Actinomycetes are Gram-positive microorganisms widely distributed in nature (Barka et al., 2016). The most dominant genus within actinobacteria is Streptomyces belongs to the family Streptomycetaceas which are highly abundant at different ecological niches (Hamid et al., 2020). Morphologically, it is different from other microorganisms by forming a layer of hyphae which differentiate into the chain of spores (Koepff et al., 2018). The most peculiar characteristic of Streptomyces is their ability to produce verities of secondary metabolites (Čihák et al., 2017). Biogeographically, the wide range of distribution of Streptomyces species in the most diverse habitats, like deserts (Idris et al., 2017), highlands (Malviya et al., 2009), and marine sediments (Dalisay et al., 2013) make them available to a great extent. Among these, terrestrial surface soil is the eminent habitat of this genus, but, strains producing novel compounds becomes difficult from easily accessible soil samples (Komaki et al., 2016). Due to this fact, there is a continual requirement for the search for new bioactive compounds from unexplored environments (Bull and Stach, 2007). It is also important to target such environments that could be highly potent sources of novel bioactive compounds. However, there has been a significant decline in the rate of discovery of novel actinobacteria in recent years (Zotchev, 2012). Therefore, the exploration of potential actinobacteria from unexplored habitats is an important approach in discovering novel antibiotics to meet the current needs (Poulsen et al., 2011). Nepal is rich in biodiversity and known for its unexplored biological resources. There are few studies that have been reported on the diversity of actinobacteria in the high land soil of Nepal (Khadayat et al., 2020), but very few studies have been reported on their genetics, biosynthesis potential, and biochemical activities.

The demand for new antibacterial compounds for the effective treatment of microbial infection is of immediate concern in the scientific community (Cheesman et al., 2017). The rising global issues of increasing multidrug-resistant pathogens and insensitivity towards existing antibiotics are some of the issues of immediate concern. This led to the immediate need for the identification of the robust secondary metabolites producing microorganisms. Streptomyces species are not only known for the production of secondary metabolites, but for the production of industrially important enzymes and therapeutics as well. Hence, search for Streptomyces species and their isolates from different altitude and geographic locations are important (Sevillano et al., 2016). Nepal, centrally located in the Himalayan range is one of the biodiversity hotspots/s. Nepal’s rich biodiversity comes from its unique geographical location with altitude and climatic variations (Pradhan et al., 2020). From the range of low land (60 MASL) to the high altitude (8,848 MASL), there is an immense possibility of having diverse microbial communities. However, screening and identification of Streptomyces species from such wide geographic variation have not been carried out in detail. In such altitude variation, there are immense possibilities to have a diverse Streptomyces species, which can produce novel secondary metabolites. The objective of this study was to molecular characterization of Streptomyces species from high altitude (4,384 MASL) soils collected from Nepal.

Chemicals and reagents

Glucose, sucrose, yeast extract, tryptone, magnesium chloride, potassium dihydrogen phosphate, potassium sulfate, calcium chloride, sodium hydroxide, TES buffer, Difco casamino acids, Difco yeast extract, ethylenediamine tetraacetic acid (EDTA), tris-hydrochloride, ethyl acetate, nalidixic acid, L-proline, and cycloheximide were purchased from HIMEDIA. HPLC grade methanol purchased from Fisher Scientific.

Sample collection

The samples were collected from soil sediment precipitates of high-altitude Gosaikunda Lake of Langtang National Park, Langtang, Nepal, situated at 4,384 m above sea level. Soil sediment samples from four different sites; northern (28°05'02"N, 85°24'42"E), southern (28°04'52"N, 85°24'51"E), eastern (28°05'00"N, 85°24'57"E), and western (28°04'58"N, 85°24'37"E) aspects of Gosaikunda lake were collected. The soil sample collection permit was taken from the Department of National Park and Wildlife Conservation, Ministry of Forest and Environment, Babarmahal, Kathmandu, Nepal with reference letter number 077/78 ECO/102-1503. The samples were placed in sterile plastic bags and kept in an icebox for transportation and further use. The collected samples were brought to the Kathmandu University, Department of Chemical Science and Engineering lab and air-dried for two weeks. The texture of soil samples was identified and classified as loamy soil from the texture analysis in the soil testing laboratory of Kathmandu University. The continuous air-drying of soil samples was performed in order to reduce the population of Gram-negative bacteria. The samples were then passed through a sieve for further analysis.

