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, 2017. 4Vol 6, Issue www.wjpr.net 116

Qasim et al. World Journal of Pharmaceutical Research

ANTI-TUMOR AND ANTIMICROBIAL ACTIVITY OF ANTIBIOTIC

PRODUCED BY STREPTOMYCES SPP

Bassam Qasim*1 and Mohsan H. Risan

2

1College of Science, Al-Nahrain University, Baghdad-Iraq.

2College of Biotechnology, Al-Nahrain University, Baghdad-Iraq.

ABSTRACT

Actinomycetes produce a wide range of secondary metabolites and

more than 70% of the naturally derived antibiotics that are currently in

clinical use are derived from soil. Actinomycetes and increasing of the

pathogens resistant against antimicrobial agents becomes a health

problem, so the current study was aimed to isolation of local

Streptomyces spp. that is capable of producing secondary metabolite

and investigate their biological antimicrobial and anticancer activities.

All isolates were tested for antimicrobial activities. Among all

suspected Streptomyces (Bag3) was able to inhibit the growth at least

two of tested microorganisms. The MICs potential against

Staphylococcus aureus and Escherichia coli were 8 and 16 µl/ml respectively, the

extracellular crude show strong inhibitory activity against Gram-positive bacteria and

moderate inhibitory activity against both gram-negative bacteria. The antitumor activities

were determined by MTT assay, which indicated Bag3 possesses a powerful effect against

used cancerous cell lines, especially against (Hep G2) by inhibiting 65.75% (400 µg /ml) of

cancer cells after 72 hours of incubation, while against the (SUN16) 42.84% (400 µg /ml)and

against the WRL-68 (normal cell line) 13.43% (400 µg/ml) this indicate the effect of

extracellular crude extract was achieved with concentration -dependent manner (dose

dependent). This means that the isolation bacteria (Bag3) have little effect on normal cells,

because we need high concentrations to effect on the normal cells.

KEYWORDS: Anti-tumor, Antimicrobial, Streptomyces.

World Journal of Pharmaceutical Research SJIF Impact Factor 7.523

Volume 6, Issue 4, 116-128. Research Article ISSN 2277– 7105

*Corresponding Author

Dr. Bassam Qasim

College of Science, Al-

Nahrain University,

Baghdad-Iraq.

Article Received on 31 Jan. 2017,

Revised on 21 Feb. 2017, Accepted on 14 March 2017

DOI: 10.20959/wjpr20174-8121

mailto:[email protected]



INTRODUCTION

Actinomycetes are a group of prokaryotic organisms grouped as gram-positive bacteria with

high guanine and cytosine in their DNA which have linear chromosome, complex

morphological differentiation (Ponmurugan and Nithya, 2008). Actinomycetes are class

Actinobacteria, subclass Actinobacteridae, order actinomycetales comprising of 14 suborders,

49 families and over 140 genera (Adegboye and Babalola, 2012). Actinomycetes produce a

wide range of secondary metabolites and more than 70% of the naturally derived antibiotics

that are currently in clinical use are derived from soil actinomycetes (Elardo et al., 2009). The

Actinomycetes which produced bioactive compounds which including anthracyclins,

glycopeptides, aminoglycosides, macrolides, polyenes, β-lactums, peptides, nucleosides,

terpenes, polyethers and tetracycline’s, which have a broad variety of the biological activities

(Raja and Prabakarana, 2011 ). The most interesting property of Streptomyces is the ability to

produce bioactive secondary metabolites such as antibacterials, antifungals, antivirals,

antitumoral, anti-hypertensives and mainly antibiotics and immunosuppressives (Patzer and

Volkmar, 2010). So, main objective of this study to isolation of Streptomyces strains with

study Antitumor and Antimicrobial.

MATERAILS AND METHODS

Bacterial isolates

Bacterial isolates identified as Streptomyces spp were obtained from soil of Iraq. This isolates

were maintained on Brain Heart Infusion agar medium.

Isolation and identification of Streptomyces

Bacterial isolates were identified according Oskay et al. (2004) 1g of dried and treated soil

samples were used to make suspension, by adding it in 99 ml of sterile distilled water (stock

suspension) and they were shacked in a shaker at 160 rpm for 30 minutes at room

temperature, Serial dilutions from 101 to 10

3 were made from the stock suspension and left

for 10 minutes. After shaking, 0.1 ml of each dilution were culture on Yeast Extract and

Malt Extract (YEME) with Streptomycin 50 ug/ml, then spread by sterile swab for making

uniform distribution of the suspension on the surface of the media. The inoculated plates

were incubated at 28°C for 7 to 10 days. Based on cultural characteristics, suspected colonies

of Streptomyces were selected which are characterized as small, white, pin-point, rough,

chalky and a clear zone of inhibition around them, these colonies was confirmed their

identification by (types of Gram’s stain, aerial and substrate mycelium color, pigment

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production and pigment color). These colonies were transferred from the mixed culture into

separate agar plates and incubated at 28±1°C for 7 days. In order to obtain a pure growth of

Streptomyces were re-streaked on International Streptomyces project (ISP) to obtain pure

colonies used for identification (Nonoh et al., 2010).

