CHARACTERIZATION OF ANTIBACTERIAL COMPOUNDS PRODUCED BY STREPTOMYCES AUREOFACIENS A3

• Main Author: Asritafriha, Lillah
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/76104
• Institution: Institut Teknologi Bandung

<p align="justify"> The problem of antibiotic resistance is spreading faster than the discovery of new antibiotics so it become a severe concern for infectious diseases. In a previous study, Streptomyces aureofaciens A3 was isolated from a sponge from Ranca Buaya Beach, West Java. Streptomyces aureofaciens A3 has antibacterial activites against Escherichia coli, Staphylococcus aureus, Bacillus subtilis, and Pseudomonas aeruginosa. A total of 85 drug-candidate metabolites have been successfully identified by GC-MS. It is required to do downstream optimization to isolate possible antibacterial compounds since the substances from the prior investigation are still mixed substances derived from crude extract Streptomyces aureofaciens A3. The aims of this study were: 1) optimizing the downstream process to obtain groups of strong antibacterial compounds using silica gel column chromatography; 2) determine the metabolite fractions that have strong antibacterial activity with the best MIC50 and MIC90 values against E. coli, S. aureus, B. subtilis, and P. aeruginosa; and 3) determine the results of separation of antibacterial compounds by GC-MS analysis. Utilizing ISP-4 A media, Streptomyces aureofaciens A3 isolate was used to produce antibacterial compounds with a cell density of ~105 CFU/mL, an initial concentration of 10% (v/v), a temperature of 26 ± 2oC, an incubation duration of 14 days, and agitation of 150 rpm. The crude extract was separated using silica gel column chromatography with a flow rate of 0.4 ml/min and chloroform: methanol (19:1, 9:1, 4:1, 7:3: 1:1, 100%v/v) as the extraction solvent. The antibacterial activity test against Escherichia coli, Staphylococcus aureus, Bacillus subtilis, and Pseudomonas aeruginosa was carried out using the Kirby-Bauer method with an extract concentration of 10 mg/ml, while the MIC test was conducted using the microdilution method. Metabolite identification was carried out using GC-MS. The highest antibacterial test of ethyl acetate extract was obtained on day 9 (D9) and day 12 (D12). Column chromatography separation obtained the final fraction of D9 as many as 5 fractions (9.I - 9.V) and D12 as many as 8 fractions (12.I - 12.VIII). Fraction with strong antibacterial activity against S. aureus (9.II, 9.IV, 9.V, 12.I, 12.II, 12.III, 12.IV, 12.VI, and 12.VII), E. coli ( 12.IV), P. aeruginosa (12.II and 12.VI), and B. subtilis (9.V, 12.II, and 12.III). The best MIC50 value of D9 and D12 against S. aureus, E. coli, P. aeruginosa, and B. subtilis were obtained from 9.IV (28.125 ?g/ml) and 12.VII (162.49 ?g/ml); 9.IV (28.125 ?g/ml) and 12.VI (45.83 ?g/ml), 9.II (109.68 ?g/ml) and 12.III (169.46 ?g/ml); and 9.II (80.21 ?g/ml) and 12.VII (160.02 ?g/ml) respectively. The best MIC90 from D9 and D12 against S. aureus, E. coli, P. aeruginosa, and B. subtilis were obtained from 9.IV (28.125 ?g/ml) and 12.VI (45.83 ?g/ml); 9.IV (28.125 ?g/ml) and 12.VI (45.83 ?g/ml); 9.IV (67.61 ?g/ml) and 12.VII (85.42 ?g/ml); and 9.II (80.21 ?g/ml) and 12.IV (60.42 ?g/ml) respectively. The results of the GC-MS analysis showed that the triazine identified as N,N-Diethyl-N',N'-Diphenyl-6-Pyroll-1-YL-[1,3,5]Triazine-2,4-Diamine had a positive correlation which is significant to E. coli (p < ,05). This substance's discovery marks the first known marine Streptomyces origin, which can be a useful strategy for accelerating the invention and development of novel antibiotics.