The Kathlekanu swamp forest is one of the unique types of forests and that is microbiologically not explored by the researchers. Therefore, the actinomycetes from swamp soil may have increased antimicrobial capability by producing different kinds of novel compounds. The antagonistic potential could be attributed to various bioactive secondary metabolites produced by the isolate under optimum growth conditions. The earlier reports also suggested about the possibility of novel bioactive compound associated with the organism from rare habitats; the extreme or unexplored regions may harbour more number of potential actinomycetes with novel bioactive compounds (Chakraborty et al. 2021; Mohamed et al. 2021). The isolate KF15 was selected for mass culture using SC broth media and ethyl acetate solvent was used to extract the fermented culture broth. The earlier reports recommended the use of ethyl acetate for the extraction of secondary metabolites as it was recommended as the best solvent for the extraction of bioactive compounds produced by actinomycetes (Kumar et al. 2012; Vijayakumar et al. 2014).
Among the thirty one isolates, notable differences were observed in case of colony structure, aerial and substrate mycelia colour. The Streptomyces species were suspected based on the characteristic moist earthy odour, powdery and chalky appearance of colonies and formation of filamentous, heavily branched mycelia. These observations were in accordance with the earlier descriptions of genus Streptomyces reported in Bergey’s manual of determinative bacteriology (Goodfellow and Williams, 1983; Goodfellow et al. 2012). As for the utilization of different substrates, it was noted that the isolate KF15 was capable of utilizing almost all the examined carbon sources; it was also eminent that the isolate exhibited the potential to produce various enzymes. Similar reports about utilization of carbon and nitrogen sources were recorded in the earlier reports on the biochemical characterization of many Streptomyces species (Taddei et al. 2006; Hussain et al. 2021). The gene sequence of the isolate Streptomyces sp. strain KF15 was blasted with the aid of nucleotide blast in NCBI database to collect matching strains and sequences. The isolate exhibited 97.22% sequence similarity with Streptomyces intermedius strain WYM39; the confirmation was attained by the construction of phylogenetic tree of related strains to understand evolutionary relationship. Similar report was recorded, where the molecular identification of the isolate Streptomyces paradoxus strain KUASN-7 was done via 16S rRNA gene sequencing along with the analysis of phylogenetic tree (Muthuraj et al. 2021).
The functional groups depicted by the FTIR analysis showed that, the bioactive compounds of KF15 may be classified into alkanes, alkenes, carboxylic acids, phenols and benzene derivatives. Similar reports about the FTIR analysis of ethyl acetate extracts from Streptomyces sp. CRB46 (Ambarvati et al. 2020), and Actinomycetes sp. (Alqahtani et al. 2022) suggested the presence of various functional groups such as alkanes, amines, phenols, carboxylic acids, and aromatic compounds. HPTLC fingerprinting analysis revealed the existence of overall chemical moiety in the extract. The major bands represented the possible bioactive compounds at different Rf values. Similar results were obtained in the earlier studies on HPTLC analysis suggesting HPTLC as a reliable chromatographic technique widely used for analysing many samples of dissimilar nature and composition; it is also used to identify and quantify bioactive compounds (Shekar et al. 2016; Anusree et al. 2019).
The GC-MS analysis revealed the peaks for 12 major compounds and among them, 6 compounds were reported to be biologically active in nature. The phenolic compound 2,4-di-tert-butylphenol was recommended to be having very good antifungal activity (Belghit et al. 2016), and the hydrocarbons namely, tetradecane, docosane, octadecane, and eicosane were reported to be having known antibacterial activity against pathogenic bacteria of both gram positive and gram negative nature (Ahsan et al. 2017; Marimuthu et al. 2020; Lammers et al. 2021; Nandhini et al. 2015). The organobromine compound 2-bromo dodecane was reported as a bioactive compound that was used for various biological activities (Al-Rubaye et al. 2020). Similar reports about bioactive compounds recognized by the GC-MS analysis of Streptomyces and other actinomycetes crude ethyl acetate crude extract demonstrated the presence of long chain alkanes, fatty acid methyl ester, fatty alcohol, pyrrolizidine and piperazinedione with well-known biological activities including broad spectrum antimicrobial activity and antioxidant potential (El-Naggar et al. 2017; Krishnamoorthy et al. 2020).
