Potential secondary metabolite from Indonesian Actinobacteria (InaCC A758) against Mycobacterium tuberculosis

Document Type : Original Article


1 Doctoral Program in Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada, Yogyakarta 55281, Indonesia

2 Department of Biomedical Sciences, Faculty of Medicine, Universitas Muhammadiyah Semarang, Semarang 50273, Indonesia

3 Department of Microbiology, Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada, Yogyakarta 55281, Indonesia

4 Research Center for Biotechnology, Indonesian Institute of Sciences, Kabupaten Bogor, West Java 16911, Indonesia

5 Department of Pharmacology and Therapy, Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada, Yogyakarta 55281, Indonesia

6 Research Division of Natural Product Technology, Indonesian Institute of Sciences, Yogyakarta 55861, Indonesia


Objective(s): This study explored Indonesian Actinobacteria which were isolated from Curcuma zedoaria endophytic microbes and mangrove ecosystem for new antimycobacterial compounds.
Materials and Methods: Antimycobacterial activity test was carried out against Mycobacterium tuberculosis H37Rv. Chemical profiling of secondary metabolite using Gas Chromatography-Mass Spectroscopy (GC-MS) and High Resolution-Mass Spectroscopy (HR-MS) was done to the ethyl acetate extract of active strain InaCC A758. Molecular taxonomy analysis based on 16S rRNA gene and biosynthetic gene clusters analysis of polyketide synthase (PKS) and non-ribosomal peptide synthetase (NRPS) from InaCC A758 have been carried out. Bioassay guided isolation of ethyl acetate extract was done, then structural elucidation of active compound was performed using UV-Vis, FT-IR, and NMR spectroscopy methods.
Results: The chemical profiling using HR-MS revealed that InaCC A758 has the potential to produce new antimycobacterial compounds. The 16S rRNA gene sequencing showed that InaCC A758 has the closest homology to Streptomyces parvus strain NBRC 14599 (99.64%). In addition, InaCC A758 has NRPS gene and related to S. parvulus (92% of similarity), and also PKS gene related to PKS-type borrelidin of S. rochei and S. parvulus (74% of similarity). Two compounds with potential antimycobacterial were predicted as 1) Compound 1, similar to dimethenamid (C12H18ClNO2S; MW 275.0723), with MIC value of 100 µg/ml, and 2) Compound 2, actinomycin D (C62H86N12O16; MW 1254.6285), with MIC value of 0.78 µg/ml.
Conclusion: Actinomycin D has been reported to have antimycobacterial activity, however the compound has been predicted to resemble dimethenamid had not been reported to have similar activity.


