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Isolasi, Karakterisasi, Aktivitas anti-Vibrio dan Klaster Gen Penyandi Senyawa Bioaktif dari Pseudoalteromonas xiamenensis STKMTI.2

DESY PUTRI HANDAYANI, Dr. Ir. Alim Isnansetyo, M.Sc; Indah Istiqomah, S.Pi., M.Si., Ph.D; Prof. Drs. Jumina, Ph.D

2022 | Disertasi | DOKTOR ILMU PERTANIAN

Vibriosis adalah ancaman serius bagi budidaya laut sehingga perlu dikendalikan agar tidak menimbulkan kerugian besar. Pseudoalteromonas merupakan bakteri laut yang dapat menghasilkan senyawa bioaktif untuk kepentingan manusia seperti senyawa antibakteri. Penelitian ini bertujuan untuk mengidentifikasi secara fenotipik dan molekuler, produksi senyawa anti-Vibrio melalui fermentasi cair, isolasi, karakterisasi dan elusidasi struktur kimia senyawa anti-Vibrio, uji aktivitas anti-Vibrio secara in vitro melawan V. alginolyticus (strain S4UA, S5UC, BCSA, and BSCP 1C), V. harveyi (strain GK18, SB 25, BT1H, SL2, SH2, A1, and A2), dan V. parahaemolyticus (strain SMI1A, CJ10, and CJ5), uji biokontrol V. harveyi GK18 oleh strain STKMTI.2 dan analisis genom lengkap serta gen penyandi senyawa bioaktif pada bakteri laut STKMTI.2. Isolat STKMTI.2 diidentifikasi menggunakan kit Kb007 dan analisis sekuen gen 16S rDNA. Isolat STKMTI.2 difermentasi pada medium Zobell 2216E agar dan cair dengan lama inkubasi 0-120 jam untuk menghasilkan senyawa antibakteri. Setelah diketahui medium dan lama inkubasi, selanjutnya dilakukan proses fermentasi pada skala yang lebih besar. Etil asetat dan ammonium sulfat digunakan untuk ekstraksi senyawa dari supernatan dan etanol untuk ekstraksi senyawa dari sel pellet. Permurnian senyawa anti-Vibrio menggunakan metode kromatografi lapis tipis, kolom kromatografi, dan Preparative Layer Chromatography, sedangkan karakterisasi senyawa menggunakan metode kromatografi lapis tipis dan Gas Chromatography Mass Spectrometry. Uji aktivitas antibakteri terutama anti-Vibrio dilakukan dengan metode difusi cakram pada medium double layer agar. Uji bioautografi dilakukan menggunakan kromatografi lapis tipis. Minimum Inhibitory Concentration dilakukan dengan metode pengenceran pada Microplate 96 well. Aktivitas penghambatan in vitro P. xiamenensis STKMTI.2 terhadap V. harveyi dilakukan dengan metode co-culture pada medium cair Zobell 2216E selama 48 jam. Analisis sekuen genom lengkap dilakukan dengan teknologi Oxford GridION. Analisis Klaster gen penyandi senyawa bioaktif dianalisis menggunakan aplikasi antiSMASH dan BAGEL4. Hasil analisis sekuen 16S rDNA menunjukkan bahwa STKMTI.2 memiliki kemiripan dengan P. xiamenensis. Senyawa anti-Vibrio dari isolat STKMTI.2 dihasilkan ketika difermentasi menggunakan medium cair Zobell 2216E selama 96 jam. Senyawa tersebut merupakan produk ekstraseluler. Isolat STKMTI.2 dengan kepadatan 105 dan 106 CFU mL-1 mampu menurunkan pertumbuhan V. harveyi hingga 50%. Kolom kromatografi memisahkan ekstrak partisi kloroform menjadi 148 fraksi. Senyawa bioaktif dari isolat STKMTI.2 secara spesifik menghambat Vibrio spp. pada dosis 2.500 �µg disc-1 untuk ekstrak kasar, 1.300 �µg disc-1 untuk partisi kloroform, dan 1.000 �µg disc-1 untuk fraksi kolom kromatografi. Analisis GC-MS pada senyawa aktif subfraksi PLC memprediksi 4 senyawa, yaitu trietoksiboran; 1,3-difenil-1,3,5,5-tetrametilsiklotrisiloksana; 1,6-diazaspiro-4.4-nonan-2,7-diona; and 1,3-bis-4-metoksifenil-â��5-fenil- 1,3,5-triazinan-2-tion. Nilai MIC dari senyawa subfraksi adalah â�¤0,78 �µg mL-1, lebihrendah dibandingkan antibiotik komersial. Isolat STKMTI.2 memiliki genom berukuran 4.563.326 bp (GC content: 43,2%), yang terdiri dari 1 kromosom, 2 plasmid sirkuler (unnamed1 dan unnamed2), dan 2 plasmid linear (unnamed4 and unnamed5), 4,824 sekuen penyandi protein dan 1 clustered regularly interspaced short palindromic repeated (CRISPR). Genom bakteri STKMTI.2 memiliki beberapa gen penyandi senyawa antibakteri, yaitu bmp8, bmp9, non ribosomal peptide synthase, polyketide-like butyrolactone, Lant class I, RiPP-like terdeteksi pada kromosom dan prodigiosin terdeteksi pada plasmid linear Unnamed5. Penelitian ini berhasil mengeksplorasi potensi dari isolat STKMTI.2 sebagai bakteri laut penghasil senyawa anti-Vibrio dan kandidat biokontrol untuk mengatasi vibriosis.

