Alwi, Muhammad (2019) Keragaman dan Potensi Actinomycetes Rhizosfer Eucalyptus deglupta Blume. dan Diospyros celebica Bakh. di Taman Nasional Lore Lindu Sulawesi Tengah Sebagai Antibakteri Patogen Manusia. Doktor thesis, Universitas Brawijaya.
Abstract
Peningkatan kasus penyakit infeksi dan penggunaan antibiotik yang tidak tepat serta tidak terkontrol, telah mengakselerasi timbulnya strain bakteri yang bersifat resisten terhadap antibiotik. Oleh sebab itu eksplorasi mikroba penghasil antibiotik baru menjadi penting. Actinomycetes banyak dikaji potensinya karena memiliki nilai ekonomi yang tinggi sebagai penghasil antibiotik. Indonesia yang beriklim tropis memiliki keanekaragaman ekosistem dan Actinomycetes yang tinggi. Actinomycetes yang hidup di daerah rhizosfer tumbuhan Leda (Eucalyptus deglupta Blume.) dan Ebony (Diospyros celebica Bakh.) mempunyai potensi menghasilkan senyawa antibakteri patogen pada manusia. Salah satu ekosistem yang diduga di huni oleh Actinomycetes penghasil senyawa antibakteri patogen pada manusia adalah rhizosfer tumbuhan khas Leda dan Ebony di kawasan Taman Nasional Lore Lindu, Sulawesi Tengah. Tujuan penelitian ini adalah (1) mengisolasi berbagai isolat Actinomycetes rhizosfer tumbuhan Leda dan Ebony di TNLL sebagai penghasil senyawa antibakteri patogen manusia, (2) menyeleksi isolat-isolat Actinomycetes dari rhizosfer tumbuhan Leda dan Ebony di TNLL yang memiliki aktivitas tertinggi sebagai antibakteri patogen manusia, (3) mengisolasi dan mengidentifikasi antibiotik dari Actinomycetes yang potensial penghasil senyawa antibakteri patogen manusia, (4) menganalisis aktivitas daya hambat metabolit isolat Actinomycetes terhadap berbagai bakteri patogen manusia, dan (5) mengidentifikasi isolat Actinomycetes yang terbaik sebagai penghasil antibakteri patogen manusia berdasarkan sekuen 16S rDNA. Penelitian ini dibagi tiga tahap. Tahap pertama sampling, isolasi, dan skrining Actinomycetes penghasil senyawa antibakteri patogen manusia. Sampel tanah rhizosfer diambil dengan metode purposive sampling yang dipadukan dengan metode kuadran dengan mengukur berbagai faktor lingkungan. Tahap kedua merupakan seleksi untuk menganalisis potensi dan efikasi isolat Actinomycetes sebagai penghasil senyawa antibakteri terhadap bakteri patogen manusia. Uji optimasi Actinomycetes potensial pada variasi suhu, waktu inkubasi, dan pH pertumbuhan; serta uji produksi melanin, hemolisis, dan berbagai fraksi pelarut. Efikasi senyawa antibakteri diuji dalam bentuk massa sel (suspensi sel) dan crude metabolit terhadap bakteri patogen manusia Gram positif dan negatif. Senyawa antibakteri yang efektif menghambat pertumbuhan bakteri patogen ditentukan konsentrasi hambat minimum (KHM) dan berat molekulnya ditentukan dengan LC-MS. Tahap ketiga adalah karakterisasi dan identifikasi filogenetik isolat Actinomycetes yang terbaik sebagai penghasil senyawa antibakteri patogen manusia berdasarkan similaritas sekuen 16S rDNA. Hasil penelitian menunjukkan bahwa kondisi tegakan tumbuhan Leda di daerah Anaso-Rorekatimbu dengan kondisi tegakan tumbuhan Ebony di daerah Sedoa-Poso pada umumnya berbeda, namun densitas Actinomycetes relatif sama. Actinomycetes rhizosfer tumbuhan Leda dan Ebony yang berhasil diisolasi secara berurutan masing-masing sebanyak 15 dan 17 isolat. Isolat Actinomycetes L213 dari rhizosfer Leda paling potensial, memiliki daya hambat spektrum luas, dan mampu menghambat keempat bakteri uji S. ix aureus, MRSA, V. cholera, dan EPEC; dengan daya hambat berturut-turut sebesar 18,94 ± 0,34 mm, 19,90 ± 1,40 mm, 21,10 ± 0,26 mm, dan 22,24 ± 0,24 mm. Isolat Actinomycetes E512 dari rhizosfer Ebony juga memiliki daya hambat spektrum luas dan mampu menghambat keempat spesies bakteri patogen V. cholera, EPEC, MRSA, dan S. aureus dengan daya hambat berturut-turut sebesar 18,55 ± 0,46 mm, 18,73 ± 1,02 mm, 19,81 ± 0,47 mm, dan 20,06 ± 0,75 mm. Crude antibakteri isolat L213 (Leda) memiliki konsentrasi hambat terendah (KHM) dalam menghambat pertumbuhan bakteri patogen S. aureus, EPEC, MRSA, dan V. cholerae berturut-turut adalah 125 ppm, 125 ppm, 250 ppm, dan 250 ppm. Crude antibakteri isolat E512 (Ebony) memiliki KHM terhadap bakteri patogen S. aureus, EPEC, MRSA, dan V. cholerae berturut-turut adalah 250 ppm, 250 ppm, 500 ppm, dan 500 ppm. Crude antibakteri isolat L213 dan E512 hasil ekstraksi menggunakan pelarut kloroform dan memiliki aktivitas antibakteri lebih tinggi dibandingkan dengan ekstrak metanol, etil asetat, dan n-heksan. Senyawa dominan dalam isolat L213 adalah antibakteri yang memiliki massa molekul 371,647 m/z dengan rumus molekul C20H20O7 yang merupakan senyawa Aurantine, sedangkan senyawa antibakteri yang dihasilkan isolat E512 memiliki massa molekul 575,60 m/z tetapi belum diketahui nama senyawanya. Meskipun demikian, ada senyawa yang memiliki karakteristik yang mirip dengan senyawa isolat E512 dengan berat molekul 606 g/mol dengan rumus molekul C27H38N6O10 sebagai senyawa Levomycin. Senyawa antibakteri isolat ini dikategorikan sebagai senyawa baru karena memiliki aktivitas antibakteri yang berbeda dengan temuan sebelumnya sebagai antikanker dan antifungi. Kedua senyawa ini dikategorikan sebagai antibakteri yang berspektrum luas yang efektif terhadap bakteri patogen serta tidak toksik terhadap manusia. Telah berhasil diisolasi 32 isolat Actinomycetes rhizosfer tumbuhan Eucalyptus deglupta Blume. dan Diospyros celebica Bakh., namun yang potensial sebagai penghasil antibakteri patogen manusia yaitu isolat L213, L411, L433, E416, dan E512. Pelarut terbaik untuk mengekstrak senyawa antibiotik Actinomycetes terpilih sebagai antibakteri patogen manusia adalah khloroform. Crude isolat L213 memiliki aktivitas terbaik diikuti isolat E512 dengan daya hambat spektrum luas dan efektif menghambat keempat jenis bakteri patogen uji. Berdasarkan similaritas sekuen 16S rDNA isolat L213 diidentifikasi sebagai Streptomyces aurantiacus LMG 19358 (similaritas 99 %), sedangkan isolat E512 sebagai Streptomyces globisporus KCTC 9026 dengan similaritas 99 %.
English Abstract
The increased cases of infectious diseases and inappropriate uncontrolled use of antibiotics have accelerated the emergence of bacterial strains with antibiotic resistance. Therefore, the exploration of new antibiotic-producing microbes becomes important. Actinomycetes are popular and considered as potential research objects as they have high economic value due to their antimicrobial compounds. Indonesia has a diversity of ecosystems and Actinomycetes due to its tropical climate. Actinomycetes that live in the rhizosphere of certain plants such as Eucalyptus deglupta Blume. and Diospyros celebica Bakh. has the potency to produce antibacterial compounds to inhibit pathogenic bacteria in humans. Actinomycetes samples were collected from the rhizosphere of Leda and Ebony plants in the Lore Lindu National Park (TNLL), Central Sulawesi. This study aimed to 1) isolate various Actinomycetes from rhizosphere of Leda and Ebony plants in TNLL as a producer of human pathogen antibacterial compounds, 2) screening Actinomycetes isolated from Leda and Ebony rhizosphere which have the highest antibacterial activity against pathogenic bacteria, 3) evaluate the best solvents for the extraction of Actinomycetes that can support the antibacterial activity of the isolates, 4) analyze the potency inhibitory activity of Actinomycetes isolates for various human pathogenic bacteria, and 5) identify the best Actinomycetes isolate that produce antibacterial compounds for pathogenic bacteria based on 16S rDNA sequences. This study was conducted in three stages. The first stage included sampling, isolation, and screening of Actinomycetes. The samples of the rhizosphere (soil) were collected according purposive sampling method combined with the quadrant method by measuring various environmental factors. The second stage was analyzed the potency and efficacy of Actinomycetes isolate to produce antibacterial compounds. The optimization test of Actinomycetes was covered out of variations of temperature, incubation time, and growth pH, as well as melanin production test, hemolysis, and solvent fraction. The efficacy of antibacterial compounds was tested as bacterial cell mass and crude cell-free supernatant against Gram-positive and