INTRODUCTION:

Antibiotics, the natural wonder drugs are one of the greatest benefits to mankind. The bacteria from clinical settings are becoming increasingly resistant to standard antibiotics. The ever increasing resistance of antibiotics is mainly due to the widespread and uncontrolled application of antibiotics^[1]. Nature acts as a fruitful treasure for novel therapeutics. Natural products and bioactive secondary metabolites are the key sources to new and novel therapeutics in our society. Microbial metabolites represents diverse array of natural therapeutic agents. The filamentous actinomycetes are the most frequent and versatile producers of bioactive metabolites. Actinomycetes are Gram positive, free-living, saprophytic bacteria capable of producing more than 10,000 bioactive compounds. The Streptomyces genera contribute 50% of bioactive compounds from the total population of soil actinomycetes^[2].

Received on 17.01.2015 Modified on 24.01.2015

Research J. Pharm. and Tech. 8(3): Mar., 2015; Page 300-309

DOI: 10.5958/0974-360X.2015.00050.5

The Sterptomyces genus exhibit a great numbers of diverse and versatile biological active compounds possessing antitumor^[3], antibacterial^[4], antiparasitic^[5], antifungal and immunosuppressive^[6]. In the search for new microbial metabolites, the isolation of novel Streptomyces species is in great need^[7] as they are prolific source of bioactive metabolites. It is very important to explore new areas to isolate novel species for antibiotic research^[8]. More than 80% of bioactive metabolites have been produced by soil actinomycetes. There are other remaining unexplored terrestrial areas for new and novel antibiotic research. The isolation of potent Streptomyces species producing bioactive compounds employs several approaches including cultural methods, genomic and metagenomic analysis. Pre-treatment of soil samples with calcium carbonate^[9], phenol^[10]. Yeast extract and sodium dodecyl sulfate^[11] are culture dependent methods used extensively to isolate potent actinomycetes strain.

In the era of drug discovery, molecular docking plays an imperative role in designing of drugs which implies the prediction and orientation of one molecule to a second when bound to each other to form a stable complex. According to Hamed et al.^[12]and Maria et al.^[13]molecular docking has been described as an optimization problem, which would describe the ‘‘best-fit’’ orientation of a ligand that binds to a particular protein of interest. In the search for novel terrestrial actinomycetes, Streptomyces xanthochromogenes JAR5 strain possessing good antimicrobial activity among forty isolates of unexplored Kodaikanal forest has been isolated using calcium-carbonate pre-treatment method. The present investigation reveals isolation, extraction, characterization, biological evaluation and in silico molecular docking studies in accordance to in vitro antibacterial activity of bioactive metabolites produced by Streptomyces xanthochromogenes JAR5.

Materials and Methods:

Sample collection

Soil samples were collected from different sampling sites (deep forest soil, highly polluted area, manure soil) in Kodaikanal forest, Tamil Nadu, India in the month of January 2012. The samples were brought to laboratory in sterile bags and were pre-treated with calcium carbonate for 7-10 d.

Isolation

Streptomyces xanthochromogenes JAR5 was isolated from CaCO₃pre-treated soil samples on starch casein nitrate agar medium (soluble starch 10 g^-1, casein 0.3 g^-1, NaCl 2 g ^-1, KNO₃2 g^-1, K₂HPO₄ 2 g^-1, MgSO₄.7H₂O 0.5 g^-1 , CaCO₃ 0.02 g^-1, FeSO_4.7H₂O 0.01 g^-1) at pH 7.0 , incubated at 28°C for 10 d. The isolated strain was further maintained on yeast extract-malt extract-dextrose (YMD) agar medium at 4°C^[14].

Phenotypic and taxonomic studies

The cultural characteristics of mature sporulating aerial and substrate mycelium of Streptomyces xanthochromogenes JAR5 were observed after 14 d of incubation on International Streptomyces project (ISP) and non-ISP medium described by Shirling and Gottlieb^[15]. The utilization of different carbon sources by the isolated strain was examined using Gottlieb^[16]protocol. The melanin production was observed on peptone-yeast extract iron agar (ISP medium 6) and tyrosine (ISP medium 7) agar. The sensitivity of isolated strain was determined against various antibiotics using Kirby-Bauer method Cappuccino and Sherman^[17]. The micro morphology of the strain JAR5 cultured on ISP-2 medium for 7 d at 28°C was examined by scanning electron microscopy (HITACH, Model S-3400N) coated with gold to avoid charging^[18].

