Streptomyces sp. Strain PBR11, a Forest-Derived Soil Actinomycetia with Antimicrobial Potential

Novel antibiotic breakthroughs are urgently required to combat antimicrobial resistance. Actinomycetia are the principal producers of antibiotics. ABSTRACT The Actinomycetia isolate PBR11 was isolated from the forest rhizosphere soil of Pobitora Wildlife Sanctuary (PWS), Assam, India. The isolate was identified as Streptomyces sp. with 92.91% sequence similarity to their closest type strain, Streptomyces atrovirens NRRL B-16357 DQ026672. The strain demonstrated significant antimicrobial activity against 19 test pathogens, including multidrug-resistant (MDR) clinical isolates and dermatophytes. Phenol, 2,5-bis(1,1-dimethylethyl), is the major chemical compound detected by gas chromatography-mass spectrometry in the ethyl acetate extract of PBR11 (EtAc-PBR11). The presence of the PKS type II gene (type II polyketide synthases) and chitinase gene suggested that it has been involved in the production of antimicrobial compounds. Metabolic profiling of the EtAc-PBR11 was performed by thin-layer chromatography and flash chromatography resulted in the extraction of two bioactive fractions, namely, PBR11Fr-1 and PBR11Fr-2. Liquid chromatography-tandem mass spectrometry analysis of both the fractions demonstrated the presence of significant antimicrobial compounds, including ethambutol. This is the first report on the detection of antituberculosis drug in the bioactive fractions of Streptomyces sp. PBR11. EtAc-PBR11 and PBR11Fr-1 showed the lowest MIC values (>0.097 and >0.048 μg/mL, respectively) against Candida albicans MTCC 227, whereas they showed the highest MIC values (>0.390 and >0.195 μg/mL, respectively) against Escherichia coli ATCC BAA-2469. The effects of PBR11Fr-1 were investigated on the pathogens by using a scanning electron microscope. The results indicated major morphological alterations in the cytoplasmic membrane. PBR11Fr-1 exhibited low cytotoxicity on normal hepatocyte cell line (CC-1) and the percent cell viability started to decline as the concentration increased from 50 μg/mL (87.07% ± 3.22%) to 100 μg/mL (81.26% ± 2.99%). IMPORTANCE Novel antibiotic breakthroughs are urgently required to combat antimicrobial resistance. Actinomycetia are the principal producers of antibiotics. The present study demonstrated the broad-spectrum antimicrobial potential of an Actinomycetia strain Streptomyces sp. strain PBR11 isolated from the PWS of Assam, India, which represents diverse, poorly screened habitats for novel microorganisms. The strain displayed 92.4% sequence similarity with genes of the closest type strain, indicating that the strain may represent a novel taxon within the phylum Actinomycetota. The metabolomics studies of EtAc-PBR11 revealed structurally diverse antimicrobial agents, including the detection of the antituberculosis drug ethambutol, in the bioactive fraction of Streptomyces sp. PBR11 for the first time. The PBR11 strain also yielded positive results for the antibiotic synthesis gene and the chitinase gene, both of which are responsible for broad-spectrum antimicrobial activity. This suggests that the untouched forest ecosystems have a tremendous potential to harbor potent actinomycetia for future drug discovery.

various metabolic engineering strategies and sophisticated methods can help in enhancing the value of these bacteria in terms of productivity or creating diverse biologically active products (20). The discovery of novel microbes and products derived from poorly explored areas such as Northeast India, China, Australia, Antarctica, and Jordan suggests that careful investigation of new habitats may continue to be beneficial (21)(22)(23)(24). A successful approach for finding new pharmacological leads or chemical scaffolds is investigating novel taxa from untapped sources (25,26). Actinomycetia have the potential to produce a variety of structurally diverse secondary metabolites for drug development; however, only a small portion of these bacteria have been cultured. The present investigation aimed to isolate Streptomyces from the poorly explored protected forest ecosystem of Assam with the capability of producing secondary metabolites with high antimicrobial action. We emphasize here the exploration of Streptomyces from the uninvestigated forest ecosystem of Assam, India, for natural products in drug discovery.

RESULTS
Morphological identification of Streptomyces sp. PBR11 strain. Streptomyces sp. strain PBR11, which is aerobic and filamentous in nature, was isolated from the forest rhizosphere soil of Pobitora Wildlife Sanctuary (PWS), Assam, India. The PBR11 strain was morphologically identified by observing the colony morphology. The aerial mycelium was light purple, whereas the vegetative substrate mycelium produced a light brown color (Fig. 1A). The pure culture of the PBR11 strain grown in GLM agar (described in Materials and Methods) produced an earthy odor.
Antimicrobial potential of PBR11 strain. The PBR11 strain demonstrated promising antimicrobial activity during bioactive screening for the production of extracellular secondary metabolites in GLM agar medium by the spot inoculation technique. The PBR11 strain exhibited maximum antifungal activity against Candida albicans MTCC 227 (47.0 6 1.0), followed by Aspergillus fumigates MTCC 1811 (44.7 6 0.6), Aspergillus  Table 1. The antimicrobial activity of the ethyl acetate extract of PBR11 (EtAc-PBR11), along with the controls (10% dimethyl sulfoxide [DMSO]; negative control and antibiotics; positive control) against Micrococcus luteus MTCC 1538 and GNR19 (Escherichia coli) are shown in Fig. 2.
