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Molecular Profiling and Anti-Infective Potency of Endophytic Actinomycetes Inhabiting Madhuca Insignis Radlk., from Western Ghats of India Soma Mondal and V

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Molecular Profiling and Anti-Infective Potency of Endophytic Actinomycetes Inhabiting Madhuca Insignis Radlk., from Western Ghats of India Soma Mondal and V Mondal and Ravishankar Rai Journal of Genetic Engineering and Biotechnology Journal of Genetic Engineering (2021) 19:36 https://doi.org/10.1186/s43141-021-00135-0 and Biotechnology RESEARCH Open Access Molecular profiling and anti-infective potency of endophytic actinomycetes inhabiting Madhuca insignis Radlk., from Western Ghats of India Soma Mondal and V. Ravishankar Rai* Abstract Background: Endophytic actinomycetes are well known for their diverse bioactive entities and considered as an important source for drug development research. Results: We isolated and identified four potential endophytic Streptomyces species, i.e., Streptomyces misionensis MI22, Streptomyces roietensis MI24, Streptomyces glaucescens MI29, and Streptomyces sp. MI04 inhabiting Madhuca insignis by its characteristic morphological features and 16S rRNA gene sequence analysis. S. misionensis MI22 exhibits a broad spectrum of anti-microbial activity against methicillin-resistant Staphylococcus aureus (25.00 ± 1.00 mm) followed by Bacillus subtilis (23.66 ± 0.57 mm), Escherichia coli (22.00 ± 0.00 mm), and Candida albicans (18.00 ± 0.00 mm). Minimum inhibitory concentrations of the ethyl acetate fraction of S. misionensis MI22 against test pathogens were ranged from 25 to 100 μg/mL. Indeed, strain MI22 also exhibited significant anti-proliferative activity against HeLa cell line with IC50 value 98 μg/mL and showed no cytotoxicity effect to the normal human embryonic kidney cell line in the MTT assay. The anti-microbial metabolites from strain MI22 were detected at Rf 0.55 as depicted by the inhibition zone on the intensive band in TLC-bioautography assay. Conclusion: The study indicates that, anti-microbial metabolites of these endophytic Streptomyces species, especially S. misionensis MI22 as a prolific source to discover novel bioactive metabolites to combat multidrug- resistant pathogens. Keywords: Endophytic actinomycetes, Streptomyces, Madhuca insignis, Drug discovery, Anti-microbial activity, TLC bioautography Background has always been essential throughout human history In the last few decades, more than 30 new infectious to combat infectious diseases. diseases have been emerged ranging from the rota- Natural products or secondary metabolites originate virus to Middle East respiratory syndrome coronavirus from plants, bacteria, marine, and fungal sources are [1, 2]. The increasing people movement across diverse the most successful source of potent lead natural borders, the rapid expansion of air traffic and human drugs [3]. They serve as a continuing potent source population have modified the ecosystem has made of novel bioactive agents, retaining an immense im- these new pathogens can easily spread all over the pact on modern medicine [4]. Antibiotic drugs like globe. Therefore, the hunt for new potential drugs erythromycin, methicillin, and penicillin are used in a great extent to treat various diseases, but currently * Correspondence: [email protected] turning to be less potent as a result of many mi- Department of Studies in Microbiology, University of Mysore, Manasagangotri, Mysuru, Karnataka 570006, India crobes become resistant to certain antibiotics [5]. The © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Mondal and Ravishankar Rai Journal of Genetic Engineering and Biotechnology (2021) 19:36 Page 2 of 10 right solution to cover the problem of drug-resistant Methods pathogens is to explore and discover new Selection of plant and study site characteristics biomolecules. Leaf, stem, and root samples of M. insignis were col- Microbes are considered as a potent source of nat- lected from Agumbe (13.5027° N, 75.0903° E), Western ural products as evidenced by the discovery of Penicil- Ghats of India for the isolation of endophytic actinomy- lin from the fungus Penicillium rubens by Alexander cetes. The collected plant tissue samples were sealed Fleming in 1928 [6]. They have the ability to bio- using parafilm to keep its endophytic nature and synthesize novel compounds that can be used for brought to the laboratory within 24 h for the isolation of medical and agricultural applications [7, 8]. Indeed, endophytes. plants with ethnomedicinal values