Genotypic characterization

The genomic DNA extraction of Streptomyces xanthochromogenes JAR5 was performed by using Rainey et al.^[19]protocol. The amplification of 16S rRNA gene was carried out by using forward primer of 400 ng 5′-AGAGTRTGATCMTYGCTWAC-3′ and reverse primer of 400 ng 5′- CGYTAMCTTWTTACGRCT-3′, 2.5 mM each of dNTPs, 10X Taq polymerase assay buffer and Taq DNA polymerase enzyme keeping the reaction volume upto 100 µl. The amplification reaction was further followed by initial denaturation at 94°C for 5 min to improve the denaturation of the DNA 5% (v/v) DMSO was added to the reaction mixture. After denaturation, annealing at 55°C for 30 s was carried out leading to final extension at 72°C using MgCl₂with 1.5 mM final concentration. The amplified product was sequenced with the primer using ABI 3730xl genetic analyzer (Amnion Biosciences Pvt. Ltd.). The phylogenetic position of the Streptomyces xanthochromogenes JAR5 was determined by performing a nucleotide sequence database search using the BLAST program from National Centre for Biotechnology Information (NCBI) GenBank. The nucleotide sequencing result was submitted to the GenBank NCBI and accession number obtained was KC509578.

Optimization of culture medium

To determine the ability of strain to produce active metabolites in the presence of various carbon and nitrogen sources was examined using inorganic salt medium composed of (NH₄)₂SO₄ 2.64 g^-1; KH₂PO₄ 2.38 g^-1; MgSO₄.7H₂O 1.00 g^-1; CuSO₄.5H₂O 0.0064 g^-1; FeSO₄.7H₂O 0.0011 g^-1; MnCl₂.4H₂O 0.0079 g^-1; ZnSO₄.7H₂O 0.0015 g^-1 at pH 8^[20].250 ml of Erlenmeyer flask containing 50 ml of basal medium was sterilized using autoclave. The different carbon sources including glucose, lactose, starch, sucrose, maltose, mannitol and nitrogen sources including peptone, sodium nitrate (NaNO₃), ammonium chloride (NH₄Cl), yeast extract, casein and soybean meal were added to basal medium at 1% of concentration. Spore suspension was prepared from 10 d well grown culture of Streptomyces xanthochromogenes JAR5 in 0.05% of Tween 20 solution. 5% of spore suspension was added to 50 ml basal medium supplemented with various carbon and nitrogen sources and were incubated on rotary shaker at 28°C for 15 d.

The inorganic salt medium without carbon and nitrogen sources served as control. The biomass was of bioactive metabolite was recorded at 600 nm (optical density) and antimicrobial activity was determined against clinical pathogens after 3, 6, 9, 12 and 15 d.

Test organisms

The Gram negative bacteria including Escherichia coli, Shigella sp., Proteus mirabilis, Pseudomonas aeruginosa, Klebsiella pneumonia, Salmonella sp. and Gram positive bacteria Staphylococcus aureus, Enterococcus sp. and fungal strains including Candida tropicalis, Fusarium sp., Aspergillus terreus strain JAS1, Scedosporium sp. JAS1, Ganoderma sp. JAS4 were procured from Microbial Biotechnology Laboratory, SBST, VIT University, Vellore, India. Bacterial and fungal clinical isolates were maintained on Nutrient agar and Potato Dextrose Agar respectively. The antibiosis studies of clinical and plant pathogenic isolates were determined against standard antibiotics vancomycin (30 µg/disc), tigecycline (15 µg/disc), erythromycin (15 µg/disc), ciprofloxacin (30 µg/disc), penicillin (10 µg/disc), ofloxacin (5 µg/disc) and fungal isolates were screened against flucanazole (25 µg/disc) and voriconazole (5 µg/disc) by disc-diffusion method^[17].

Fermentation, isolation and extraction of bioactive metabolites

A loopful culture of Streptomyces xanthochromogenes JAR5 was cultivated in YMD broth as seed medium and incubated in rotary shaker at 220 rpm at 28°C for 48 h. After the incubation of 48 h, 10% of the seed medium was inoculated into the optimized fermentation medium for the production of bioactive metabolites consisting of soluble starch 10 g^-1, casein 0.3 g^-1, soybean meal 10 g^-1 NaCl 2 g^-1, KNO₃2 g^-1, K₂HPO₄ 2 g^-1, MgSO₄.7H₂O 0.5 g^-1, CaCO₃ 0.02 g^-1, FeSO_4.7H₂O 0.01 g^-1 at pH 7.2. The fermentation was carried out for 144 h at 28°C in fermentor with continuous agitation at 260 rpm. The Streptomyces xanthochromogenes JAR5 culture of 2.5 L after 7 d was obtained after the fermentation was completed. The culture filtrate was centrifuged at 2000 × g at 4°C for 10 min. The organic solvent and culture filtrate was vigorously shaken for an hour in separating funnel and kept stationary for another 30 min to separate the aqueous layer. 1.24 g of the brownish gummy metabolic product was recovered from separating funnel. For the separation of active metabolite silica gel column chromatography was performed using CHCl₃: MeOH (90:10) as a mobile phase. The bands were observed on silica gel coated TLC plate in iodine vapor chamber. The active elutes fractions were characterized by UV-Vis absorption spectra, fourier-transform infrared spectroscopy (FT-IR), gas chromatography mass spectrometry (GC-MS).