1. Miggiano R, Rizzi M, and Ferraris DM. Mycobacterium tuberculosis pathogenesis, infection prevention and treatment. Pathogens 2020; 9:10–13.
2. WHO. Global tuberculosis report 2019. Geneva; 2019.
3. Nahid P, Mase SR, Migliori GB, Sotgiu G, Bothamley GH, Brozek JL, et al. Treatment of drug-resistant tuberculosis. Am J Respir Crit Care Med 2019; 200:1208–1218.
4. Cox E and Laessig K. FDA approval of bedaquiline-The benefif-risk balance for drug-resistant tuberculosis. N Engl J Med 2014; 371:689–691.
5. Shen B. A new golden age of natural products drug discovery. Cell 2015; 163:1297–1300.
6. Lahlou M. The success of natural products in drug discovery. Pharmacol Pharm 2013; 4:17–31.
7. Lee H and Suh JW. Anti-tuberculosis lead molecules from natural products targeting Mycobacterium tuberculosis ClpC1. J Ind Microbiol Biotechnol 2016; 43:205–212.
8. Chen C, Wang J, Guo H, Hou W, Yang N, Ren B, et al. Three antimycobacterial metabolites identified from a marine-derived Streptomyces sp. MS100061. Appl Microbiol Biotechnol 2013; 97:3885–3892.
9. Wardani IGAAK, Andayani DGS, Sukandar U, Sukandar EY, and Adnyana IK. Study on antimicrobial activity of Nocardia sp. strain TP1 isolated from Tangkuban Perahu Soil, West Java, Indonesia. Int J Pharm Pharm Sci 2013; 5:713–716.
10. Muharni, Fitrya, Oktaruliza M, and Elfita. Antibacterial and anti-oxidant activity testing of pyranon derivated compound from endophytic fungi Penicillium sp of kunyit putih (Curcuma zedoaria (Berg) Roscoe). Tradit Med J 2014; 19:107–112.
11. Retnowati Y, Moeljopawiro S, Djohan TS, and Soetarto ES. Antimicrobial activities of actinomycete isolates from rhizospheric soils in different mangrove forests of Torosiaje, Gorontalo, Indonesia. Biodiversitas 2018; 19:2196–2203.
12. Jiang ZK, Tuo L, Huang DL, Osterman IA, Tyurin AP, Liu SW, et al. Diversity, novelty, and antimicrobial activity of endophytic actinobacteria from mangrove plants in Beilun Estuary National Nature Reserve of Guangxi, China. Front Microbiol 2018; 9:1–11.
13. Sulistiyani TR, Lisdiyanti P, and Lestari Y. Population and diversity of endophytic bacteria associated with medicinal plant Curcuma zedoaria. Microbiol Indones 2014; 8:65–72.
14. Baskaran R, Mohan PM, Sivakumar K, Kumar A. Antimicrobial activity and phylogenetic analysis of Streptomyces parvulus DOSMB-D105 isolated from the mangrove sediments of Andaman Islands. Acta Microbiol Immunol Hung 2016; 63:27–46.
15. George TK, Devadasan D, and Jisha MS. Chemotaxonomic profiling of Penicillium setosum using high-resolution mass spectrometry (LC-Q-ToF-MS). Heliyon 2019; 5:1–9.
16. Franzblau SG, Degroote MA, Cho SH, Andries K, Nuermberger E, Orme IM, et al. Comprehensive analysis of methods used for the evaluation of compounds against Mycobacterium tuberculosis. Tuberculosis 2012; 92:453–488.
17. Wang X, Huang L, Kang Z, Buchenauer H, and Gao X. Optimization of the fermentation process of actinomycete strain Hhs.015(T). J Biomed Biotechnol 2010; 2010:1–10.
18. Sengupta S, Pramanik A, Ghosh A, Bhattacharyya M. Antimicrobial activities of actinomycetes isolated from unexplored regions of Sundarbans mangrove ecosystem. BMC Microbiol 2015; 15:1–16.
19. Rakhmawatie MD, Wibawa T, Lisdiyanti P, Pratiwi WR, Mustofa. Evaluation of crystal violet decolorization assay and resazurin microplate assay for antimycobacterial screening. Heliyon 2019; 5:e02263.
20. Gontang EA, Gaudêncio SP, Fenical W,  Jensen PR. Sequence-based analysis of secondary-metabolite biosynthesis in marine actinobacteria. Appl Environ Microbiol 2010; 76:2487–2499.
