Streptomyces spiroverticillatus from the rhizospheric soil of Arnebia euchroma: its
antimicrobial and anticancer potential
M. Katoch*1, S. Bhushan2, A. Katoch, P. R. Sharma2, S. Kitchlu3
Division, 2 Cancer Pharmacology Division, 3 Biodiversity and Applied Botany
Indian Institute of Integrative Medicine (CSIR), Canal Road, Jammu-180001, India
1Biotechnology
Abstract
Streptomyces spiroverticillatus has been isolated from the rhizospheric soil of Arnebia
euchroma which has been known for its ethano-botanical uses. The isolate was characterized
by scanning electron microscopy and showed small hypae and long cylindrical smooth spores.
Interestingly, the isolate showed antibacterial activity against Gram positive bacteria (MRSA,
VRE) and antifungal activity against candida albicans. It also showed cytotoxic activity against
HL60 cells with the IC50 value 10 µg/ml. The bioactivity of each microbe was extractable in
various organic solvents. Molecular biological studies on the 16S rRNA gene sequence of the
isolate revealed that it is distinct from other genetic accessions of streptomycetes.
Key words: Streptomyces spiroverticillatus, antimicrobial activity; MTT; rRNA
INTRODUCTION
Actinomycetes are a diverse group of filamentous gram-positive bacteria well known
for their production of extensive array of chemically diverse and medicinally important
secondary metabolites [1, 2]. The plant-actinomycete interaction is a mutualistic relationship
whereby the plants provide nutrients to the actinomycete while the actiniomycete produces the
metabolites that enhance plant growth and protect the plant from invasive root pathogens [3].
Among Actinomycetes, the members of the genus Streptomyces are considered economically
*Meenu Katoch, Department of Biotechnology
Indian Institute of Integrative Medicine, Canal Road, Jammu
Email: meenusamiksha@rediffmail.com; mkatoch@iiim.ac.in
Phone No. +91 09419157224
important because they alone constituted 50% of actinomycetes population and produced 7080% of total bioactive molecules [4].
Cancer is the second common cause of death in world. Prolonged use of broad-spectrum
antibiotics has led to emergence of drug-resistant pathogens, both in medicine and agriculture.
New threats like drug-resistant pathogens, cancer and the pharmacological limitations of drugs
have increased the exigency of novel, less toxic and efficacious antimicrobial/ anticancer
agents. To develop an effective therapeutic agent, microbial isolates, cultured from nature
should be screened for a new antibiotic or anticancer agent. The secret to continued success is
the examination of hitherto under-explored habitats.
These filamentous bacteria also exist in soil/rhizospheric soil and produce active
compounds [5]. Because the plant kingdom is so large, a selection of plant to study its
rhizospheric streptomyces also might be based on an ethano-botanical approach. Rhizospheric
soil of Arnebia euchroma was chosen as a source for isolation of actinomycetes. Its roots are
antipyretic, cancer, contraceptive, emollient and vulnerary. It is used in the treatment of
measles, mild constipation, burns, frostbite, eczema, dermatitis etc. Experimentally it has
shown contraceptive action on rats, inhibiting oestrus, the fertility rate and the release of
pituitary gonadotrophin hormone and chorion gonadotrophin hormone. It inhibits the growth of
cancer cells on the chorionic membrane. The root contains shikonin, an antitumour and
bactericidal compound. It inhibits the growth of E. coli, Bacillus typhi, B. dysenteriae,
Pseudomonas sp. and Staphylococcus aureus. Shikonin also promotes the healing of wounds
on topical application [6].
Streptomycetes associated with plant, may produce some metabolites which are not
toxic to its associated organism. Thus one of the major concerns in drug discovery, relating to
the toxicity of a drug candidate, may be averted by dealing with these streptomyces and their
2
biological products. In the present study the rhizospheric soil of Arnebia euchroma was chosen
because its roots have medicinal values like antibacterial, antipyretic, anticancer, contraceptive,
emollient and vulnery properties. In present study, an attempt was made to isolate the
actinomycetes from rhizospheric soil of Arnebia euchroma and explore them for biological
activities.