Identification of bacteria by VITEK 2 system

The VITEK 2 which is recently installed at the Central Health Laboratories / Ministry of

Health is an automated microbiology system utilizing growth-based technology. A sterile

swab sample used to transfer a sufficient number of colonies of a pure culture and to suspend

them into 3 ml of normal saline (NaCl 0.45%, pH 5-7). Then turbidity adjusted by a turbidity

meter called the Densi Check to match 0.5 – 0.6 McFarland which is the proper inoculum

density for Gram-negative and Gram positive bacteria as stated by the manufacturer (Aziz et

al. 2014).

Streptomyces isolation and identification media

International Streptomyces Project (ISP2) Medium (Shirling and Gottlieb, 1966).

These medium it was prepared by dissolving amount of each component in 1000 ml distilled

water and media were sterilized by autoclaving at 121 C, (15 Ib/in) for 15 min.

The Best Media Composition for Antimicrobial Production

According to Shirling and Göttlieb, (1966) international Streptomyces projects (ISP) and

Khan and patel, (2011), different media were used for isolation and identification of

Streptomyces spp furthermore, to achieving the best types of media composition for

production of antimicrobial metabolites, different media with different composition.

No. Medial name Abbreviation

1 Tryptone-yeast extract broth ISP1

2 Yeast extract-malt extract broth ISP2

3 Inorganic salts-starch broth ISP4

4 Glycerol-asparagine broth ISP5

5 Peptone-Yeast Extract Iron agar ISP6

6 Tyrosine Agar ISP7

7 Glycerol yeast extract broth GYE

Component Quantity (g/l)

Yeast extract 4

Malt extract 10

Dextrose 4

Agar 20



Microbial Pathogens used for Antimicrobial Activities

The pathogenic microorganisms were used as a reference strains for testing the antimicrobial

activities are listed in table below,

Strains Source

Staphylococcus aureus Al-Nahrain university,

Department of Biotechnology

Al-Nahrain university,

Escherichia coli Department of Biotechnology

In vitro Antimicrobial Activities of Isolated Actinomycetes (bag3)

Extracellular crude extract of Streptomyces spp were screened for their antibacterial activity

invitro by well diffusion method (Bagamboula et al., 2004). Using sterile swabs, Mueller

Hinton agar plates inoculated with microbial pathogens and dug wells of 6mm diameter using

Pasteur pipette, the wells and the plates were incubated at 37oC for 24 hours. The plates were

observed for the inhibition zone, which recorded by a metric ruler.

Minimum Inhibitory Concentrations (MICs)

MIC was determined by taking 1ml of nutrient broth into 10 test tubes, with a negative

control and positive control tube, then antimicrobial compounds (extracellular crude extract)

were added into the first test tube and made a serial dilution to obtain a final concentration (1,

2, 4, 8, 16, 32, 64, 125, 250, 500 ul /ml), 50µl of testing microbial pathogens were added into

each test tube (except negative control) and they were incubated at 370 C for 24 hours. MICs

were determined by naked, clear tubes were considered as MIC when compared with both

positive and negative controls, adapted from with little modification (Andrews, 2001).

Streptomyces as anticancer

Cancer Cell Lines

The cell lines were used in the study for screening of cytotoxicity activity of extracellular

crude extract (Bab3) were Hepg2 (Human liver cancer), SUN1 (Human stomach cancer) and

WRL (human normal liver), these cell lines were supplied by Pharmacology

Department/Medicine College/Malayia University.

In vitro Evaluating Antitumor Activity of Extracellular Crude Extract of (Bag3)

Antitumor properties were determined by MTT assay for (Bag3) according to (Cheah et al.,

2011); Saravana Kumar et al., (2014). with little changes in the protocol and the process was

carried out at the Pharmacology Department/Medicine College/Malayia University, against



different cell lines were used such HepG2(Human liver cancer ), SUN1 (Human stomach

cancer) and WRL (human normal liver). Cell lines were seeded in 96-well tissue culture

plates (5000 cells/well), as well as, from lyophilized extracellular crude extract stock

solutions were prepared in sterile distilled water and diluted to the required concentrations by

serial dilutions (400, 200, 100, 50, 25, 12.5, 6.25ug/mL) were done via the cell culture

medium Eagles Minimum Essential Medium (EMEM) containing 10% fetal bovine serum

(FBS), in addition, the (EMEM) were mixed with cancerous cell lines which treated as a

control, however, all treated and none treated sample were done in triplicate trials.