Isolation of pure culture and physical characterization

Streptomyces strains were isolated from the soil samples in the ISP2 medium. Briefly, 1 g of soil samples collected through the sieve was dissolved in 9 ml of DW and allowed to settle for 5 min. The prepared soil samples were diluted ten times and streaks into the ISP2 solid agar plate containing antibiotics nalidixic acid (20 µg/ml) and cycloheximide (50 µg/ml). The plates were allowed to grow for two weeks at 28°C. The colony was isolated from the mixed culture and screening twice with antibiotics to confirm the pure culture. The isolated cultures were grown in the R2YE solid agar medium and characterization of Streptomyces was performed based on their Gram staining, growth pattern, and colony morphology.

Preparation of culture media

The isolated culture of Streptomyces strains were grown in R2YE medium containing 103 g sucrose, 0.25 g K₂SO₄, 10.12 g MgCl₂·6H₂O, 10 g glucose, 0.1 g casamino acids, 5 g yeast extract dissolved separately in 800 ml and 2 ml of trace elements, 10 ml TES buffer (0.5%), 10 ml KH₂PO₄ (0.5% w/v), 80 ml CaCl₂·2H₂O (3.68% w/v), 15 ml L-proline (20% w/v), and 5 ml 1 M NaOH were added separately. Trace element composition (0.004% ZnCl₂, 0.02% FeCl₃·6H₂O, 0.001% CuCl₂·2H₂O, 0.001% MnCl₂·4H₂O, and 0.001% Na₂B₄O₇·10H₂O).

Isolation of genomic DNA and 16S rRNA analysis

Isolates were inoculated in the R2YE media and incubated at 28°C for 7 days. Isolation of genetic deoxyribose nucleic acid (gDNA) was carried out using standard phenol-chloroform methods (Gumińska et al., 2018). For the identification of bacterial 16S rRNA sequence analysis was performed using universal primer 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1429R (5'-GGTTACCTTGTTACGACTT-3'). The polymeric chain reaction (PCR) condition was set at initial denaturation at 94°C for 5 min; 30 cycles at 95°C for 30 sec, 55°C for 30 sec and 72°C for 120 sec; and a final extension at 72°C for 7 min. The PCR amplified products were analyzed by 0.7% agarose gel electrophoresis. The purified PCR products were sequenced using 27F and 1429R primer with Sanger sequencing methods at Genotech.

Sequence analysis

The 16S rRNA sequence of the isolates were deposited in the National Center for Biotechnology Information (NCBI) gene bank. The homology search of the partial DNA sequence of 16S rRNA of the isolates was performed using the NCBI database and multiple sequence alignment was performed using ClustalW and the phylogenic tree was plotted using MEGA version 6 software by the neighbor-joining methods with bootstrap values calculated from 1,000 replications.

Extraction of metabolites

Isolated Streptomyces species were cultured in 50 ml R2YE medium for 7 days at 28°C. The culture broth was extracted with an equal volume of ethyl acetate. The organic layer of the extract was filtered using Whatman filter paper and dried using a vacuum evaporator for further analysis.

Analysis of antimicrobial activity

The antimicrobial activity of the isolates against four pathogenic strains, viz., Klebsiella pneumoniae, Staphylococcus aureus, Enterococcus faecalis, and Bacillus subtilis (ATCC) were examined disc diffusion method (Pandey et al., 2017). A sterile disc of 6 mm diameter was dripped with 20 μl of the ethyl acetate crude extract (1 mg/ml), placed on the surface of the Mueller-Hinton Agar that has been rubbed with a cotton swab containing test pathogen, and then, incubated for 24 h at 37°C. The antibacterial activity was, then, assessed by measuring the inhibition zone diameter (mm) on an agar plate. Ampicillin and Kanamycin were used as positive control and methanol was used as a negative control.

ABTS assay

2, 2'-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) scavenging assay was performed (Pandey et al., 2020). One ml of different concentrations crude ethyl acetate extract of isolated Streptomyces species was mixed with 3 ml of ABTS working solution. The absorbance was recorded at 720 nm using a UV-visible spectrophotometer (Shimadzu-1800) after 10 min of incubation. The 50% methanol was used as a positive control. For the antioxidant activities, Gallic acid was used as a reference. The percentage of ABTS radical scavenging capacity (RSA) was determined according to the formula.