Biochemical characterization and optimization of cultural conditions on growth and antimicrobial production. The aerial mycelia are long and branched, as confirmed by scanning electron microscopy (SEM) analysis. Spore chains are rectiflexibiles with 10 or more spores per chain. Each chain has oval-shaped spores that range in size from 848.7 to 874.6 nm in diameter (Fig. 1C). Table 2 shows the cultural traits of the PBR11 strain in different culture mediums, where the strain grew well on all media except actinomycetes isolation agar medium (Fig. 1D). The strain was found to grow at between pH 5 and pH 11. The maximum growth and highest secondary metabolite production were obtained at a pH of 8 ( Fig. 1E and Table 3). However, poor growth and low antimicrobial production were detected at pH levels above pH 11 and below pH 4. The optimum temperature for maximum growth and highest antibiotic production was 28°C. The highest production of secondary metabolites and best growth was observed on the third day under shake-flask conditions at 28°C. To evaluate the potentiality of PBR11 stain for novel antibiotics, eight standard antibiotics were used for the antibiotic susceptibility test; of these, the strain showed resistance to nitrofurantoin (300 mg), ampicillin (10 mg), and co-trimoxazole (25 mg). The complete biochemical characteristics of strain PBR11 and its antibiotic susceptibility profile are shown in Table 4.
Gas chromatography-mass spectrometry analysis. The analysis of bioactive constituents present in the crude extract EtAc-PBR11 was evaluated by gas chromatogra-  phy-mass spectrometry (GC-MS). Five major chemical compounds were detected based on molecular weight and their retention times by comparing their mass spectra with the National Institute of Standards and Technology (NIST) database. Most of the detected compounds are known to have antibacterial, antifungal, anticancer, antioxidant, and antituberculosis activities. The results are shown in Table 5, and the structures of the identified compounds and GC-MS chromatogram graph of EtAc-PBR11 are shown in Fig. 3. The peak area of the detected compound indicates the quantity of compounds in the extract. Molecular characterization and phylogenetic analysis of PBR 11 strain. The partial 16S rRNA gene sequence of the PBR11 strain was deposited into the NCBI GenBank database under the accession number MH718314. The gene size for the partial sequences is 1,370 bp. The strain showed the highest sequence similarity with Streptomyces atrovirens strain NRRL B-16357 (DQ026672). Based on a neighbor-joining approach, the phylogenetic tree also displayed its highest similarity to Streptomyces atrovirens (92.91%) (Fig. 4A). The phenotypic data and molecular identification indicated that the PBR11 strain belonged to the genus Streptomyces, and thus the strain was designated as Streptomyces sp. PBR11.
Detection and investigation of the biosynthetic gene PKS type II and chitinase gene for the prediction of chemical classes. Streptomyces sp. PBR11 showed positive results against both biosynthetic genes clusters PKS-II and for chitinase 18 Glycosyl Hydrolase family gene (GH18) (Fig. 4B). The partial sequences of the PKS-II gene and chitinase gene were deposited in GenBank under accession numbers ON911582 and ON911583, respectively. The amino acid sequence of the PKS type II gene showed close similarity to beta-ketoacyl synthase family protein and shared 99.42% similarity with their closest match Streptomyces sp. (WP_004922256) at the amino acid level. The predicted protein structure displayed the KS domain, which is similar to the structure of actinorhodin polyketide putative beta-ketoacyl synthase of Streptomyces coelicolor A3(2) (70.9% identity, PDB ID 1tqy) responsible for polyketide antibiotic biosynthesis. The predicted protein product showed maximum similarity to macrolide antibiotic megalomicin. The translated protein sequence of the chitinase GH18 gene showed the highest similarity to GH18 protein with 98.18% similarity to their closest match Streptomyces katrae (WP_045948892). The predicted protein structures were similar to the crystal structure of chitinase 40 of the thermophilic actinomycetia Streptomyces thermoviolaceus (70.9% identity, PDB ID 4w5u) involved in chitin degradation (Table 6).
Morphological effect of PBR11Fr-1 on the test pathogens. SEM analysis of C. albicans MTCC 227, E. coli ATCC BAA-2469, and A. baumannii ATCC BAA-1705 cells after treatment with 1Â MIC PBR11Fr-1 revealed significant morphological changes compared to the control cells ( Fig. 5C to H). The cytoplasmic membrane structures of treated cells displayed deformities, which led to cell shrinkage and the loss of cell integrity. The cell surfaces of the nontreated cells (control) were intact and retained their original cell structures and morphologies with an intact cell surface.