are the potent sources of broad-spectrum bioactive agents. Microbial Surface sterilization and isolation endophytes associated with medical plants play a vital Leaves, roots, and stem samples were first rinsed in run- role in secondary metabolism pathways and increase ning tap water and then with double-distilled water [25]. the ability to produce natural metabolites [9–11]. The To get rid of surface microorganisms, samples were endophytic actinomycetes constitute one of the fascin- washed with 0.1% Tween 20 for 1 min and washed with atinggroupsofmicrobesassociatedwithawiderange 75% ethanol for 5 min. The samples were rinsed with of medicinal plants. They are considered as a group of 2% sodium hypochlorite for 5 min, and rinsed with 10% actinobacteria bearing the capacity to synthesize new sodium bicarbonate for 5 min to inhibit the growth of bioactive secondary metabolites [12]. Biological diver- fungi. Later, all samples were washed with double- sity, species richness, and species distribution of endo- distilled water and kept for surface drying. The plant tis- phytic actinomycetes are significantly influenced by sue materials were cut into tiny segments (5 mm) and ecological environs [13]. The endophytic actinomy- placed on starch casein agar (HiMedia, India) and incu- cetes diversity and anti-microbial activity inhabiting bated at 30 °C for 1 month. To confirm, the effect of the medicinal plants of the tropical region have been re- surface sterilization process, final wash aliquots of the ported in the earlier studies [14–16], but the plants sterile distilled was involuted on the culture plates. Petri from Western Ghats region in India has not gained re- plates were constantly checked for endophytic actinomy- search attention. cetes growth. The actinomycetes isolates were observed The genus Streptomyces is known for a diverse under the light microscope and scanning electron micro- array of bioactive agents like anti-cancer, anti- scope. The morphological features like spiral sporo- microbial, anti-infective biomolecules, and other phores, filamentous nature, hyphe, and Gram-straining pharmaceutical important drugs [17–21]. They are were analyzed. considered as prolific producers of diverse bioactive agents like antioxidants, antibiotics, enzyme inhibitors, Molecular identification of endophytic actinomycetes and other biomolecules having therapeutic importance Genomic DNA extraction, taxon sampling, and phylogenetic [22]. Most of the endophytic actinomycetes that have affiliation been exhibited anti-microbial potential belong to the To isolate genomic DNA, the endophytic actinomycete Streptomyces genus, indicating that this particular isolates were cultured in International Streptomyces genus is of great interest to discover novel anti- Project-2 (ISP-2) broth for 7 days at 28 °C. DNA extrac- microbial drugs [23–25]. Hence, there is a need to tion was carried out following the standard protocol explore actinomycetes diversity for bioactive [32]. The amplification of the 16s rRNA gene was metabolites. carried by a PCR method using universal primers, 27F Madhuca insignis is a riparian tree that belongs to (5′-AGAGTTTGATCMTGGCTCAG-3′) and 1492R (5′ the Sapotaceae family which is mainly distributed in TACGGYTACCTTGTTACGACTT-3′)[33]. PCR con- Karnataka and Kerala states of South India [26–30]. It ditions: denaturation 95 °C for 5 min followed by 35 is a critically endangered and endemic riparian species cycles at 95 °C for 1 min; annealing 54 °C for 1 min; ex- found in the Western Ghats of India. Several reports tension 72 °C for 1 min, final extension at 72 °C for 10 are also documented on the pharmacological applica- min and cooling at 4 °C. The amplified PCR products tions of Madhuca species [31]. There are no reports were visualized in gel electrophoresis. 16S rRNA gene available in search of endophytic actinomycetes from sequence was carried out in ABI 3730 Genetic Analyzer M. insignis, despite its medicinal value. Hence, this (Applied Biosystems, USA). The nucleotide sequences of study was designed to explore endophytic actinomy- endophytic actinomycetes with similar identities were re- cetes inhabiting M. insignis and to evaluate their anti- trieved from the National Center of Biotechnology Infor- microbial and anti-proliferative potential against mation (NCBI) database using BLAST search. The human pathogens. multiple sequence alignment was carried out using Mondal and Ravishankar Rai Journal of Genetic Engineering and Biotechnology (2021) 19:36 Page 3 of 10 CLUSTAL Omega and the
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