The antimicrobial activity of ethyl acetate crude extract from KF15 exhibited varied results against the tested bacterial pathogens and it was noticed that the higher inhibition zones were observed in case of gram positive bacteria when compared to gram negative bacteria. The difference in sensitivity towards the extract was attributed the dissimilarity in cell wall arrangement; the lipopolysaccharide present in the outer membrane acts as a protective barrier for gram negative bacteria. Whereas, the devoid of lipopolysaccharide protective layer in gram positive bacteria makes them susceptible to bioactive secondary metabolites (Reygaert, 2018; Pallavi et al. 2021). The earlier studies suggested that, the mechanism of action of antimicrobial compounds includes interaction of metabolites with the cytoplasmic membrane followed by the cell membrane damage and subsequent release of K+, PO4−, and nuclear contents from the matrix of the cell. Furthermore, the gram negative bacteria are suspected to utilize antimicrobial mechanisms like, drug target modification, limiting the drug uptake, drug efflux activation and drug inactivation to overcome the effects of antimicrobials (Biswas et al. 2022; Chakraborty et al. 2022).
The swamp soil Streptomyces sp. KF15 exhibited considerable antifungal potential by effectively restricting the growth of fungal pathogens. The antagonistic nature of KF15 may be credited to the ability to produce and secrete secondary metabolites having antifungal activity that diffuses in the medium and inhibits the further growth of the fungi (Li et al. 2021). The shift in growth curve pattern towards right-side indicates that, the fungus was unable to adjust to the conditions and moreover, the cells took more time for attaining maturity and to be ready to divide. Previous study involving the turbidimetric analysis of growth curve suggested that, the delayed lag phase was correlated with lower elongation rates of the formed hyphae and germination rates of conidia than those of control. This in turn, affected the lag phase by preventing the fungus in reaching critical turbidity which is measured by spectrophotometer to give higher OD (Meletiadis et al. 2003). The study also recommended that, turbidimetry was a brisk, non-destructive and low-cost method for examining the growth of filamentous fungi.
The effect of KF15 extract on the morphology of F. oxysporum was examined with the help of SEM imaging, and the results indicated clear evidence of morphological changes in treated cells. The earlier investigations on effect of Streptomyces extract on the morphology of fungal pathogens were also stated similar kinds of deformations and distortions in treated cells. The studies reported several levels of diastrophic and fractured mycelium and spores along with pores in treated cells. The results hinted at complete collapse and degeneration of spores due to decreased exopolysaccharide formation in outer membrane (Li et al. 2021). Another study reported the morphology of treated hypha showing exfoliated flakes, shrivelling, vacuolation and blistering; thickened cell wall and septum with partly ruptured cell membrane along with swollen cell nucleus was also observed (Zou et al. 2021).
The MTT assay results depicted the potentiality of KF15 extract to obstruct the growth of HeLa cells, and IC50 value of 99.85 µg/ml suggests that even at lesser concentration it was able to restrict cancer cell proliferation. The previous studies also reported increased anticancer activity of Streptomyces sp. extract against human breast cancer (T47D) cell line (Jaroszewicz et al. 2021), human lung cancer (A549) cell line (Kumar et al. 2021), and human cervical cancer (HeLa), hepatocellular (HepG2) and breast cancer (MCF7) cell lines (Taechowisan et al. 2021). Surprisingly, there was no or minimal significant adverse effect on the normal human lung cells and murine epithelial (L929) cells were observed in those studies indicating less adverse effect of microbial extract on normal or healthy cells. The reports also mentioned that, the bioactive compounds in the extract impart cytotoxicity mainly by interacting with DNA region having high G-C, topoisomerase-II activity inhibition, and breakage of single stranded DNA. The earlier studies also suggested that, the reactive oxygen species (ROS) instigates apoptosis in the tumour cells which finally leads to cell death (Ma et al. 2021).