Cytotoxic activity of bioactive fraction PBR11Fr-1. The toxicity of the PBR11Fr-1 on normal liver cell line CC-1 was evaluated at different concentrations. The highest percentage of cell viability was recorded at 5 mg/mL (96.37% 6 1.89%). The cell viability started declining with the increase in the concentration from 50 mg/mL (87.07% 6 3.22%) to 100 mg/mL (81.26% 6 2.99%). The PBR11Fr-1 fraction exhibited a low cytotoxic effect in a normal cell line (Fig. 7).

DISCUSSION
Infectious diseases are the leading cause of death worldwide, impacting health care and socioeconomic development. Furthermore, the rapid development of drug resistance to currently available antimicrobial agents and adverse side effects from prolonged use is a major public health concern. Therefore, the development of novel antimicrobial drugs is urgently required. Actinomycetia are the most prolific producers of natural bioactive compounds, especially antibiotics and many other bioactive compounds with diverse biological properties (4,27). Isolation of microorganisms from untouched ecosystems leads to the possibility of discovering novel microbial products, particularly when it pertains to the north-eastern region of India (28). The majority of the forest area of this region is a part Indo-Burma biodiversity hot spots, and there is an excellent possibility that these forest ecosystems may contain various novel microbes capable of producing diverse secondary metabolites. PWS is located on the south bank of the Brahmaputra River (26°120 to 26°160N and 91°580 to 92°050E), about 50 kilometers east from Guwahati, the state capital of Assam, India. The climate of the PWS is subtropical monsoon. The rainy season, which lasts from May to September, has an average rainfall of 2,000 mm. This period is very humid and warm. The maximum average temperature is 25°C, and the humidity is .95% (29). The entire region is covered by the Brahmaputra flood plains. Because it is low-lying, it experiences yearly flooding. The soil is mostly made up of river alluvium, and its color ranges from reddish-brown to reddish yellow. The soil type is fertile clayey loam with silt (28). Although it is a small area, it is biologically diverse and is located in one of the world's megabiodiversity hot spots. Using serial dilution technique, a total of 65 morphologically different putative actinomycetia were isolated and purified from the soil samples of the unique niches of PWS. The serial dilution technique is a well-established microbiological technique that has been used successfully to improve culturability and facilitate the separation of molecularly detectable bacteria and archaea from freshwater, marine, and soil systems (30-34). Schoenborn and his team hypothesized in 2004 that liquid serial dilution should also enable the isolation of microbes from poorly investigated bacterial families from an unsaturated soil (35). Streptomyces sp. PBR11 was isolated from the rhizosphere soil of Cynodon dactylon, commonly known as Bermuda grass. Based on the results of in vitro antimicrobial bioassay, of 65 actinomycetia isolates, the most potent isolate, PBR11, was selected for further studies.
The aerial mycelia of the PBR11 strain are long and branching. Spore chains are rectiflexibiles, and the spore shape is globose. PWS of Assam, India, is the least-explored area for the isolation of microorganisms particularly actinomycetia, and only a few studies on their antimicrobial biosynthetic capacity have been published (28). It has been previously reported that an actinomycetia strain, Nocardia sp. PB-52, with broad spectrum bioactivity was isolated from the PWS (28). Our present work represents the first report on the isolation of a Streptomyces genus from this unscreened habitat that demonstrated broad-spectrum action against 19 different test pathogens, including multidrug-resistant clinical isolates and dermatophytes. Fungi are eukaryotic microorganisms, and they utilize mechanisms similar to those of higher animals for synthesizing proteins and nucleic acids. Therefore, it is exceedingly challenging to identify antifungal agents that specifically inhibit fungal metabolism without being toxic to people (36). There is evidence that the dermatophytes, which cause skin infections (dermatophytosis) in humans, have developed resistance to some antimycotic medications (37,38). The PBR11 strain exhibited strong antifungal activity against all the tested fungal pathogens responsible for causing fungal infections. It has been already reported that Streptomyces species remains a prominent source of antimicrobial agents with low cytotoxic effects (39,40).
The production medium and its growing parameters needed to be optimized to increase the production of antimicrobial metabolites. Streptomyces sp. strain PBR11 is a mesophilic bacterium, and its highest antibiotic production capability was recorded at 28°C and pH 8. GLM medium was found to be the most effective growth medium for evaluating the antagonistic activity of the strain while it showed the least growth on actinomycetes isolation agar. The strain showed diffusible pigments on ISP6 and ISP7 media. The ideal incubation period for the synthesis of antimicrobial metabolites was noted on the third day of growth. Our findings slightly deviate from previous reports. Earlier, a report stated that Streptomyces sp. isolated from pristine habitats produced maximum antimicrobial metabolites after the fifth day of culture at a pH of 7 (41). Similarly, the actinomycetia strain Streptomyces sp. VITSVK5 isolated from the marine sediment of the Bay of Bengal, India, showed maximum antifungal activity at pH 8.2 (42). Growth-limiting nutrients have a significant impact on the interaction between growth metabolism and product secretion in the formation of secondary metabolites (42). Carbohydrates such as starch, glycerol, maltose, sucrose, xylose, and mannose have been associated with the synthesis of maximal antimicrobial metabolites (43). Our recent work on Streptomyces sp. PBR11 also illustrates positive results for utilizing all the above-mentioned carbohydrates including trehalose, dextrose, L-arabinose, insulin, salicin, trehalose, mannitol, arbitol, erythritol, adonital, L-methyl-D-glucoside, rhamnose, cellubiose, melezitose, L-methyl-D-mannoside, xylitol, ONPG (o-nitrophenylb-D-galactopyranoside), esculin hydrolysis, D-arabinose, and sorbose. Eight different types of antibiotics from different classes such as quinolones, cephalosporins, beta-lactam first-generation cephalosporins, aminoglycosides, quinolones, nitrofurans, penicillin beta-lactam antibiotics, and sulfonamides were used to check the antibiotic resistant and susceptibility profiles of the strain using a disc diffusion method. The strain exhibited high sensitivity to norfloxacin (10 mg), cefotaxime (30 mg), cephalothin (30 mg), gentamicin (10 mg), and nalidixic acid (30 mg), whereas it showed resistance to nitrofurantoin (300 mg), ampicillin (10 mg), and co-trimoxazole (25 mg), respectively.