21. Ayuso-Sacido A, Genilloud O. New PCR primers for the screening of NRPS and PKS-I systems in actinomycetes: Detection and distribution of these biosynthetic gene sequences in major taxonomic groups. Microb Ecol 2005; 49:10–24.
22. Kumar S, Stecher G, and Tamura K. MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for bigger datasets. Mol Biol Evol 2016; 33:1870–1874.
23. Ahsan T, Chen J, Wu Y, Irfan M, Shafi J. Screening, identification, optimization of fermentation conditions, and extraction of secondary metabolites for the biocontrol of Rhizoctonia Solani AG-3. Biotechnol Biotechnol Equip 2017; 31:91–98.
24. Saitou N and Nei M. The neighbor-joining method: A new method for reconstructing phylogenetic trees. Mol Biol Evol 1987; 4:406–425.
25. Felsenstein J. Confidence limits on phylogenies: An approach using the bootstrap. Evolution (N Y) 1985; 39:783–791.
26. Tamura K and Nei M. Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. Mol Biol Evol 1993; 10:512–526.
27. Abd-Elnaby H, Abo-Elala G, Abdel-Raouf U, Abd-Elwahab A, and Hamed M. Antibacterial and anticancer activity of marine Streptomyces parvus: Optimization and application. Biotechnol Biotechnol Equip 2016; 30:180–191.
28. Gonzalez-Pimentel JL, Jurado V, Laiz L, and Saiz-Jimenez C. Draft genome sequence of a granaticin-producing strain of Streptomyces parvus isolated from a Roman Tomb in the Necropolis of Carmona, Spain. Microbiol Resour Announc 2019; 8:4–5.
29. Schoenian I, Spiteller M, Ghaste M, Wirth R, Herz H,  Spiteller D. Chemical basis of the synergism and antagonism in microbial communities in the nests of leaf-cutting ants. Proc Natl Acad Sci U S A 2011; 108:1955–1960.
30. Hu D, Chen Y, Sun C, Jin T, Fan G, Liao Q, et al. Genome guided investigation of antibiotics producing actinomycetales strain isolated from a Macau mangrove ecosystem. Sci Rep 2018; 8:1–12.
31. Shetty PR, Buddana SK, Tatipamula VB, Naga YVV, and Ahmad J. Production of polypeptide antibiotic from Streptomyces parvulus and its antibacterial activity. Brazilian J Microbiol 2014; 45:303–312.
32. Rahman MM, Ahmad SH, Mohamed MTM, and Ab Rahman MZ. Antimicrobial compounds from leaf extracts of Jatropha curcas, Psidium guajava, and Andrographis paniculata. Sci World J 2014; 2014:1–8.
33. Yu M, Li Y, Banakar SP, Liu L, Shao C, Li Z, et al. New metabolites from the co-culture of marine-derived actinomycete Streptomyces rochei MB037 and fungus Rhinocladiella similis35. Front Microbiol 2019; 10:1–11.
34. Olano C. Hutchinson’s legacy: Keeping on polyketide biosynthesis. J Antibiot (Tokyo) 2011; 64:51–57.
35. Altameme H, Hameed IH, Kareem M. Analysis of alkaloid phytochemical compounds in the ethanolic extract of Datura stramonium and evaluation of antimicrobial activity. African J Biotechnol 2015; 14:1668–1674.
36. Isnaeni I, Kusumawati I, Suwito MF, Darmawati A, Mertaniasih NM. Antimicrobial activity of Streptomyces spp. isolated from vegetable plantation soil. J Biol Res 2016; 21:69–74.
37. Morbidoni HR, Vilchèze C, Kremer L, Bittman R, Sacchettini JC, Jacobs WR. Dual inhibition of mycobacterial fatty acid biosynthesis and degradation by 2-alkynoic acids. Chem Biol 2006; 13:297–307.
38. Zhao WY, Zhu TJ, Fan GT, Liu HB, Fang YC, Gu QQ, et al. Three new dioxopiperazine metabolites from a marine-derived fungus Aspergillus fumigatus Fres. Nat Prod Res 2010; 24:953–957.
39. Reina JC, Pérez-Victoria I, Martín J, and Llamas I. A quorum-sensing inhibitor strain of vibrio alginolyticus blocks Qs-controlled phenotypes in Chromobacterium violaceum and Pseudomonas aeruginosa. Mar Drugs 2019; 17:1–18.