DPPH assay

Using standard protocol, 1,1-diphenyl 1-2-picryl-hydrazyl (DPPH) radical scavenging potential of isolated Streptomyces crude extract was assessed (Pandey et al., 2020). Briefly, one ml of different concentrations crude ethyl acetate extract of isolated species was added in 3 ml of the DPPH solution (100 µM) followed by 30 min of incubation in the dark and absorbance was measured at the 517 nm in a UV-visible spectrophotometer. Gallic acid was used as a reference and methanolic DPPH was used as a positive control. The percentage RSA was calculated by using the following formula.

The inhibition concentration at which absorbance is reducing by 50% (IC₅₀) were calculated by using average RSA.

Screening for amylase activity

Isolated Streptomyces species were spot inoculated on starch agar media (SAM) with the composition, g/L: soluble starch 10.0, Meat extract 3.0, DW (50% v/v) 1,000 ml, pH 7.0 ± 0.2, agar 15.0 and incubated for 7 days at 28°C. The final growth inoculation was flooded with povidone-iodine solution and left for 5 min. The zone of clearance or decolorization against the blue color background indicates the production of amylase by the organisms (Wang et al., 2016).

Screening for protease activity

The protease activity of the isolates was studied using milk casein agar with the composition in g/L: peptone 1.0, sterile skimmed milk (10%), agar 15.0, pH 7.0 ± 0.2, DW (50% v/v) 1,000 ml. Test Streptomyces strains were streaked and incubated for 7 days at 28°C. Following incubation, a clear zone surrounding the Streptomyces growth organisms indicates the secreting protease enzyme exhibit a zone of proteolysis (Suganthi et al., 2013).

Screening for cellulolytic activity

The isolates of Streptomyces species were center streaked and grown at 28°C for 7 days on CMC agar medium with the composition, g/L: carboxymethyl cellulose 10.0, potassium nitrate 2.0, potassium dihydrogen phosphate 4.0, disodium phosphate 4.0, calcium chloride 0.001, magnesium sulfate 0.2, ferrous sulfate 0.004, agar 15.0, DW (50% v/v) 1,000 ml, pH 7.0 ± 0.2. After incubation, the plates were analyzed for cellulolytic ability by flooding with 1 mg/ml of congo red solution for 15 min. Decant the dye, and re-flood the plates with NaCl (1 M) for 15 min. Positive colonies are detected to be surrounded by a pale orange to clear zone against the red background confirmed the cellulolytic activity (Shaik et al., 2017).

Screening for L-asparaginase activity

The isolates of Streptomyces species were center streaked on the asparagine dextrose salts agar plates (ADS agar) with the composition, g/L: L-asparagine 10.0, dextrose 2.0, dipotassium phosphate 1.0, magnesium sulfate 0.5, agar 15.0, seawater (50% v/v) 1,000 ml, pH 7.0 ± 0.2, phenol red 0.009% (in ethanol). After 7 days of incubation at 28°C, a change in the color of the medium from yellow to pink indicates the extracellular L-asparaginase production by the actinomycetes isolates (Shaik et al., 2017).

Statistical analysis

The significant difference in mean value of zone of inhibition of Streptomyces isolates against 4 bacterial strains were analyzed by independent sample t-test and significant difference in mean of IC₅₀ values of antioxidant activities of isolates against both ABTS and DPPH was analyzed using one-way ANOVA by SPSS version 26.

Isolation and identification of Streptomyces species

Soil sample collected from Gosaikunda located in Rasuwa District of Nepal was used for the screening of the Streptomyces species. The selective ISP2 agar medium supplemented with nalidixic acid and cycloheximide was used to prevent the growth of Gram-negative bacteria and fungus strains. During the screening, microbial colonies showing distinct morphology were selected for further characterization. The isolates were identified as Streptomyces based on the mycelial growth and cellular morphology as shown in Fig. 1. The culture characteristics of the isolated species were analyzed in ISP2 and R2YE medium. All these four isolates revealed significant microbial growth as well as secondary metabolites formation evident by distinct pigment formation.

Fig. 1. Colony morphology of the isolated Streptomyces species from soil samples of Goshaikunda Lake, Nepal. Culture agar plate were incubated at 28°C for 14 days in R2YE medium.