MATERIAL AND METHODS
Site and Sampling Procedures
The rhizospheric soil of Arnebia euchroma (Royle) Johnst was collected from
Rangdum, Zhanskar Valley of Laddakh, Jammu & Kashmir, India, located at 34.06°N 76.35°E
(an altitude of 3675 m) for this study. No history exists indicating that the plant had ever been
previously studied for microorganisms. These samples were immediately placed in plastic bags,
labeled and taken to the laboratory and either stored in 4ºC or processed within three days of
collection.
Actinomycetes isolation
Actinomycetes isolation was mainly carried out as described by Hayakawa [7]. Dry soil
(1 g) was stirred with 100 ml of sterile distilled water containing 2-3 drops of Tween-80 in the
shaker at 40 rpm for 1h. Well mixed 0.2 ml samples of different dilutions from 10-1 to 10-5 (in
sterile deionized water) were plated in triplicate on to different microbiological media such as
the Starch Casein Agar (SCA) and Water yeast extract (WYE) agar. WYE agar consisted of
yeast extract (0.25 g/L) as a sole carbon and nitrogen source, plus agar (18 g/L). The medium
was buffered with K2HPO4 (0.5 g/L) and adjusted to pH 7.2 prior to autoclaving. Nalidixic acid
(20 µg/ml) and cyclohexamide (100 µg/ml) were added to the media to reduce fungal and
bacterial contamination. Plates were incubated at 28°C for 15 days. Isolated pure colonies were
preserved on Bennett’s agar slants at 4°C. After the proper incubation of the plates, seven day
3
old cultures were preserved by placing pieces of filamentous growth in 15% glycerol and
storing at -70°C. All the media and chemicals were obtained from Hi Media. The cultures were
also submitted to the IIIM Microbial repository for lyophilization and preservation.
Characterization of Actinomycetes by SEM
The culture was observed through SEM. The methods mainly referred to Castillo et
al., [8] with slight modifications. The specimen was first fixed with 2.5% glutaraldehyde in
phosphate buffer (PBS, pH 7.0) for 24 h at 4°C, post fixed in 1% (w/v) osmium tetroxi (OsO4)
in PBS for 4 h. The specimens were dehydrated by a graded series of acetone (50, 70, 80, 90,
95, and 100%) for about 15-20 min at each step, and cleared with amyl acetate. The
dehydration process was done slowly to discourage hyphal shriveling. Ultimately, the samples
were dehydrated in critical point dryer (Bio-Rad) with liquid CO2 and coated with gold using
sputter coater (Polaron). Scanning images of the surface of the specimen were recorded with
JEOL 100 XII electron microscope with ASID at 40KV.
16s rRNA gene sequencing
Total genomic DNA was extracted using a modified cetryl-trimethylammonium
bromide (CTAB)-NaCl protocol [9]. A nested PCR was employed to identify the isolated
cultures [10]. The primers of the first PCR were 101F (5’-GTT TGA TCC TGG CTC AGG
AC – 3’) and 102R (5’ – GGT GTT CCT CMH GAT ATC TG – 3’), while those of the second
PCR were 103F (5’ – GAA CGC TGG CGG CGT GCT- 3’) and 104R (5’ - GCG CAT TYC
ACC GCT ACA CC -3’). The reaction was performed in a final volume of 20ul containing 20
ng of DNA, 10 pmol of each primer, 200 µM dNTP mix, 1 U of Taq DNA polymerase
(Bangalore Geneii, India) with reaction buffer supplied by the manufacturer. Amplifications
were then performed in Gene Amp PCR system 9700 (ABI, Applied Biosystems) according to
the following profile: 1 min at 95oC and 14 cycles of 30 s at 94oC, 30 s at 65oC and 30 s at 72
4
o
C and 21 cycles of 30 s at 94oC, 30 s at 58oC and 30 s at 72oC, followed by 2 min at 72oC final
extension. The second PCR reaction was annealing temperature 84oC for first 14 and 79oC for
last 21 cycles. The PCR product was analyzed in a 2% agarose gel and purified from the gel
using the gel extraction kit (Qiagen). Direct purified PCR-amplified DNA was sequenced using
an automatic DNA Sequencer (310 Genetic Analyser; Applied Biosystems, Foster city, CA).