Vibriosis is a prevalent disease in almost all mariculture spesies worldwide caused by Vibrio spp. Pseudoalteromonas is a marine bacterium that can produce secondary metabolites for human uses, such as antibacterial compounds. This study aimed to identify the STKMTI.2 isolate, conduct the isolation, purify and characterize the anti-Vibrio compounds, conduct in vitro anti-Vibrio activity tests on V. alginolyticus (strain codes S4UA, S5UC, BCSA, and BSCP 1C), V. harveyi (strain codes GK18, SB 25, BT1H, SL2, SH2, A1, and A2), and V. parahaemolyticus (strain codes SMI1A, CJ10, and CJ5), perform a biocontrol test against V. harveyi GK18, and analyze the complete genome and the biosynthetic gene cluster of STKMTI.2 isolate. STKMTI.2 isolate was identified using the Kb007 biochemical test kit and 16S rDNA sequencing analysis. STKMTI.2 was inoculated on the Zobell 2216E agar and broth medium with incubation time of 0-120 hours to produce antibacterial compounds. After knowing the medium and incubation time, a large-scale fermentation was processed. Isolation of anti-Vibrio compound was carried out using ethyl acetate and ammonium sulphate of supernatan and etanol of the cell pellet. Characterization of anti-Vibrio compounds was carried out by thin layer chromatography, column chromatography, preparative layer chromatography and gas chromatography mass spectrometry methods. Antibacterial activity test was conducted by disc diffusion on the double layer agar medium. Bioautography was carried out by the TLC. Minimum Inhibitory Concentration test was carried out by microdilution method on the 96 well microplate. In vitro test of STKMTI.2 was conducted by co-culture test on the Zobell 2216E broth medium for 48 hours. Complete genome sequence analysis was conducted by Oxford Nanopore GridON technology. Clusters of genes encoding secondary metabolites synthesis was analysed using antiSMASH and BAGEL4. The results showed that STKMTI.2 was closest to P. xiamenensis based on the 16S rDNA sequencing analysis. Anti-Vibrio compounds from STKMTI.2 were produced after 96 hours of incubation on the Zobell 2216E broth medium. These compounds are extracellular products. The isolate of STKMTI.2 with 105 and 106 CFU mL-1 inhibited 50% of V. harveyi growth. Column chromatography separated the crude extract into 148 fractions. Bioactive compound from STKMTI.2 inhibited Vibrio spp. at a dose of 2.500 �µg disc-1 for crude extract, 1.300 �µg disc-1 for the chloroform extract, and 1.000 �µg disc-1for column chromatography fraction. The results of GC-MS analysis on the active compound sub-fraction PLC were predicted 4 compounds, namely triethoxy-borane; 1,3-diphenyl- 1,3,5,5-tetramethyl-cyclotrisiloxane; 1,6-diazaspiro-4.4-nonane-2,7-dione; and 1,3-bis-4- methoxyphenyl-5-phenyl-1,3,5-triazinane-2-thione. The MIC value of the partially purified compound was â�¤ 0.78 �µg mL-1, lower than commercial antibiotics. The genome size of STKMTI.2 was 4,563,326 bp (GC content: 43.2%) consisted of 1 chromosome, 2 circular plasmids, and 2 linear plasmids, 4,824 protein-coding sequences and 1 clustered, regularly interspaced, short palindromic repeated (CRISPR). The genome of STKMTI.2 has several biosynthetic gene clusters, namely bmp8, bmp9, non ribosomal peptide synthase, polyketide-like butyrolactone, lant class I, RiPP-like was detected in chromosome1 and prodigiosin was detected in plasmid linear unnamed5. This research is a pioneer to explore further the potential of STKMTI.2 isolate as a marine bacterium producing anti-Vibrio compounds and a candidate for vibriosis biocontrol.

Kata Kunci : Anti-Vibrio, Kromatografi, Pseudoalteromonas, WGS

  1. S3-2022-436460-abstract.pdf  
  2. S3-2022-436460-bibliography.pdf  
  3. S3-2022-436460-tableofcontent.pdf  
  4. S3-2022-436460_title.pdf