Molecular identification and phylogenetic analysis of isolated Streptomyces species

Molecular characterization of the isolates was carried out using 16S rRNA sequence analysis. The PCR amplified 16S rRNA were sequenced using the set of universal primer 27F and 1429R and amplification products were visualized using 0.7% agarose gel electrophoresis. The 16S rRNA sequence analysis revealed that four isolated strains, G-10, G-14, G-18, and S4L belong to the Streptomyces species. The sequence of the four isolates was deposited in the NCBI GenBank. The local alignment with the NCBI nucleotide database revealed the sequence similarity of the isolated species with the partial 16S rRNA sequences of deposited Streptomyces species in the database (Table 1). Further, phylogenetic tree analysis of 16S rRNA gene sequences using neighbor-joining methods, revealed that G-18 isolates closely related to Streptomyces albidoflavus strain PAS-12. Whereas, G-14 closely related to Streptomyces species GS13 and S4L closely related to Streptomyces species strain QJSt1 (Fig. 2). Streptomyces are one of the taxonomically complex groups of species, the majority of the species share similar 16S rRNA sequences. Further, most of the available databases contain the partial 16S rRNA sequence, which might not be sufficient enough to predict the new species. Moreover, sequence variations between multiple 16S rRNA gene copies can also exist within the same species. Hence, multilocus sequence analysis based on housekeeping genes and chemotaxonomic characterization are required to identify the new species.

Table 1

16S rRNA sequence analysis of four isolated Streptomyces species with the selected Streptomyces species in NCBI database

Isolates (Gene bank)	Match in the database	% Identity	Accession number
G-10 (MW663768.1)	Streptomyces champavatii strain GMR01	100	MK134629.1
	Streptomyces sampsonii strain ATCC 25495	100	MH196556.1
	Streptomyces graminearus strain NM1	99.79	MT071574.1
G-14 (MW663770.1)	Streptomyces species GS13	99.93	MN052663.1
	Streptomyces coelicoflavus strain 205517-2	99.79	JX465725.1
	Streptomyces species strain EYSt4	99.78	KX950858.1
G-18 (MW663767.1)	Streptomyces violascens strain EA28	99.71	KU973983.1
	Streptomyces albidoflavus strain PAS-12	99%	KP122209.1
	Streptomyces species strain EYSt4	99.84	KX950858.1
S4L (MW663769.1)	Streptomyces species strain QJSt1	99.84	KX950900.1
	Streptomyces violascens strain WZS031	99.76	MH482912.1
	Streptomyces koyangensis strain VK-A60T	99.84	CP031742.1

Fig. 2. Neighbor-joining phylogenetic tree of 16S rRNA from four isolates. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1,000 replicates).

Antimicrobial activities

The isolated strains of Streptomyces revealed the range of antimicrobial activities with the zone of inhibition varies from 9 mm to 21 mm (Fig. 3). The isolates G-10 showed a relatively higher zone of inhibition toward Bacillus substilis ATCC 6051, whereas G-14 extract toward Staphylococcus aureus ATCC 12600. Among the analyzed extract of the isolates, S4L revealed higher inhibitory potential toward Enterococcus species. We performed the independent sample t-test with a 95% confidence interval. Statistical analysis showed a significant difference in the mean zone of inhibition of individual Streptomyces isolates against all four bacterial strains except for S4L against Klebsiella pneumoniae and Bacillus substilis. It is proven that most of the actinomycetes species isolated from different environments produced bioactive secondary metabolites. Maiti et al. (2020) have reported that the Streptomyces species SM01 isolated from soil sample of Kashmir, India produces the novel antibiotic Picolinamycin and revealed strong antimicrobial activities against pathogenic strains such as Staphylococci and Enterococci with minimum inhibitory concentration (MIC) value of 0.04 to 5.12 µg/ml. It clearly indicates that, isolated Streptomyces from high altitude Himalaya could be the potential source of new antimicrobial drugs

Fig. 3. Antimicrobial activities of Streptomyces species isolated from soil sample. The inhibitory potential was measured as zone of inhibition (mm). All the experiments were carried out triplicates and values are presented as mean ± SD. Mean values within a single bacterial strain with different letters (a–d) are significantly different for p < 0.05, Independent sample t-test.

(Maiti et al., 2020). Most of the bioactive secondary metabolites isolated from Streptomyces species act as antibacterial agents, antifungal, anticancer agents, immunosuppressants, and antiparasitic agents (Salwan and Sharma, 2020). Furthermore, it has been reported that among the bacterial produced known commercial antibiotics, two-third is reported from Streptomyces species alone (Fair and Tor, 2014). Some of the commercially used antibiotics such as Cephalosporin, Tetracycline, Streptomycin, Neomycin, Kanamycin, and Chloramphenicol are derived from S. clavuligerus, S. aureofaciens, S. griseus, S. fradiae, S. kanamyceticus, and S. venezuelae for the treatment of different human ailments (Quinn et al., 2020). Despite the success, the demand for new antibiotics is increasing as many pathogens are showing upsurging resistance to the existing antibiotics. Hence, lots of scientific efforts are ongoing for the search of novel antibiotics through natural resources. Further purification and identification of the secondary metabolites from these species are important to characterize the isolated Streptomyces species and their biochemical potentials.