40. Nuñez A, Sapozhnikova Y, and Lehotay SJ. Characterization of MS/MS product ions for the differentiation of structurally isomeric pesticides by high-resolution mass spectrometry. Toxics 2018; 6:1–12.
41. Kottege J. Evaluation of the active dimethenamid – P in the product frontier – P herbicide. Canberra; 2007.
42. Rhee KH. Isolation and characterization of Streptomyces sp. KH-614 producing anti-VRE (vancomycin-resistant enterococci) antibiotics. J Gen Appl Microbiol 2002; 48:321–327.
43. Kresge N, Simoni RD, and Hill RL. Selman Waksman: The father of antibiotics. J Biol Chem 2004; 279:101–103.
44. Khieu TN, Liu MJ, Nimaichand S, Quach NT, Chu-Ky S, Phi QT, et al. Characterization and evaluation of antimicrobial and cytotoxic effects of Streptomyces sp. HUST012 isolated from medicinal plant Dracaena cochinchinensis Lour. Front Microbiol 2015; 6:1–9.
45. Hurwitz J, Furth JJ, Malamy M, and Alexander M. The role of deoxyribonucleic acid in ribonucleic acid synthesis. III. The inhibition of the enzymatic synthesis of ribonucleic acid and deoxyribonucleic acid by actinomycin D and proflavin. Proc Natl Acad Sci U S A 1962; 48:1222–1230.
46. Ciulli A, Scott DE, Ando M, and Reyes F. Inhibition of Mycobacterium tuberculosis pantothenate synthetase by analogues of the reaction intermediate. ChemBioChem 2015; 9:2606–2611.
47. Yang Y, Gao P, Liu Y, Ji X, Gan M, Guan Y, et al. A discovery of novel Mycobacterium tuberculosis pantothenate synthetase inhibitors based on the molecular mechanism of actinomycin D inhibition. Bioorganic Med Chem Lett 2011; 21:3943–3946.
48. Munir MU, Ahmed A, Usman M, and Salman S. Recent advances in nanotechnology-aided materials in combating microbial resistance and functioning as antibiotics substitutes. Int J Nanomedicine 2020; 15:7329–7358.
49. Khameneh B, Iranshahy M, Vahdati-Mashhadian N, Sahebkar A, and Fazly Bazzaz BS. Non-antibiotic adjunctive therapy: A promising approach to fight tuberculosis. Pharmacol Res 2019; 146:1–12.
50. Barka EA, Vatsa P, Sanchez L, Nathalie Gaveau-Vaillant CJ, Klenk H-P, Clément C, et al. Taxonomy, physiology, and natural products of actinobacteria. Microbiol Mol Biol Rev 2016; 80:1–43.
51. Schmitzer PR, Graupner PR, Chapin EL, Fields SC, Gilbert JR, Gray JA, et al. Ribofuranosyl triazolone: A natural product herbicide with activity on adenylosuccinate synthetase following phosphorylation. J Nat Prod 2000; 63:777–781.
52. Õmura S and Crump A. The life and times of ivermectin - A success story. Nat Rev Microbiol 2004; 2:984–989.
53. Singh V and Tripathi CK. Isolation and characterization of actinomycin V and D from a new isolate of Streptomyces sp. In: International Conference and Exhibition on Pharmacognosy, Phytochemistry, and Natural Products. Hyderabad; 2013; 261.
54. Chandrakar S, Gupta AK. Actinomycin-Producing Endophytic Streptomyces parvulus Associated with Root of Aloe vera and Optimization of Conditions for Antibiotic Production. Probiotics Antimicrob Proteins 2019; 11:1055–1069.
55. Zhang X, Ye X, Chai W, Lian XY, Zhang Z. New metabolites and bioactive actinomycins from marine-derived Streptomyces sp. ZZ338. Mar Drugs 2016; 14:1–9.
56. Praveen V and Tripathi CKM. Studies on the production of actinomycin-D by Streptomyces griseoruber - A novel source. Lett Appl Microbiol 2009; 49:450–455.
57. Srinu M, Kumar MMK, and Shankar GG. Actinomycin D from marine sediment associated Streptomyces capillispiralis MTCC10471. Asian J Pharm Res Heal Care 2013; 5:16–23.