The class Actinomycetia continues to offer novel secondary metabolites with diverse biological activities, including antimicrobial, anticancer, and anthelmintic actions. These compounds have the potential to emerge as therapeutic guides for the synthesis of new chemical compounds. Several published reports have been documented that describe the GC-MS studies to identify the chemical constituents of bioactive compounds and natural product discovery (44,45). Here, GC-MS analysis was performed in which five major compounds were detected in EtAc-PBR11 with various retention times. Phenolic compounds are potent antioxidants and exhibited significant antibacterial activity because of their lipophilic nature, which enhances their antimicrobial activity by promoting their contact with the cell membrane (46). GC-MS fractions with

Bioactive compounds Produced by Streptomyces
Microbiology Spectrum a more area percentage of phenolic compounds demonstrated strong antimicrobial activity (47). Phenol, 2,5-bis(1,1-dimethylethyl), is the major chemical compound detected in the EtAc-PBR11 with 33.03% (area %). This phenolic compound may significantly influence the inhibitory action against various test pathogens. Other major compounds include 1-tetradecanol, n-pentadecanol, 1-nonadecene, and pyrrolo[1,2-a]pyrazine-1,4-dione hexahydro-3-(phenylmethyl). These compounds have demonstrated significant antibacterial (48)(49)(50), antituberculosis, anticancer, antioxidant, antifungal, and antimicrobial activities, respectively (51)(52)(53)(54)(55). The identification of the isolate PBR11 was done using the recommended international standards. The polyphasic approach has recently become the most widely accepted system for classification and identification (56)(57)(58). This method incorporates many different types of data, including phenotypic, chemotaxonomic, genotypic, and phylogenetic data (59). The phenotypic, microscopic, and biochemical properties of PBR11 were determined in accordance with the International Streptomyces Project (ISP) (60) and Bergey's Manual of Systematic Bacteriology (61). The isolate was identified to the genus level by comparing the morphology of spore bearing hyphae with the entire spore chain and structure of spore as described by Li et al. (59). The opaque, rough, nonspreading morphology of Streptomyces colonies, which are frequently embedded and adhere to agar medium, make them easy to identify, and almost every colony of Streptomyces produced an earthy odor (geosmin), which is also an important characteristic of Streptomyces (62). Moreover, the phylogenetic analysis by a neighborjoining approach showed that the PBR11 forms a separate subclade from the other members of Streptomyces sp., indicating that it may represent a novel taxon, and demonstrated 92.91% sequence similarity with the closest match, Streptomyces atrovirens NRRL B-16357. Therefore, the phenotypic, biochemical, and molecular traits of PBR11 revealed that the isolate possessed typical characteristics of the genus Streptomyces; thus, the isolate was designated Streptomyces sp. PBR11.These findings suggested that the PWS is a repository of novel Streptomyces sp. from the phylum Actinomycetota with promising antagonistic properties against various disease-causing pathogens. Biosynthetic gene cluster PKS-II plays a central role in the biosynthesis of bioactive secondary metabolites (63). PBR11 strain exhibits positive results for the PKS type II gene. Aromatic polyketides, such as anthracycline, angucycline, and tetracycline, are commonly synthesized by polyketide synthase type II (64). The amino acid sequence of the PKS-II gene showed close similarity to beta-ketoacyl synthase (KS) family protein (99.42%) of Streptomyces sp. The predicted protein product showed maximum similarity to macrolide antibiotic megalomicin. The KS domain is the most conserved catalytic domain involved in type II polyketide synthesis. They are responsible for producing antimicrobial compounds with broad-spectrum antimicrobial activity (65).