The 16S rRNA gene sequence submitted to gene bank (Accession no. JX570583) was
compared with the available sequences from GenBank using the blast program
(http://www.ncbi.nlm.nih.gov/BLAST/) to determine approximate phylogenetic affiliations.
Fermentation and extraction
Each isolate was cultured in starch casein medium at 28°C and was shaken at 180 rpm
for 10-12 days. After 7-12 days of cultivation, the fermentation broth of the culture was
homogenized with 10% methanol, extracted thrice with methylene chloride (DCM). After
DCM extraction, culture was extracted with ethyl acetate (EA). Process was repeated three
times and extract was pooled together. Each extract was then evaporated under reduced
pressure to yield dried extract. Dried extract was dissolved in DMSO (dimethyl sulphoxide) at
a concentration of 100 µg/µl. Remaining culture was filtered and filtrate termed as water
extract. Water extract was freeze dried and dissolved in water at a required concentration. All
these three extracts were used for different bioactivities.
Antimicrobial Bioassay
The test organisms used were the Gram positive bacteria Staphylococcus aureus
(MRSA 15187), Enterococus (VRE) (ATCC700221) and the Gram negative bacteria
Pseudomonas aeruginosa (ATCC 27853). The test yeast/fungus used was Candida albicans
(ATCC10231). By Agar well-diffusion method extracts at 500 µg/well were tested for both
antibacterial and antifungal activity [11]. Ciprofloxacin (5 µg/well) and Amphotericin-B (1
5
µg/well) were used as a standard antibacterial and antifungal agent respectively in this study
whereas di-methyl sulphoxide (DMSO) was used as negative control.
Determination of Anticancer activity
This assay is a quantitative colorimetric method for determination of cell survival and
proliferation [12]. For in-vitro cytotoxic activity, Central nervous system-SF295, Lung A-549,
and Leukemia-THP-1 cancer cell lines were procured from National Centre for Cell Sciences
(NCCS), Pune, India. Cells were grown in RPMI-1640 medium containing 10% FCS, 100U
penicillin/100 µg per mL streptomycin of medium in CO2 incubator (Thermo-con Electron
Corporation, USA) at 37°C with 98% humidity and 5% CO2 gas environment. The cells were
plated in 96-well plates at a density of 2.0 x 104 in 200 µL of medium per well. Cultures were
incubated with different concentrations of test extract (100 µg per mL) and incubated for 48 h.
The medium was replaced with fresh medium containing 100 µg per mL of 3-(4, 5dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) for 3h. The supernatant was
aspirated and MTT-formazan crystals were dissolved in 200 μL DMSO and the OD of the
resulting solution was measured at λ540nm (reference wavelength, λ620nm) on ELISA reader
(Thermo Labs, USA). Cell growth was calculated by comparing the absorbance of treated
versus untreated cells. All vehicle controls will contain the same concentration of DMSO.
Clinical drugs like 5-Fu, Paclitaxel, Adriamycin were included as positive controls. IC50 value
will be calculated by Curvfit software.
RESULT AND DISCUSSION
Isolation of actinomycetes
Rhizospheric soil of Arnebia euchroma was chosen as a source for isolation of
actinomycetes. The pure and isolated colony appeared on day seven were transferred to SCA
medium plates and coded as A3E. The actinomycetes showed yellowish brown colonies when
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culture was young and later appeared dark colored (Fig1). SEM studies of A3E culture showed
smooth surfaced cylindrical long spores. Various novel streptomyces viz. Streptomyces
marokkonensis, Streptomyces thinghirensis were isolated from rhizospheric soil of different
plants [13, 14]. This is the first report of isolation of Streptomyces from rhizospheric soil of
Arnebia euchroma.
The 16S rRNA analysis has proved to be a very important tool in Streptomyces
systematic, also helpful in assigning the newly isolated strain to the genus Streptomyces [11].
