Actinomycetes’ secondary metabolites have received considerable attention due to their many beneficial biological activities. However, many biosynthetic gene clusters in actinomycetes remain silent as they are not transcribed under standard laboratory conditions. Therefore, this study aims to introduce antibiotic elicitors to activate cryptic secondary metabolites in soil actinomycetes and screen them for antibacterial potential. A total of 20 cave and 10 mangrove soil actinomycete isolates were exposed to streptomycin or erythromycin at subinhibitory concentration (0.5–1048 μg/mL) in minimal media. The ethyl acetate extracts were subjected to high-performance liquid chromatography (HPLC) profiling to observe the effect of elicitors towards secondary metabolite production. As a result, 61.7% of the isolates showed a positive impact (appearance of ‘new’/increase in metabolite production) when elicitors were supplemented. These changes were more pronounced in erythromycin-induced media (63.3%) than in streptomycin (60.0%). Two isolates (CS3PT50 and CS3PT53) exhibited significant changes in the profile, with additional peaks detected at 210 and 245 nm, which may indicate the production of new metabolites. More antibacterial activities were observed from stimulated (26.7%) as opposed to non-stimulated isolates (10.0%), including 6 new activities, 1 improved, and 1 decrease in inhibitory. Furthermore, isolate CS3PT53 (0.5 mg/disc) displayed broad-spectrum activities, inhibiting S. aureus ATCC 25923 and S. Typhimurium ATCC 14028. The hit actinomycete isolates belonged to the genus Streptomyces (55.6%), Norcardia (22.2%), Norcardiopsis, and Saccharomonospora (11.1%). Overall, this study demonstrated that incorporating antibiotic elicitor at subinhibitory concentration could effectively trigger the production of cryptic secondary metabolites with antibacterial properties in soil actinomycetes.

• Abdelmohsen, U. R., Grkovic, T., Balasubramanian, S., Kamel, M. S., Quinn, R. J., & Hentschel, U. (2015). Elicitation of secondary metabolism in actinomycetes. Biotechnology Advances, 33(6), 798–811. https://doi.org/10.1016/j.biotechadv.2015.06.003

• Akhter, N., Liu, Y., Auckloo, B., Shi, Y., Wang, K., Chen, J., Wu, X., & Wu, B. (2018). Stress-driven discovery of new angucycline-type antibiotics from a marine Streptomyces pratensis NA-ZhouS1. Marine Drugs, 16(9), Article 331. http://dx.doi.org/10.3390/md16090331

• Ariffin, S., Abdullah, M. F., & Mohamad, S. A. S. (2017). Identification and antimicrobial properties of Malaysian Mangrove Actinomycetes. International Journal on Advanced Science, Engineering and Information Technology, 7(1), Article 71. https://doi.org/10.18517/ijaseit.7.1.1113

• Balagurunathan, R., Radhakrishnan, M., Shanmugasundaram, T., Gopikrishnan, V., & Jerrine, J. (2020). Bioassay-guided isolation and characterization of metabolites from Actinobacteria. In Protocols in actinobacterial research (pp. 147-163). Springer. https://doi.org/10.1007/978-1-0716-0728-2_8

• Barka, E. A., Vatsa, P., Sanchez, L., Gaveau-Vaillant, N., Jacquard, C., Klenk, H. P., Clément, C., Ouhdouch, Y., & van Wezel, G. P. (2016). Taxonomy, physiology, and natural products of actinobacteria. Microbiology and Molecular Biology Reviews, 80(1), 1–43. https://doi.org/10.1128/mmbr.00019-15

• Begani, J., Lakhani, J., & Harwani, D. (2018). Current strategies to induce secondary metabolites from microbial biosynthetic cryptic gene clusters. Annals of Microbiology, 68(7), 419–432. https://doi.org/10.1007/s13213-018-1351-1

• Belknap, K. C., Park, C. J., Barth, B. M., & Andam, C. P. (2020). Genome mining of biosynthetic and chemotherapeutic gene clusters in Streptomyces bacteria. Scientific Reports, 10(1), Article 2003. https://doi.org/10.1038/s41598-020-58904-9

• Breijyeh, Z., Jubeh, B., & Karaman, R. (2020). Resistance of gram-negative bacteria to current antibacterial agents and approaches to resolve it. Molecules, 25(6), Article 1340. https://doi.org/10.3390/molecules25061340

• Caboche, S. (2014). Bioinformatics bolster a Renaissance. Nature Chemical Biology, 10(10), 798–800. https://doi.org/10.1038/nchembio.1634

• CLSI. (2021). M100 Performance Standards for Antimicrobial Susceptibility Testing (31st ed.). Clinical and Laboratory Standards Institute. Wayne, Pennsylvania.