Furthermore, the strain was also found positive for the chitinase GH18 family gene. Chitinases belong to the chitinase GH18 family gene, and they catalyze the hydrolysis of b-1,4-linkages in chitin which exhibited an inhibitory action on fungal growth (66). The amino acid sequence of the chitinase gene showed maximum similarity to the chitinase GH18 protein (98.18%) of Streptomyces katrae, and the predicted protein structures showed similarity to the crystal structure of chitinase 40 from thermophilic actinomycetia, Streptomyces thermoviolaceus, involved in the hydrolysis of the b-1,4-linkage between N-acetyl-D-glucosamine residues of chitin. The broad-spectrum antimicrobial activity of the Streptomyces sp. PBR11 strain may be controlled by the expression of the biosynthetic PKS type II gene and the chitinase GH18 family gene. DMSO is an ideal solvent for preparing samples for evaluating biological activity and dissolving bioactive compounds in in vitro drug discovery (67,68). Because of its ability to dissolve both polar and nonpolar compounds, as well as its miscibility with water and culture media, DMSO is an excellent solvent for bioassays (69). It also improves the accuracy of the concentrations of test compound or drug by reducing room temperature and evaporation and can be used to maintain stock solutions of test compounds (69,70). For our studies, DMSO was used as a positive vehicle control and also for the preparation of crude extracts and bioactive fractions. Fractionation of the crude ethyl acetate extract EtAc-PBR11 produced two bioactive fractions, PBR11Fr-1 and PBR11Fr-2. Nonetheless, PBR11Fr-1 was the most promising fraction for antimicrobial activity. LC-MS/MS analysis of both bioactive fractions demonstrated the presence of antimicrobial and bioactive compounds. Three biologically active compounds -N-depyridomethyl-indinavir, crustecdysone (20-hydroxyecdysone), and ethambutol -were detected in both fractions. Indinavir, an antiretroviral drug, is a potent and specific inhibitor of HIV-1 (71,72). According to a recent report (73), crustecdysone (20hydroxyecdysone) demonstrated in many in vitro and in vivo models numerous biological properties, including anabolic, anti-inflammatory, antioxidant, immunomodulatory, antidiabetic, and anti-obesity activities, in addition to serving as a neuroprotective and hepatoprotective drug. It also has antiparasitic activity, inhibits caspase activity, and induces autophagy (74,75). Ethambutol is a first-line drug therapy recommended by the World Health Organization for treating tuberculosis (76,77). In the present investigation, ethambutol was detected for the first time in the bioactive fraction of Streptomyces sp. PBR11. This is the most significant finding that emerged from our study. Synthesizing this antituberculosis drug from any natural resources has not been reported so far. Other bioactive compounds detected in the fraction PBR11Fr-1 were arecoline, 10-nitro-9Z,12Z-octadecadienoic acid, campestanol, SM(d18:0/0:0), ethoxyquin, phendimetrazine, colforsin, 10-deoxymethymycin, anandamide (20:2, n-6), and miltefosine. Arecoline is reported to have anthelmintic activity (78,79), while 10-nitro-9Z,12Z-octadecadienoic acid (80) and colforsin (81,82) are known for displaying antiinflammatory and apoptosis action. Campestanol (83), SM(d18:0/0:0) (84), ethoxyquin (85,86), phendimetrazine (87), 10-deoxymethymycin (88), anandamide (20:2, n-6) (89), and miltefosine (90) have hypocholesterolemic, cell signaling, antioxidant, anticarcinogenic, anorexigenic, antibacterial, fatty acid neurotransmitter, and antileishmanial effects, while no bioactivity has been reported for 11-amino-undecanoic acid, D-pantetheine 49-phosphate, and 3-hydroxytetradecanedioic acid. The biologically active compounds diethylcarbamazine, acetylsalicylic acid (aspirin), 2-propyl-9Z-octadecenoic acid, and oleandrin (72,(91)(92)(93)(94)(95) detected in the bioactive fraction of PBR11Fr-2 exhibited anti-inflammatory effects, nonsteroidal anti-inflammatory action, antiplasmodial activity, anticancer effects, novel antiviral activity, and anorexigenic effects. However, there is no bioactivity reported for 12,14-pentacosadiynoic. Our results confirmed that natural product research continues to be important in discovering and developing novel bioactive compounds for clinical and therapeutic applications.
The metabolites isolated from the Streptomyces sp. exhibited significant antimicrobial activity with low MIC values, making it one of the best sources of effective antimicrobial agents for treating infectious diseases (96). The MIC values exhibited by the crude extract EtAc-PBR11 and bioactive fraction PBR11Fr-1 were found within the range of $0.048 to $0.390 mg/mL. EtAc-PBR11 had the lowest MIC of $0.097 g/mL against C. albicans MTCC 227, while PBR11Fr-1 showed an MIC of $0.048 mg/mL. Similarly, against E. coli ATCC BAA-2469, the lowest MIC shown by EtAc-PBR11 was $0.390 mg/mL, and for PBR11Fr-1 it was $0.195 mg/mL, respectively. Against A. baumannii ATCC BAA-1705, the crude extract exhibited an MIC of $0.195 mg/mL, and the bioactive fraction showed an MIC of $0.097 mg/mL. We found that the MICs displayed by the crude extract and the bioactive fraction PBR11Fr-1 were significantly lower than the MICs obtained from the standard antimicrobial drug. According to an earlier report (97), a compound isolated from the actinomycetia strain M. auratinigra showed promising antimicrobial activity with low MICs compared to the reference antibiotic neomycin sulfate.