negative bacteria. The effectivity of antibacterial compounds to inhibit the growth of pathogenic bacteria was determined by the minimum inhibitory concentration (MIC) and the molecular weight was determined by LC-MS. The third stage was the characterization and identification of the best Actinomycetes isolate for the antibacterial agent based on the similarity of the 16S rDNA sequence. The habitus condition of Leda (Eucalyptus deglupta) in Anaso-Rorekatimbu area was different from the Ebony (Diospyros celebica) in Sedoa-Poso. However, the density of Actinomycetes was relatively the same. Actinomycetes were successfully isolated from the Leda and Ebony rhizospheres, as many as 15 isolates and 17 isolates, respectively. The isolate L213 from Leda rhizosphere was the most potency, had a broad spectrum and it was able to inhibit the four test bacteria (S. aureus, MRSA, V. cholera, and EPEC), with inhibition diameter are 18.94±0.34 mm, 19.90±1.40 mm, 21.10±0.26 mm, and 22.24±0.24 mm, respectively. In addition, the isolate E512 from Ebony rhizosphere also has broad- xi spectrum inhibitory and it was able to inhibit all pathogenic bacteria (V. cholera, EPEC, MRSA, and S. aureus) wide 18.55±0.46 mm, 18.73±1.02 mm, 19.81±0.47 mm, and 20.06±0.75 mm, respectively. The crude metabolite of L213 isolate had the lowest Minimum Inhibitory Concentration (MIC) to inhibit the growth of S. aureus, EPEC, MRSA, and V. cholerae, with the concentrations were 125 ppm, 125 ppm, 250 ppm, and 250 ppm, respectively. The crude antibacterial compound of E512 isolate had MIC on the pathogenic bacteria of S. aureus, EPEC, MRSA, and V. cholerae were 250 ppm, 250 ppm, 500 ppm, 500 ppm, and 500 ppm, respectively. Furthermore, the crude metabolite of L213 and E512 isolates extracted using chloroform solvents had the highest antibacterial activity compared to methanol, ethyl acetate, and n-hexane solvents. The antibacterial metabolite of L213 had a molecular weight of 371,647 m/z, molecular formula of C20H20O7 which is assumed as Aurantine compound, while the antibacterial compound produced by E512 isolate had a molecular weight of 575.60 m/z but the name of the compound is still unknown. Nonetheless, there was compound that had characteristics similar to isolate E512 with a molecular weight of 606 g/mol, it was molecular formula of C27H38N6O10 as a Levomycin. The antibacterial compound of this isolate is categorized as a new compound as it has different antibacterial activity than the previous findings as anticancer and antifungal. Both of these compounds are categorized as broad-spectrum antibacterial and are not toxic to humans. Based on observations toward 32 Actinomycetes from rhizosphere isolates of Eucalyptus deglupta Blume. and Diospyros celebica Bakh. however, the potential as an antibacterial producer of human pathogens are isolates L213, L411, L433, E416, and E512. Chloroform was the best solvent for Actinomycetes metabolites. The crude metabolite of L213 isolates had the best activity followed by E512 isolate with broad-spectrum inhibitory and it was able to inhibit wide types of pathogenic bacterial tests. Based on 16S rDNA sequence similarity, isolate L213 was identified as Streptomyces aurantiacus LMG 19358 and isolate E512 as Streptomyces globisporus KCTC 9026 with 99% similarity.
Other obstract
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| Item Type: | Thesis (Doktor) |
|---|---|
| Identification Number: | DIS/615.329 37/ALW/k/2019/061911234 |
| Uncontrolled Keywords: | Actinomycetes Rhizosfer Eucalyptus deglupta Blume, Diospyros celebica Bakh, Taman Nasional Lore Lindu |
| Subjects: | 600 Technology (Applied sciences) > 615 Pharmacology and therapeutics > 615.3 Organics drugs > 615.32 Drugs derived from plants and mikroorganisms > 615.329 37 Drugs derived from microorganisms, fungi, algae (Lactobacillus) |
| Divisions: | S2/S3 > Doktor Biologi, Fakultas MIPA |
| Depositing User: | Endang Susworini |
| Date Deposited: | 25 May 2022 03:29 |
| Last Modified: | 25 May 2022 03:29 |
| URI: | http://repository.ub.ac.id/id/eprint/190749 |
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