The cells were incubated at 37°C with 5% CO2 and 95% air in 100% relative humidity in

(Thermo Forma Series II Water Jacketed CO2 incubator), After 72 hrs; of incubation the

solution in the medium was removed. An aliquot amount of 100 μl of medium containing 1

mg/ml of 3-(4, 5-dimethylthiazol- 2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT) was

loaded to the plates. The cells were cultured for 4 hrs in a dark chamber for the conversion of

MTT to formazan and then the solution in the medium was removed, well was loaded an

aliquot amount of 100 μl of DMSO were added to the plates, then shaken was done until the

formed crystals were dissolved.

The cytotoxicity against cancer cells line was determined by measuring the absorbance of the

converted dye at 570 nm in a (Hidex Chameleon plate reader). Cytotoxicity of each

concentration was expressed as % inhibition values, and the equation which used for

calculating the inhibition rate of the cells on the basics that the (MTT assay) was reduced,

their absorbance in metabolically active cells to yield an insoluble purple formazan product

as an end product, by calculating the optical density between control well and treated well

with extracellular crude extract as follow.

Cell viability % = (mean OD control x mean OD treated)/ (mean OD control)*100%

RESULTS AND DISCUSSION

Streptomyces producing secondary metabolite that able to kill or inhibit the growth of other

microorganisms the result shown in figure (1) and table (1) reveled that in vitro activities of

antimicrobial for locally isolated Streptomyces (Bag3) has been studied, the highest results

achieved against gram positive Staphylococcus aureus (22mm) inhibition while against gram

negative Escherichia coli which recording only 18mm.



Figure (1): The inhibition zone on locally isolated Streptomyces (bag3) against (A)

Staphylococcus aureus, (B) Escherichia coli.

The obtained results from other research showed the best inhibition zone on gram positive

bacteria Staphylococcus aureus from that of Boudjelal et al., (2011) got 24mm inhibition also

Naine et al., (2015) obtain 24 mm also comparing our results against bacteria Escherichia

coli (gram negative) were less than Gurung et al., (2009) results, which obtained 17mm

inhibition zone; also Attimarad et al., (2012) obtained 25mm inhibition.

Table (1) Estimating minimum inhibition concentrations (MICs) of extracellular crude

extract of isolated strain (Bag3), against different tested microbial pathogens, after 24

hours of incubation at 37 °C

Extracellular Crude

concentration ul /ml

Staphylococcus

aureus Escherichia coli

1 + +

2 + +

4 + +

8 + +

16 + +

32 + Mic +Mic

64 - -

125 - -

250 - -

500 - - (MIC)



NC (broth +crude) - -

PC(broth +pathogen) + +

Bacteriostatic assay + +

Bactericidal assay - -

(NC; Negative control, PC; positive control, +; growth, - ;No growth (inhibition).

Antitumor activity of extracellular crude extract (bag3) by MTT assay

Determining the antitumor activities of extracellular crude extract (bag3) werecarred out

using MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium

reduction assay against different cancerous cell lines, like Hep G2 (human liver cancer),

SUN16 (human stomach cancer) and WRL-68 (human normal liver), through using different

concentrations (400, 200, 100, 50, 25, 12.5 and 6.25 ug/mL) after incubating the cell lines

with the mentioned concentrations for 72 hrs., at 370C the optical density were recorded at, as

well as, the results presented in table (2) summarized the mean percent cell viability with its

standard deviation for all used cell lines.

Table (2) Antitumor activity (cell viability) of extracellular crude extract (bag3) on Hep

G2,SUN16 and WRL-68 cell lines using MTT assay for 72 hrs incubation.

Concentration

ug / Ml

Cell viability%

Hep G2 SUN16 WRL-68

Mean St. Dev. Mean St. Dev. Mean St. Dev.

400 34.25 8.660 57.16 4.272 86.57 2.478

200 54.60 4.401 76.08 1.372 91.30 0.7372

100 78.71 2.191 83.65 4.363 94.61 1.343

50 85.25 5.977 94.06 2.376 95.83 2.183

25 84.22 1.002 96.20 3.466 98.45 0.7675

12.5 97.16 1.701 96.90 1.456 99.14 0.9807

6.25 96.52 2.575 97.70 0.3342 99.30 0.9910

As the results only highlighted concentrations (400, 200, 100, 50, ug/mL) that gave variable

results, while the rest of other results remain somewhat constant values. For all used cell lines

as formerly mentioned by increasing the concentration of the crude extract their cell viability

rate were decreased which means the rate of grown cancer cell lines will be inhibited

(decreased) and can’t be able to convert MTT compound to forming insoluble formazan

product.