Biological assays

Primary and secondary screening

The isolated strain Streptomyces xanthochromogenes JAR5 was streaked horizontally onto modified nutrient agar medium consisting glucose 5 g^-1, peptone 5 g^-1, beef extract 3 g^-1, NaCl 3 g^-1, agar 15 g^-1and incubated for 5 d at 28°C^[21].The pathogenic Gram negative and Gram positive bacteria were streaked perpendicular to the isolated strain on the modified nutrient agar plates and then incubated at 37°C for 24 h. The active metabolite inhibition was determined by measuring the zone of inhibition against test organism.

Secondary screening of the isolated strain was examined by Kirby-Bauer method on Muller-Hinton Agar. The Muller-Hinton agar plates were seeded with 100 µl of test organism, 6 mm diameter of four wells were punctured onto agar plates. The active elute with different concentration of 25 µl, 50 µl, 75 µl and 100 µl was added into four wells. The agar plates were further incubated at 37°C for 24 h and zone of inhibition was measured.

Minimum Inhibitory concentration

The minimum inhibitory concentration (MIC) against bacteria clinical pathogens was determined by using Boruwa et al. protocol^[22]. The MIC of bioactive metabolite produced by strain JAR5 was determined in the culture tube containing nutrient broth and the final volume was adjusted to 5 ml. The nutrient broth without active compound served as control. The bacterial pathogens were adjusted to a final inoculums size of 3 × 10⁵ colony forming units (cfu/mL). After inoculation the culture tubes were shaken well and then incubated at 37°C for 24 h and were observed for turbidity. Turbidity was observed in all the tubes including control tubes and to determine the MIC of bacterial pathogens 10 µl content from each tube was spread onto nutrient agar plates at different intervals for 24 h. MIC of bioactive compound was defined as the lowest concentration at which the pathogens were inhibited 100% as against control. The cell survivability rate of inhibited test organism with time dependent studies of the metabolite was determined using Dubey et al^[23] protocol.

Molecular docking studies

In silico molecular docking studies were carried out by Autodock vina 4.2 program which is recently introduced by Scripps Research Institute^[24]. The 3D crystal structure of Topoisomerase II chain A (PDB ID: 3ILWA) was selected and downloaded from PDB (www.rcsb.org/pdb). The PDB 3ILWA is a crystal structure of DNA gyrase^[25].In Autodock vina 4.2, crystallographic water molecules and non polar hydrogen atoms were removed from A chain of Topoisomerase II and was used as receptor where bioactive metabolites produced by JAR5 were used as ligand for docking studies. Topoisomerase A chain was first modified by adding polar hydrogen atoms and kollman charges using Autodock tools (ADT)^[26]. The torsional bonds of ligand were set free by ligand module in Autodock tools- ADT^[26]. Grid points of 40 × 40 × 40 with 0.375 A.U. spacing were calculated around the docking area for all the ligand atom types using Autodock vina 4.2 default optimization parameters. Docking results from each calculation were clustered on the basis of root mean square deviation (RMSD) between the cartesian coordinates of ligands and were ranked according to binding energy. The conformer of each ligand with lowest binding free energy was chosen for docking.

Results and Discussion:

Morphology and cultural characteristics

In the present study, a new terrestrial actinomycetes strain Streptomyces xanthochromogenes JAR5 has been isolated from deep forest of Kodaikanal. The phenotypic characteristics of Streptomyces xanthochromogenes JAR5 was observed after 14 d on ISP1-7 medium. The isolated strain JAR5 developed good aerial and vegetative mycelium on 7 d. The color of aerial mycelium appeared to be creamish white after 6 d of incubation and remain white althrough 14 d whereas substrate mycelium changed color from light to dark violet brown. The cultural characteristics of the JAR5 strain are represented in Table 1. The strain JAR5 produced diffusible pigment on IPS-2, ISP-3, ISP-5, ISP-7 and Maltose-tryptone agar medium. Melanin production was observed on ISP-2, ISP-7 and Maltose-tryptone agar. Figure 1 depicts the spore morphology of the strain JAR5 to be spiny type, the strain can be placed in the spira group of the family Streptomycetaceae and the genus Streptomyces.

