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AKTINOMISETES PENGHASIL ANTIBIOTIK DARI HUTAN BAKAU TOROSIAJE GORONTALO

YULIANA RETNOWATI, PROF. DR. A. ENDANG SUTARININGSIH SOETARTO, M.SC; PROF. DR. SUKARTI MOELJOPAWIRO, M.APP.SC; PROF. DR. TJUT SUGANDAWATY DJOHAN, M.SC

2019 | Disertasi | DOKTOR BIOLOGI

Peningkatan resistensi mikroba patogen terhadap antibiotik dan penemuan kembali antibiotik sejenis yang dihasilkan aktinomisetes terestrial, mendorong upaya eksplorasi aktinomisetes penghasil antibiotik di lingkungan ekstrem. Hutan bakau Torosiaje, Gorontalo berada di ekosistem kars, terdiri atas tipe overwash dan fringe yang berbeda dalam status hidrologi yang direspon distribusi pohon bakau dalam pola zonasi. Penelitian ini bertujuan untuk mengungkap distribusi, kemelimpahan, dan diversitas aktinomisetes penghasil antibiotik di rizosfer berbagai jenis pohon bakau pada tipe hutan berbeda, dan potensinya sebagai penghasil antibiotik. Tanah rizosfer dikoleksi berdasarkan purposive sampling method pada pohon bakau Rhizophora mucronata dan Bruguiera gymnorhiza di zona upper overwash; Rhizophora apiculata dan Bruguiera gymnorhiza di zona middle fringe; Avicennia marina, Soneratia alba, Ceripos tagal, dan Xylocarpus sp. di zona upper fringe. Distribusi dan kemelimpahan aktinomisetes di tanah rizosfer berbagai jenis pohon bakau ditentukan melalui tahapan isolasi aktinomisetes berdasarkan teknik kultur diperkaya. Diversitas aktinomisetes penghasil antibiotik dianalisis berdasarkan karakter fenetik colour grouping, profil elemen BOX, dan profil ARDRA; dan karakter molekular sekuen gen 16S rRNA. Aktivitas antibiotik ditentukan melalui tahapan percobaan sintesis antibiotik berdasarkan shake flask culture method, ekstraksi antibiotik berdasarkan metode solvent, dan uji aktivitas antibiotik berdasarkan Kirby-Bueur test melawan bakteri Gram-positif dan Gram-negatif, serta fungi. Antibiotik diidentifikasi berdasarkan karakter profil KLT, LCMS, GCMS, bioautografi, dan deteksi gen pengkode sintesis antibiotik. Identitas isolat aktinomisetes penghasil antibiotik ditentukan berdasarkan pendekatan sistematika polifasik. Hasil penelitian menunjukkan bahwa aktinomisetes ditemukan terdistribusi pada rizosfer seluruh jenis pohon bakau dengan kemelimpahan yang bervariasi. Kemelimpahan tertinggi pada rizosfer R.mucronata di zona upper overwash, dan terendah pada rizosfer R.apiculata di zona middle fringe. Sebanyak 167 isolat aktinomisetes berhasil diisolasi, 77 isolat diantaranya berpotensi penghasil antibiotik. Isolat aktinomisetes penghasil antibiotik menunjukkan keanekaragaman fenetik yang tinggi, baik karakter morfologi, maupun karakter elemen BOX, dan profil ARDRA. Diversitas aktinomisetes penghasil anibiotik berdasarkan karakter molekular 16S rRNA menunjukkan komunitas aktinomisetes penghasil antibiotik tersusun atas genus Streptomyces, Nocardiopsis, Saccharomonospora, dan Amycolatopsis. Anggota genus Streptomyces mendominasi seluruh lokasi dan jenis bakau, Nocardiopsis dan Saccharomonospora spesifik ditemukan di zona upper overwash, sedangkan Amycolatopsis spesifik ditemukan di zona upper fringe. Antibiotik yang dihasilkan isolat aktinomisetes terpilih menunjukkan aktivitas antibakteri bersifat broad spectrum dan narrow spectrum, juga antifungi. Aktivitas antibiotik tertinggi dihasilkan dua isolat aktinomisetes anggota genus Streptomyces, BUFA-2 dan BMFB-9, dengan nilai MIC 0,0625�0,5 mg/ml terhadap E.coli, S.aureus, B.subtilis, A.niger dan C.albicans. Antibiotik merupakan senyawa campuran yang tersusun atas β-laktam, aminoglikosida, namun bukan golongan alkaloid. Sebagian antibiotik merupakan asam lemak bebas yang diidentifikasi sebagai asam palmitat, asam Di-n-oktil ptalat, dan asam risinoleat; juga merupakan kelompok poliketida aromatik dan non-ribosimal polipeptida. Isolat aktinomisetes penghasil antibiotik diidentifikasi sebagai Streptomyces sp. strain BUFA-2, Streptomyces sp. strain BMFB-9, Streptomyces sp. strain BMFR-4, Saccharomonospora sp. strain BUOB-1, Amycolatopsis sp. strain BUFX-4 and Nocardiopsis sp. strain BUOR-2, memiliki peluang sebagai spesies baru (novel species). Hutan bakau Torosiaje Gorontalo merupakan habitat aktinomisetes anggota genus Streptomyces dan rare actinomycetes yang berpotensi menghasilkan antibiotik dengan karakter khusus.

