialigeti. 2013. Practical Microbiology: based on the Hungarian practical notes entitled “Mikrobiológiai Laboratóriumi Gyakorlatok.” Eötvös Loránd University, HUngary.

Wang, A.R., C. Ran, E. Ringø & Z.G. Zhou. 2018. Progress in fish gastrointestinal microbiota research. Rev. Aquac. 10: 626-640. doi:https://doi.org/10.1111/raq.12191

Xia, Y., E. Yu, M. Lu & J. Xie. 2020. Effects of probiotic supplementation on gut microbiota as well as metabolite profiles within nile tilapia, Oreochromis niloticus. Aquaculture. 527: 735428. doi:https://doi.org/10.1016/j.aquaculture.2020.735428

Zainuddin, M., W.A. Setyati & P.P. Renta. 2018. Zona hidrolisis dan pertumbuhan bakteri proteolitik dari sedimen ekosistem mangrove Rhizophora mucronata Telukawur-Jepara. Akuatik J. Sumberd. Perair. 11: 31-35. doi:10.33019/akuatik.v11i2.241.

DOI: https://doi.org/10.22146/jfs.64072

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Jurnal Perikanan Universitas Gadjah Mada (print ISSN 0853-6384; online ISSN 2502-5066) is published by Department of Fisheries, Universitas Gadjah Mada in collaboration with Semnaskan UGM (Seminar Nasional Tahunan Hasil Perikanan dan Kelautan) and ISMFR (International Symposium on Marine and Fisheries Research).

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