• Covington, B. C., Spraggins, J. M., Ynigez-Gutierrez, A. E., Hylton, Z. B., & Bachmann, B. O. (2018). Response of secondary metabolism of hypogean Actinobacterial genera to chemical and biological stimuli. Applied and Environmental Microbiology, 84(19), Article e01125-18. https://doi.org/10.1128/AEM.01125-18

• Davies, J., Spiegelman, G., & Yim, G. (2006). The world of subinhibitory antibiotic concentrations. Current Opinion in Microbiology, 9(5), 445-453. https://doi.org/10.1016/j.mib.2006.08.006

• Doroghazi, J. R., Albright, J. C., Goering, A. W., Ju, K. S., Haines, R. R., Tchalukov, K. A., Labeda, D. P., Kelleher, N. L., & Metcalf, W. W. (2014). A roadmap for natural product discovery based on large-scale genomics and metabolomics. Nature Chemical Biology, 10(11), 963–968. https://doi.org/10.1038/nchembio.1659

• El-Hawary, S. S., Hassan, M. H., Hudhud, A. O., Abdelmohsen, U. R., & Mohammed, R. (2023). Elicitation for activation of the actinomycete genome’s cryptic secondary metabolite gene clusters. RSC Advances, 13(9), 5778-5795. https://doi.org/10.1039/D2RA08222E

• Ezeobiora, C. E., Igbokwe, N. H., Amin, D. H., Enwuru, N. V., Okpalanwa, C. F., & Mendie, U. E. (2022). Uncovering the biodiversity and biosynthetic potentials of rare actinomycetes. Future Journal of Pharmaceutical Sciences, 8(1), 1-9. https://doi.org/10.1186/s43094-022-00410-y

• Imai, Y., Sato, S., Tanaka, Y., Ochi, K., & Hosaka, T. (2015). Lincomycin at subinhibitory concentrations potentiates secondary metabolite production by Streptomyces spp. Applied and Environmental Microbiology, 81(11), 3869-3879. https://doi.org/10.1128/AEM.04214-14

• Janardhan, A., Kumar, A. P., Viswanath, B., Saigopal, D. V., & Narasimha, G. (2014). Production of bioactive compounds by actinomycetes and their antioxidant properties. Biotechnology Research International, 2014, Article 217030. https://doi.org/10.1155/2014/217030

• Jiang, Y., Li, Q., Chen, X., & Jiang, C. (2016). Isolation and cultivation methods of actinobacteria. In D. Dhanasekaran & Y. Jiang (Eds.), Actinobacteria - Basics and Biotechnological Applications (pp. 39-58). InTechOpen. https://doi.org/10.5772/61457

• Lee, L., Zainal, N., Azman, A., Eng, S., Goh, B., & Yin, W. (2014). Diversity and antimicrobial activities of actinobacteria isolated from tropical mangrove sediments in Malaysia. The Scientific World Journal, 2014, Article 698178. https://doi.org/10.1155/2014/698178

• Maier, R. M., & Pepper, I. L. (2015). Bacterial growth. In I. L. Pepper, C. P. Gerbal & T. J. Gentry (Eds.), Environmental Microbiology (3rd ed.; pp. 37–56). Academic Press.