SEM is a powerful tool for investigating changes in the bacterial surface structure and cell damage (98). In this study, the SEM result confirms the disruptive action of fraction PBR11Fr-1. It caused considerable morphological changes in the cellular membrane of the selected pathogens, including the shrinkage of the cells and surface deformation compared to untreated cells. This further indicates that the mode of bactericidal action of the bioactive fraction against the pathogens is through membrane disruption mechanism and prevents further cell development.
The drug toxicity of the bioactive fraction PBR11Fr-1 was evaluated against normal liver cell line CC1 using an MTT assay. The toxicity of the fraction was found to be dose dependent and demonstrated a low cytotoxic effect. The percentage of the cell viability started declining with the increase in the concentration from 50 mg/mL (87.07% 6 3.22%) to 100 mg/mL (81.26% 6 2.99%). Similar findings have been reported (39,99), suggesting that Streptomyces spp. have fewer cytotoxic effects against normal cell lines.

MATERIALS AND METHODS
Soil sample collection site. Samples of rhizosphere soil were collected from various sites of PWS (26°120 to 26°160N and 91°580 to 92°050E) of Assam, which is in the northeastern part of India (Fig. 8). The entire area falls under the Brahmaputra flood plains. Soil samples were taken from various depths on the earth's surface, ranging from layers just beneath the upper surface to a 10-cm depth after removing the upper layer of the topsoil. The samples were collected in sterile zip lock bags, labeled correctly with the collection date, and moved to the laboratory on the same day. The collected soil samples were stored at 4°C until they were analyzed.
Isolation of actinomycetes from soil samples. The PBR11 strain was isolated from soil by the serial dilution technique (4). A soil suspension was made from the collected soil sample by dissolving 5 g of soil in 100 mL of normal saline water (NaCl 9g/l), which was then incubated at 28°C for 24 h at 180 rpm with continuous shaking. The mixtures were allowed to settle, and dilutions were made up to a concentration of 10-4 with sterile saline water and thoroughly mixed by vortexing at maximum speed. Aliquots (0.1 mL) from each dilution were spread onto plates of isolation media, followed by incubation at 28°C and checked after 48, 72, and 96 h (23). To inhibit the growth of fungi and bacteria, the isolation plates were supplemented with amphotericin B (75 mg/mL) and rifampicin (2.5 mg/mL), respectively. The culture plates were then incubated for 5 days at 28°C. Repeated streaking of the isolate on GLM (yeast extract, 3 g; malt extract, 3 g; peptone type I, 5 g; starch, 10 g; agar, 20 g; distilled water, 1,000 mL; pH 7.5) agar plates resulted in the purification of the PBR11 isolate, which was stored at -80°C in 20% glycerol for future use.
Evaluation of antimicrobial activity. (i) Test pathogens. (ii) Antimicrobial activity assessment. Preliminary screening for in vitro antimicrobial bioassay of PBR11 strain against the test pathogens was carried out by the spot inoculation method (100). The zone of inhibition was evaluated after 24 to 48 h of incubation at 37°C for bacteria and at 28°C for yeasts and fungi. Each experiment had three replicates. The secondary screening for the antimicrobial assay was done by the disc diffusion method (101). The strain was grown in 250-mL flasks containing 100 mL of GLM broth and incubated for 5 days at 28°C and pH 7.5 in a rotary shaker (180 rpm) with continuous shaking. By centrifuging (Sigma 3K30) the culture broth at 7,000 Â g for 20 min, the cell mass was separated. The supernatant was then extracted for 30 min in a separating funnel with ethyl acetate (EtAc) at a 1:1 (vol/vol) ratio. A rotary evaporator (Rotavapor R-215; BUCHI, Labortechnik AG, Flawil, Switzerland) was used to evaporate the EtAc extract to dryness under reduced pressure at 45°C. The dried extract (EtAc-PBR11) was prepared before the antimicrobial bioassay by dissolving it in 10% dimethyl sulfoxide (DMSO) at a concentration of 1 mg/mL. Then, 30 mL of the extract was loaded onto sterile discs (6 mm in diameter) placed on Mueller-Hinton agar plates seeded with bacterial pathogens (0.5 McFarland turbidity standards). For fungal pathogens, this was carried out on Sabouraud dextrose agar plates. Rifampicin (30 mg/disc) for bacteria and amphotericin B (30 mg/disc) for fungal species were used as positive controls, while a 10% DMSO-loaded disc served as a negative control. After 24 h of incubation at 37°C for bacteria and 28°C for Candida, antimicrobial activity was observed.