Table 1. Morphological and Cultural characterististics of Streptomyces xanthochromogenes JAR5.

S. No.	Culture Medium	Growth	Aerial mycelium	Substrate mycelium	Diffusable pigment	Melanoid Pigment
1	Tryptone-yeast agar medium (ISP-1)	Moderate	White	Pale brown	-	-
2	Yeast extract malt-extract agar (ISP-2)	Very Good	White	Brown	Dark brown	+
3	Oatmeal agar (ISP-3)	Good	White	None	Brown	-
4	Inorganic salt-starch agar (ISP-4)	Good	Pale violet	None	-	-
5	Glycerol asparagine agar (ISP-5)	Good	White	Pale yellow	Voilet	-
6	Peptone yeast iron agar (ISP-6)	Good	NG	Light brown	-	+
7	Tyrosine agar (ISP-7)	Moderate	White	Brown	Dark voilet	+
8	Starch-casein nitrate agar	Good	White	Light brown	-	-
9	Sabourad agar	Poor	White	None	-	-
10	Maltose-tryptone agar	Good	White	Brown	Dark	+

*Note: + Present, - Not Present

Table 2. Phenotypical characteristics of Streptomyces xanthochromogenes JAR5.

S. no.	Utilization of carbon sources	Strain JAR5	Antibiotic	Zone of inhibition in mm
1	D-glucose	P	Tigecycline (15 µg/disc)	S (40)
2	D-sucrose	W	Penicillin (10 µg/disc)	R
3	D-mannitol	P	Streptomycin (10 µg/disc)	S (33)
4	D-lactose	W	Chloramphenicol (30 µg/disc)	S (29)
5	D-Fructose	P	Vancomycin (30 µg/disc)	S (38)
6	Arabinose	W	Gentamicin (10 µg/disc)	S (30)
7	D-xylose	P	Ampicillin (10 µg/disc)	R
8	Maltose	W	Kanamycin (30 µg/disc)	S (33)
9	Inositol	W	Ciprofloxacin (30 µg/disc)	S (30)
10	Rhamanose	W	Erythromycin (15 µg/disc)	S (20)
11	H₂S production	N	Methicillin (10 µg/disc)	R
12	Citrate utilization	N	Tetracycline (30 µg/disc)	S (40)
13	Gelatin	N	Fluconazole (25 µg/disc)	R
14	Urease	N	Voriconazole (5 µg/disc)	S (37)

Note: P-positive, W-weak, N-negative result, R-Resistant, S-Sensitive, mm-millimeters

Fig 2: Phylogenetic relationship based on 16S rRNA gene nucleotide sequences between the Streptomyces xanthochromogenes. JAR5 and reference sequences retrieved from NCBI Gen Bank constructed through the neighbor joining method.

Fig 3(a): Effect of the carbon sources on the antibiotic production by Streptomyces xanthochromogenes JAR5 (b) Effect of the nitrogen sources on the antibiotic production by Streptomyces xanthochromogenes JAR5.

Fig 4(a): UV-spectra of active eluted fraction isolated from the strain JAR5 (b) FT-IR spectrum of active eluted fraction isolated from the strain JAR5.

Fig 5: Mass spectrum of 3-N-Hexylthiolane, S,S-dioxide in active elute isolated from Streptomyces xanthochromogenes JAR5.

Fig 6: Mass spectrum of Phenol,2,4-bis (1,1-Dimethylethyl) in active elute obtained from Streptomyces xanthochromogenes JAR5.

Fig 7: Mass spectrum of 1-Heptacasanol in the active elute of strain JAR5.

Table 3. Antibiogram studies of clinical pathogens.