Antioxidant activities

Antioxidant activities of the ethyl acetate extract of four isolated Streptomyces species were analyzed using DPPH and ABTS assay. The scavenging activities of the extract are concentration dependence as shown in Fig. 4A and B. Results revealed a range of antioxidant activities with IC₅₀ values 221.75 to 511.32 µg/ml using DPPH assays and 137.73 to 192.27 µg/ml using ABTS assays (Table 2). Results were compared with standard gallic acid and found to be significantly different with IC₅₀ values of extracts. Our results showed that the isolated Streptomyces species are the potential source of antioxidant compounds. Secondary metabolites produced by the isolated Streptomyces species might be responsible for the antioxidant activities.

Table 2

IC₅₀ values for antioxidant activities of the isolated Streptomyces species measured using DPPH and ABTS assay

Samples	DPPH (µg/ml)	ABTS (µg/ml)
G-14	221.75 ± 8.17b	192.27 ± 4.74b
G-10	511.32 ± 39.85c	149.09 ± 3.60c
S4L	415.75 ± 32.01d	137.73 ± 0.58d
G-18	480.44 ± 119.17e	181.18 ± 26.89e
Reference (Gallic Acid)	5.512 ± 0.126a	1.967 ± 0.056a

All experiments were carried out triplicates and values are presented as mean ± SD. Mean values within a column with different letters (a–e) are significantly different for p < 0.05, One-way ANOVA, Post-hoc Tukey multiple comparison test.

Fig. 4. Radical scavenging percentage of the isolated Streptomyces species (A) DPPH scavenging activities and (B) ABTS scavenging activities.

Screening of the enzyme

Enzymes produced by the Streptomyces species are of industrial importance. A wide range of enzymes such as amylase, cellulase, L-asparaginase, and protease are of commercial importance. Amylase and cellulase are used in pharmaceutical, laundry, food, fermentation, textile, paper, and pulp industries (Vermelho et al., 2012). Whereas, L-asparaginase enzymes produced from the microbial hosts are used for the treatment of human cancers (Muneer et al., 2020). Although many species are known for the production of these enzymes, strains isolated from different geographic niches are important for the expression of these enzymes in variable conditions. The isolated Streptomyces species were subjected to the screening of the industrially important enzymes in a suitable agar medium as described in ‘Material and Methods.’ Our result revealed that among the four isolates, G-18 and S4L revealed significantly higher production of amylase enzymes. Whereas G-18 isolate also revealed higher cellulase activity. Furthermore, G-14 and S4L isolates shows L-asparaginase activity. However, in our preliminary analysis, none of the strains show protease activity (Table 3). Our results open up the possibility in the future to optimize the production of these enzymes as well as identification of genes responsible for the higher production of these enzymes.

Table 3

Qualitative test for the screening of the enzyme by the isolated Streptomyces species

Isolates	Amylase	Protease	Cellulase	L-Asparaginase
G-10	+	-	-	-
G-14	-	-	+	+
G-18	+++	-	+++	-
S4L	+++	-	-	+

Where +, ++, and +++ are relative higher activities, whereas (-) indicates no activity.

Four Streptomyces species were isolated and characterized as Streptomyces species based on 16S rRNA sequence analysis. The crude extract of the isolates revealed good antioxidant and antimicrobial activities. Furthermore, these isolates are an important source of commercial enzymes such as alpha-amylase and cellulase. We noticed that 16S rRNA sequence analysis might not be sufficient to identify the species. Hence, whole-genome sequencing and chemotaxonomic analysis are required for the identification of the species. Future research should direct the identification of bioactive metabolites and their potential application.

This research was supported by grant support from University Grant Commission, Nepal (Award No. FRG'73/74'S&T'10). We would like to acknowledge Prayon Joshi, Anil Upreti, Muna Fuyal, Bibek Byanju, Apekshya Chettri, and Dr. Dipesh Dhakal for their contribution in preliminary works.

The authors have no conflicts of interest to report.