(iii) Biochemical characterization and optimization of cultural conditions on growth and antimicrobial production. The PBR11 strain was grown in nine different growth mediums to assess its cultural characteristics. Light microscopy and SEM were used to examine the pattern of the mycelium and ornamentation of the spore chain (16). The carbohydrate utilization by the strain was tested by using a Hicarbo kit (HiMedia). The PBR11 strain was grown in the Hicarbo kit and incubated at 28°C for 24 to 48 h. A disc diffusion method with Octa-Disc (HiMedia) was used to detect the susceptibility of the PBR11 strain to eight standard antibiotics. To evaluate the growth response and antibiotic synthesis, the strain was inoculated in GLM broth at 28°C for 8 days at various pH values ranging from acidic to alkaline (pH 3 to pH 12). The impact of temperature on the growth and production of the antimicrobial agent was investigated on GLM at different temperatures (15, 20, 25, 28, 32, 35, 40, and 45°C) at pH 8. The antimicrobial activity of PBR11 was assessed against C. albicans by the disc diffusion method (101) because C. albicans proved to be a more sensitive test organism during the evaluation of antimicrobial production by spot inoculation and well diffusion techniques.
(iv) Gas chromatography-mass spectrometry analysis. The crude extract (EtAc-PBR11) was dissolved in HPLC-grade methanol and filtered through a 0.2-mm syringe filter. The bioactive metabolites present in the EtAc-PBR11 were identified using GC-MS (16) with slight modifications. The peaks were identified by comparing the mass spectra to the NIST (USA) library.
Molecular characterization. (i) 16S rRNA gene amplification and phylogenetic analysis. A Genetix kit was used for genomic DNA extraction of the PBR11 strain. The universal eubacterial primers 27F (59-AGA GTT TGA TCC TGG CTC AG-39) and 1492R (59-GGT TAC CTT GTT ACG ACT T-39) were used to amplify the 16S rRNA gene (102). PCRs were carried out in a Proflex PCR System (Applied Biosystems, USA) by the method described by Sharma et al. (28). Then, a 1.8% (wt/vol) agarose gel prepared in 1Â TAE buffer was used to confirm the amplified products. The PCR bands were analyzed using a Bio-Rad Gel Doc XR1 system (Hercules, Richmond, CA). BLASTN (103) and EzTaxon server (http://www .ezbiocloud.net) (104) were used for phylogenetic analysis based on the 16S rRNA gene sequence of the PBR11 strain. The top 40 nucleotide sequences of 16S rRNA genes with the highest nucleotide similarity were selected for multiple sequence alignment by the CLUSTAL W program (105). A phylogenetic tree was constructed by the neighbor-joining method (106) using MEGA X (107). A bootstrap analysis with 1,000 replications was used to assess the support of each clade (108).
(ii) Detection and analysis of the PKS-II gene and chitinase gene for prediction of bioactive chemical classes. To detect the antimicrobial metabolite-producing biosynthetic gene in Streptomyces sp. PBR11 strain, PCR amplification of polyketide synthase II (PKS-II) pathway-encoded genes was done. The PKS-II gene was amplified using the degenerate primers KS a F (59-TSG CST GCT TCG AYG GCS ATC-39) and KS b R (39-TCG CCG BAA GCC GCC NAA GGT-59) (109). Group A bacterial chitinase gene of glycoside hydrolase family 18 was amplified by using a degenerate set of primers, GA1F and GA1R, as previously described by (110). The PCRs for the PKS-II and chitinase genes were performed according to previously described methods (24,111). The amplified products for the PKS-II and chitinase genes were evaluated in a 1.8% (wt/vol) agarose gel made in 1Â TAE buffer. The PCR bands were examined under UV light and documented using a Bio-Rad Gel Doc XR1 system (Hercules). The PCR-amplified product of the PKS-II gene and the chitinase gene of the Streptomyces sp. PBR11 was sequenced at First BASE Laboratories, Malaysia. The web tool ORF FINDER was used to translate the nucleotide sequences of the PKS-II and chitinase genes into amino acid sequences, which were further used as queries to search their gene products in the NCBI database using the protein BLAST tool with default settings. The predicted pathway product of the biosynthetic gene of strain PBR11 was identified using SBSPKSv2 (112). The secondary structures of the polyketide ketosynthase domains and the chitinase gene were created using the PDBsum tool (113).
(iii) Cultivation and fermentation of Streptomyces sp. PBR11 for the production of active metabolites. The fermentation process of the strain Streptomyces sp. PBR11 was already discussed in the antimicrobial assay section. The fermentation was carried out for 3 days at pH 8 under identical conditions (28). A total of 20 L of culture broth was prepared by continuing the fermentations, in the same way, to extract and purify the active metabolites. The harvested cell mass of Streptomyces sp. PBR11 was separated by centrifugation at 7,000 Â g for 15 min, and the supernatant was extracted twice with equal volumes of EtAc-fermented broth (1:1 [vol/vol]) for 30 min in a separating funnel. A rotary evaporator (Rotavapor-R 210; BUCHI) was used to dry the EtAc extract of Streptomyces sp. PBR11 (EtAc-PBR11) under reduced pressure at 45°C. The dried crude extract was collected and stored in a refrigerator for further investigation.
(iv) Bioactivity-guided fractionation and chemical profiling of Streptomyces sp. PBR11. Three chromatographic methods-TLC, flash chromatography, and LC-MS/MS-were used to investigate the metabolomic profile of EtAc-PBR11.