S. No.	Clinical pathogens	Diameter zone of inhibition (mm) against antibiotic disc
S. No.	Clinical pathogens	Ofloxacin (5mcg/disc)	Erythromycin (15mcg/disc)	Tigecycline (15mcg/disc)	Vancomycin (10mcg/disc)	Ciprofloxacin (30mcg/disc)	Tetracycline (30mcg/disc)
1	E. coli	S (13)	R	S (20)	R	S (18)	R
2	Salmonella sp.	S (20)	R	S (16)	R	S (13)	S (14)
3	S. aureus	S (22)	S (18)	S (26)	R	S (19)	S (17)
4	P. mirabilis	S (25)	R	R	R	R	S (18)
5	Shigella sp.	S (29)	R	S (20)	R	R	S (21)
6	Enterococcus sp.	S (17)	S (23)	R	R	S (19)	R
7	K. pneumoniae	S (19)	R	S (21)	R	S (19)	R
8	P. aeruginosa	S (20)	S (22)	S (18)	R	S (16)	R

Note R-Resistant, S-Sensitive, mm-millimeters, mcg-microgram

The terrestrial actinomycetes are regarded as the most economical and biotechnologically valuable microbial source for drug screening programme in the past fifty years. Among actinomycetes Streptomyces genera are the largest producers of biological active compounds. A new terrestrial actinomycetes strain Streptomyces xanthochromogenes JAR5 has been isolated from deep forest of Kodaikanal. The JAR5 strain exhibited very good growth on ISP-2, ISP-7 and Maltose-tryptone agar medium with melanin production. The aerial mycelium of the strain JAR5 remains white throughout 14 d of incubation representing spiny surface. The strain JAR5 utilized most of the sugars provided indicating the wide pattern of carbon assimilation. The morphological and physiological characteristics were in close agreement to identification pattern of Williams et al^[28]. The strain JAR5 was found to be resistant to antibiotics including methicillin (10 µg/disc), ampicillin (10 µg/disc) and penicillin (10 µg/disc) which indicates that bioactive metabolites produced by the isolated strain may be responsible for the resistance to these antibiotics. The results of antibiotic sensitivity test of strain JAR5 are similar to the finding of Kavitha et al^[18]. The isolated strain was able to tolerate up to 5% of sodium chloride for abundant growth. The strain JAR5 was not able to utilize citrate and showed negative result for H₂S production, urease and gelatin liquefaction. According to Kampfer et al^[27] the biochemical characterization of the isolated strain is indispensable tools for the classification of Actinobacteria. The morphological, phenotypical and biochemical characterization of the isolated strain indicated the isolated strain to be in Streptomyces genera. The strain showed 99% similarity Streptomyces xanthochromogenes based on 16S rRNA gene sequences. S. xanthochromogenes has been reported to produce only DNA-binding compounds. However there is a lacunae regarding the production of antimicrobial compounds from S. xanthochromogenes. In the present investigation, an attempt has been made for the first time to confirm that S. xanthochromogenes JAR5 is a potential microbial source for the production of antimicrobial agents against clinical pathogens and in vitro studies has been verified with in-silico molecular docking studies with topoisomerase II (3ILWA).

The bioactive metabolites were recovered from the optimized fermented broth medium of strain JAR5. The active elute obtained after preparative-TLC was characterized through UV-visible spectrophotometer, FT-IR and GC-MS. The FT-IR of active elute depicted a strong strech at 3450.65cm^-1 represents the phenolic components present in the active elute. The small strech at 1242.16cm^-1 indicates the presence of S=O in the active elute. The presence of C-F bond is indicated by strong strech at 1093.64 cm^-1. Another strong strech at 3186.40cm^-1 suggests the presence of aliphatic ‘CH’ strech. The molecular weight of the compounds present in the active elute were identified through GC-MS by their molecular ion peaks. The mass spectra of the compounds was confirmed by the fragmentation pattern of the respective compounds. The results showed that bioactive metabolites from S. xanthochromogenes JAR5 was successfully able to inhibit Enterococcus sp. and Candida tropicalis. The 80 µg/ml of the active elute was able to inhibit Enterococcus sp. and the cell viability was effected by the bioactive metabolites produced by strain JAR5. The present investigation is the first study with in vitro antimicrobial potential of Streptomyces xanthochomogenes JAR5 with reference to in silico molecular docking studies. In the present work, we have isolated, characterized and have optimized the culture medium for the production of antimicrobial agents from strain JAR5. The partially purified fraction from crude extract of strain JAR5 exhibited good antimicrobial effect against vancomycin resistant Enterococcus sp. with relevance to molecular docking studies. Therefore, furthermore purification studies should be employed to determine the antimicrobial potential of bioactive metabolites separately for the pharmaceutical applications at large scale.

The authors report no conflicts of interest.

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S. No.	Test organisms	Fluconazole (25mcg/disc)	Voriconazole (5mcg/disc)
1	Scedosporium sp. JAS1	R	S (42)
2	Asperigillus tereus strain JAS1	R	S (38)
3	Ganoderma sp. JAS4	R	S (15)
4	Candida tropicalis	R	S (28)
5	Fusarium sp.	S (32)	S (23)