Blast analysis of the 16S rRNA nucleotide gene sequence of strain A3E (600bp) with the
corresponding Streptomyces sequences clearly showed that the organism form a distinct
position between the Streptomyces spp. (Fig 1). The isolate was closely related to the type
strain of Streptomyces spiroverticillatus (Gene bank Accession Number DQ487019) sharing a
16S rRNA gene sequence similarity of 99% which was also supported by high bootstrap value
(98). The phylogenetic analysis suggested that S. spiroverticillatus from NBRC collections are
closer with S. cinnamonensis, whereas sj33 strain of S. spiroverticillatus is entirely different
with other strains of S. spiroverticillatus. The results support the classification of the isolate
A3E as a novel strain.
Biological activity
Antibacterial and antifungal activity
A3E was grown in culture and then the culture was mixed with 10% methanol and
homogenized. Homogenized culture was extracted with di-chloromethane, Ethyl acetate and
water. All the three extracts of A3E were used for antibacterial and antifungal activities against
gram positive and negative pathogens (MRSA, VRE) and fungal pathogen candida albicans
(Table 1). DCM extract of A3E showed only antifungal activity whereas ethyl acetate extract
showed both antibacterial as well as antifungal activities. The EA extract showed 20 mm and
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18 mm zone of inhibition against gram positive bacteria MRSA, and VRE strains respectively
whereas against candida albicans, it showed 12 mm zone of inhibition.
Previously Streptomyces spiroverticillatus has been isolated from the soil of China,
which is a source of a new antibiotic Tautomycin [15, 16]. Tautomycin is inhibitory to various
plant pathogenic fungi, yeasts and limited species of gram-negative bacteria. In contrary,
present study showed zone of inhibition against gram positive bacteria (MRSA, and VRE
strains). These results suggesting that present strain might be producing some other molecule,
different from tautomycin.
Anticancer activity
The in-vitro cytotoxic activity of crude extract against HL-60 cancer cell lines was
determined by MTT assay. Monolayer culture of HL-60 cells was exposed to 10, 30, 100 µg/ml
concentration of each extract. At these concentrations, DCM extract showed 38%, 18% and
17% viable cells respectively (Fig 2). The IC50 value of DCM extract was found to be 10 µg/ml.
Ethyl acetate extract and water extract did not show any cytotoxic activity.
Tautomycin isolated from Streptomyces spiroverticillatus also inhibited the spreading
of human myeloid leukemia cell HL60 induced by phorbol ester at the concentration of 0.030.3 µg/ml. Similarly, present study showed in-vitro cytotoxic activity of DCM extract against
HL-60 cancer cell lines (un-induced). Tautomycin was extracted in ethyl acetate [15]. The
results of present study suggested that DCM extract had cytotoxic property, whereas ethyl
acetate extract did not show any cytotoxic activity suggesting that this activity is not because of
Tautomycin but some other molecule extractable in DCM.
The results showed that this novel streptomyces do have interesting and potentially
useful biological activities that are extractable with various organic solvents. The relationship
to the original ethnobotanical uses, at this point, is still unclear. However, this work will thus
8
serve as a prelude to more comprehensive studies on the chemistry and biology of the bioactive
natural products produced by this streptomyces and it can be further examined to learn if it can
be used as new pharmacological agent.
Acknowledgements
Authors are grateful to the Director, Indian Institute of Integrative Medicine (CSIR)
Jammu, India for giving platform, financial and technical support for accomplishing this
present research work.
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Table 1: Antimicrobial activity of A3E
S.
No.
1
2
3
4
Pathogens Tested
Bacterial
Fungal
MRSA
VRE
P. aeruginosa
C. albicans
DCM
0
0
0
10
Zone Diameter (in mm)
A3E
EA
W
20
0
18
0
0
0
12
0
Drug
Control
0
0
32
20
12
Fig 1 Unrooted Phylip Tree based on 16S rRNA gene sequences showing the position of
strain A3E and related Streptomycetes species. Numbers on nodes are bootstrap values (%).
13
Fig 3 MTT assay result of A3E as graph and IC50 value was shown on it.
100
90
% Cell viability
80
70
60
50
40
30
20
10
0
0
10
20
30
40
50
60
70
80
90
100
14