(v) Thin-layer chromatography. Silica gel-coated TLC plates (TLC silica gel 60 F254; Merck, Germany) were used to evaluate the polarity of the secondary metabolites present in EtAc-PBR11. The nature of the secondary metabolites and the pattern of separation of the various fractions formed by them were evaluated by dissolving the dried crude extract in methanol and then examining it on TLC plates. The extract was overlaid onto a TLC plate by repeated spotting with a small capillary at room temperature in a TLC chamber. The loaded plate was run in various combinations of organic solvent systems-viz. chloroform-methanol, ethyl acetate-methanol, and hexane-ethyl acetate-and visualized under visible and UV light. After development, the TLC plate was stained to identify the various colored bands of the compounds in the crude extracts using anisaldehyde/H 2 SO 4 spraying reagent.
(vii) LC-MS/MS analysis. The chemical compounds present in the bioactive fractions of PBR11Fr-1 and PBR11Fr-2 were identified using LC-MS/quadrupole time-of-flight mass spectrometry (410 Prostar Binary LC with 500 MS; Varian, Inc., USA) equipped with a photodiode array detector and a C 18 column. Both the fractions were dissolved in HPLC-grade methanol, and the injection volume was 3 mL. The mobile phases employed for the analysis were A (100% water) and B (100% acetonitrile) in a gradient elution method with a 30-min run time and a flow rate of 0.3 mL/min. Data on mass spectra were retrieved in positive ionization mode for the mass range m/z 150 to 1,000. In addition, the following conditions were set for the mass spectrometer: nebulizing gas pressure, 35 lb/in 2 ; drying gas flow rate, 13 L/min; drying gas temperature, 300°C; and nebulizing gas flow rate, 11 L/min.
(viii) Investigation of MIC of EtAc-PBR11 and PBR11Fr-1. The MIC assay was carried out according to the previous method, with a few modifications (114). A stock solution (1,000 mg/mL) of filter-sterilized (0.2 mm) EtAc-PBR11 and bioactive fraction PBR11Fr-1 was prepared in 10% DMSO, and 2-fold serial dilutions of the extracts (working solution, 50 to 0.048 mg/mL) were prepared for MIC tests. These were transferred to a 96-well plate, and each well received 10 5 CFU (0.5 McFarland turbidity standards) of freshly grown test microbial strains. The positive control, made with conventional antibiotics such as levofloxacin and amphotericin B, should be clear, while the negative control made with DMSO (10%) without antimicrobial drugs, should be turbid. The bacteria and Candida plates were incubated aseptically for 24 h at 37 and 28°C, respectively, and the absorbance at 600 nm was measured by using a UV-Vis spectrophotometer. (Varioskan Flash; Thermo Scientific, San Jose, CA). After adding 30 mL of 0.015% resazurin to each well, the plates were incubated for 2 h at room temperature. The MIC value of the extract was determined by the concentration at which blue was observed. Cells (10 mL) from the blue-colored wells were spread on Muller-Hinton agar and Sabouraud dextrose agar plates, followed by incubation overnight. MICs were defined as the values at which no visible microbial growth was seen.
(ix) Morphological effects of PBR11Fr-1 on test pathogens. The bioactive fraction PBR11Fr-1 was studied by SEM as described earlier (28) with a few modifications for its antimicrobial effects on C. albicans MTCC 227, E. coli ATCC BAA-2469 and A. baumannii ATCC BAA-1705. Test microorganisms were treated with 1Â MIC PBR11Fr-1.
(x) In vitro cytotoxic activity of bioactive fraction PBR11Fr-1. The in vitro drug sensitivity of the bioactive fraction, PBR11Fr-1, was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. In 48-well plates, normal liver cells (CC-1) were seeded at a density of 2,000 cells per well. These were allowed to grow overnight and then treated with PBR11Fr-1 at various concentrations (5,10,25,50,75, and 100 mg/mL) for 48 h at 37°C in a humidified 5% CO 2 incubator. An MTT reduction conversion assay was used to assess drug cytotoxicity. Each well received 50 mL of MTT at a concentration of 5 mg/mL and was incubated for 4 h. Formazan crystals formed by mitochondrial enzymatic action on the MTT substrate were solubilized in 200 mL of DMSO. After the plates were shaken for 20 min, the absorbance at 570 nm was measured by using a microplate reader. Each treatment was performed in triplicate. All data are presented as means 6 the standard deviations (SD). The number of healthy cells in a sample is known as the cell viability. The evaluation of cell viability is essential in toxicity studies. Cell viability assays are primarily used to test the response of cells to a drug or chemical agent (115,116). The following equation was used to calculate the percentage of cell viability: where, A = absorbance. Bioinformatics and data analysis. All experiments were performed in biological triplicate. The data are reported as means 6 the standard deviations of the mean. The chemical structure and other parameters for each compound were searched using online database software. MarvinSketch was used to draw the chemical structures.
Data availability. The partial 16S rRNA gene sequence of the PBR11 strain was deposited into the NCBI GenBank database under the accession number MH718314. The strain showed the highest sequence similarity with Streptomyces atrovirens strain NRRL B-16357 (DQ026672). The partial sequences of the PKS-II gene and the chitinase gene were deposited in GenBank under accession numbers ON911582 and ON911583, respectively.