The increasing pathogenic-microbial resistance to antibiotics, and rediscovery of antibiotic by terrestrial actinomycetes encourages a study to explore antibiotic-producing actinomycetes in extreme environments. Mangrove forest of Torosiaje in Gorontalo is located in a karst environment. This forest consists of two types, i.e., overwash and fringe mangrove, with different hydrological status represented by the distribution of mangrove tree in a zonation. The objectives of this study were to reveal the distribution, abundance, and diversity of antibiotic-producing actinomycetes in the rhizosphere of various mangroves, as well as the potential of the actinomycetes as an antibiotic producer. The rhizosphere soil as source of actinomycetes was collected based on purposive sampling method. The sampling involved selected mangrove tree species in several zonations, such as Rhizophora mucronata and Bruguiera gymnorhiza in upper overwash zonation; Rhizophora apiculata and Bruguiera gymnorhiza in middle fringe zonation, and; Avicennia marina, Soneratia alba, Ceripos tagal, and Xylocarpus sp. in upper fringe zonation. The distribution and abundance of actinomycetes in the rhizosphere soil of several species of mangrove were determined through several steps of actinomycetes isolation based on enrichment cultures technique. The diversity of the antibiotic-producing actinomycetes was analyzed based on the phenetics character of color grouping, BOX element profile, and ARDRA profile, as well as the molecular character of the 16S rRNA gene sequence. The activity of antibiotics was determined through stages of antibiotics synthesis assay based on shake flask culture method, antibiotic extraction based on solvent method, and antibiotic activity tests based on the Kirby-Bauer test against Gram-positive and Gram-negative bacteria, and fungi. The antibiotics were identified based on TLC, LCMS, and GCMS technique, bio-autography, and detection of gene encoding of antibiotics synthesis. Moreover, the identity of antibiotic-producing actinomycetes was determined based on polyphasic systematics approach. The result of the study shows that the actinomycetes were found distributed on the rhizosphere of all mangroves types with varied abundance. The highest abundance level was in the rhizosphere of R.mucronata in the upper overwash zonation, and the lowest one was R.apiculata in middle fringe zonation. Out of 167 actinomycete isolates that have been successfully isolated, 77 isolates were potential antibiotic-producers. Those isolates have significant phenetics variety, in terms of its morphological character, BOX element, and ARDRA profile. The diversity of antibiotic-producing actinomycetes based on the character of 16S rRNA gene reveals that the actinomycetes consist of several genera, namely Streptomyces, Nocardiopsis, Saccharomonospora, and Amycolatopsis. The Streptomyces genus was mostly in all sites and every species of mangrove. Both Nocardiopsis and Saccharomonospora were exclusive to upper overwash zonation, while Amycolatopsis was specifically found in upper fringe zonation. The antibiotics produced by the selected actinomycetes isolate have a broad and narrow spectrum antibacterial activity, and also antifungal activity. The highest antibiotic activity was produced by two actinomycetes isolates of Streptomyces, BUFA-2 and BMFB-9, with MIC values of 0.0625�0.5mgml-1 against E. coli, S. aureus, B. subtilis, A. niger and C. albicans. Antibiotics were mixed compounds composed of β-lactams, aminoglycosides, but the compounds are not alkaloids. Several antibiotics were free fatty acid identified as palmitic acid, Di-n-octyl phthalate acid, and ricinoleic acid; those antibiotics were also polyketide aromatics and non-ribosomal polypeptide groups. Antibiotic-producing actinomycetes isolates that identified as Streptomyces sp. strain BUFA-2, Streptomyces sp. strain BMFB-9, Streptomyces sp. strain BMFR-4, Saccharomonospora sp. strain BUOB-1, Amycolatopsis sp. strain BUFX-4, and Nocardiopsis sp. strain BUOR-21, were a potential as novel species. Mangrove forest of Torosiaje, Gorontalo is a habitat of Streptomyces and rare actinomycetes in which both hold potential to produce antibiotics with unique characteristics.

Kata Kunci : Actinomycetes, rhizosphere, mangrove, antibiotic, molecular analysis

  1. S3-2019-373983-abstract.pdf  
  2. S3-2019-373983-bibliography.pdf  
  3. S3-2019-373983-tableofcontent.pdf  
  4. S3-2019-373983-title.pdf