• Mazumdar, R., Dutta, P. P., Saikia, J., Borah, J. C., & Thakur, D. (2023). Streptomyces sp. strain PBR11, a forest-derived soil Actinomycetia with antimicrobial potential. Microbiology Spectrum, 11(2), Article e03489-22. https://doi.org/10.1128/spectrum.03489-22

• Narayani, M., & Srivastava, S. (2017). Elicitation: A stimulation of stress in in vitro plant cell/tissue cultures for enhancement of secondary metabolite production. Phytochemistry Reviews, 16, 1227-1252. https://doi.org/10.1007/s11101-017-9534-0

• Ochi, K. (2017). Insights into microbial cryptic gene activation and strain improvement: Principle, application and technical aspects. The Journal of Antibiotics, 70(1), 25-40. https://doi.org/10.1038/ja.2016.82

• Okada, B. K., & Seyedsayamdost, M. R. (2017). Antibiotic dialogues: Induction of silent biosynthetic gene clusters by exogenous small molecules. FEMS Microbiology Reviews, 41(1), 19-33. https://doi.org/10.1093/femsre/fuw035

• Quach, N. T., Nguyen, Q. H., Vu, T. H. N., Le, T. T. H., Ta, T. T. T., Nguyen, T. D., Van Doan, T., Van Nguyen, T., Dang, T. T., Nguyen, X. C., Chu, H. H., & Phi, Q. T. (2021). Plant-derived bioactive compounds produced by Streptomyces variabilis LCP18 associated with Litsea cubeba (Lour.) Pers as potential target to combat human pathogenic bacteria and human cancer cell lines. Brazilian Journal of Microbiology, 52(3), 1215–1224. https://doi.org/10.1007/s42770-021-00510-6

• Sadiran, S. H. (2011). Bioactive microbial metabolites from Malaysian rainforest soil fungi as a source of new drugs candidates. [Master thesis]. UiTM Press. https://ir.uitm.edu.my/id/eprint/65230/1/65230.pdf

• Salwan, R., & Sharma, V. (2020). Molecular and biotechnological aspects of secondary metabolites in actinobacteria. Microbiological Research, 231, Article 126374. https://doi.org/10.1016/j.micres.2019.126374

• Shentu, X., Liu, N., Tang, G., Tanaka, Y., Ochi, K., Xu, J., & Yu, X. (2016). Improved antibiotic production and silent gene activation in Streptomyces diastatochromogenes by ribosome engineering. The Journal of Antibiotics, 69(5), 406–410. https://doi.org/10.1038/ja.2015.123

• Tanaka, Y., Kasahara, K., Hirose, Y., Morimoto, Y., Izawa, M., & Ochi, K. (2017). Enhancement of butanol production by sequential introduction of mutations conferring butanol tolerance and streptomycin resistance. Journal of Bioscience and Bioenineering, 124(4), 400–407. https://doi.org/10.1016/j.jbiosc.2017.05.003

• Tomm, H. A., Ucciferri, L., & Ross, A. C. (2019). Advances in microbial culturing conditions to activate silent biosynthetic gene clusters for novel metabolite production. Journal of Industrial Microbiology and Biotechnology, 46(9–10), 1381–1400. https://doi.org/10.1007/s10295-019-02198-y

• Wang, H., Zhao, G., & Ding, X. (2017). Morphology engineering of Streptomyces coelicolor M145 by sub-inhibitory concentrations of antibiotics. Scientific reports, 7(1), Article 13226. https://doi.org/10.1038/s41598-017-13493-y

• Yagüe, P., Willemse, J., Xiao, X., Zhang, L., Manteca, A., & van Wezel, G. P. (2022). FtsZ phosphorylation pleiotropically affects Z-ladder formation, antibiotic production, and morphogenesis in Streptomyces coelicolor. Antonie van Leeuwenhoek, 116(1), 1–19. https://doi.org/10.1007/s10482-022-01778-w

• Zhang, Y., Huang, H., Xu, S., Wang, B., Ju, J., Tan, H., & Li, W. (2015). Activation and enhancement of fredericamycin A production in deepsea-derived Streptomyces somaliensis SCSIO ZH66 by using ribosome engineering and response surface methodology. Microbial Cell Factories, 14, 1-11. https://doi.org/10.1186/s12934-015-0244-2

• Zong, G., Fu, J., Zhang, P., Zhang, W., Xu, Y., Cao, G., & Zhang, R. (2022). Use of elicitors to enhance or activate the antibiotic production in Streptomyces. Critical Reviews in Biotechnology, 42(8), 1260–1283. https://doi.org/10.1080/07388551.2021.1987856