The results presented in figure (2) summarized the effects of different extracellular crude

extract concentrations on SUN16 (human stomach cancer), the concentration (400ug/mL)

exhibited the highest result (42.84%) of the SUN1 cells was inhibited which meaning it can’t



forming the final formazan product , however, 57.16 of the cells were remains viable during

the entire incubation process, on the other hand, the lowest results was recorded by (50

ug/mL) that means only (5.94) of the SUN16 cell line were inhibited during the incubation

process.

Results were agreed with that described by Panchanathan et al.,(2014) they obtained 56%

inhibition of SUN16 cell line by the activity of their isolated strain of actinomycetes after 72

hours incubation via using MTT assay.

Figure 2: Antitumor activities of extracellular crude extract (bag3) against SUN16 cell

lines by MTT assay, after 72hrs incubation at 370C.

Results showed in figure (3) represent the antitumor activities of extracellular crude extract

(Bag3) on the Hep G2 (human liver cancer), the highest result (cell inhibition ) was recorded

by (400ug/mL) exactly (65.75%) of Hep G2liver cancer cells were inhibited after 72hr

incubation which meaning only (34.25%) of Hep G2 were able to forming insoluble

formazan products which remaining as a live cells, on the other hand, the (50ug/mL)

concentration recorded the lowest results were (only inhibits 14.75% of the liver cancer

cells).

Concentration

Cel

l v

iab

ilit

y %



Figure 3: Antitumor activities of extracellular crude extract (bag3) against Hep G2 cell


These results were higher than that of Warunee.et al., (2011) they obtained 58% inhibition of

Hep G2 cell line by the activity of extracellular of Streptomyces after 72 hours incubation via

using MTT assay, while our extract inhibit 65.75% of Hep G2 cell line after 72 hours

incubation.

The results in figure (4) summarize the antitumor activities of extracellular crude extract

(bag3) on the WRL-68 (Normal cell line), the highest result cell inhibition was recorded by

400ug/mL exactly (13.43%) after 72hr incubation, which meaning only (86.57%) of WRL-

68 were able to forming insoluble formazan product which remaining as a live cells, on the

other hand, the (200, 100 and 50 ug/mL) concentrations were recorded the percentage cell

viability as (91.30%, 94.61%, 95.83respectively), as well as, the lowest results were recoeded

by (6.25 ug/mL) only inhibits (2%) as presented in (Figure 1.4). These results were in

agreement with that obtained by Saravana Kumar et al., (2014), the effect ofextracellular

crude extract was achieved with concentration -dependent manner (dose dependent) This

means that the isolation bacteria (bag3) have little effect on normal cells, because we need

high concentrations to effect on the normal cells.

Concentration

Cel

l v

iab

ilit

y %

%



Figure 4: Antitumor activities of extracellular crude extract (bag3) against WRL-68 cell


Mueller and Nicole, (2002) and Azambuja et al., (2005) they described that the Antitumor

antibiotics (antitumorantibiotics / cytotoxic) are medications that prevent and fighting the

growth of tumors and the antitumor antibiotics which produced by Streptomyces species are

precious in the medical field.

Results were agreed with that described by Ravikumar et al., (2008) and Rashad et al.,

(2015) they evaluated that some genus of actinomycetes especially Streptomyces having

energetically product compounds of functional bioactive metabolites with a broad range of

pharmaceutically application such, antimicrobial metabolites, anthelminthic, antiviral and

antitumor agents, as well as their in vitro application of cytotoxicity assay from the secondary

metabolites against different cancerous cell lines were showed significantly antitumor activity

and this effect was dependent on dose, isolated strains and types of cancerous cell lines.

Ayuso et al., (2005) also confirmed that some isolated actinomycetes strain such

Streptomyces are talent to produced dissimilar antitumor complexeswith various chemical

nature because they anchorage differentgene clusters which encoding poly-ketide and non-

ribosomalpeptide synthases, while, Bode et al., (2002) described the way which can be used

to isolate and extract these bioactive metabolites by using different organic solvents because

these compounds were secreted into culture broth, finally Wadkins et al., (1998) summarized

the mechanism action of some of these compounds isolated and extracted from some species

Concentration

Cel

l v

iab

ilit

y %



of Streptomyces through intercalating with genetic materials especially with duplex DNA

which cause harmfuleffects on fast multiplying cells by preventing the DNA dependent RNA

polymerase activities.

CONCLUSIONS

1. Streptomycse spp. screening for the from soils were successfully done and obtaining many

suspected species, with numerous bioactive properties.

2. The isolated Streptomyces show potential antimicrobial, antifungal and antitumor

activities.

3. The isolated Streptomyces appeared more susceptible to Gram-positive bacteria

(Staphylococcus aureus) than both Gram-negative bacteria and yeast (Escherichia coli and

Trychophyton spp).

4. Optimum condition for the production and extraction of the bioactive compounds were

successfully completed, for the best media, optimum incubation days, optimum pH, NaCl salt

tolerance and Temperature.

5. Analyzing by High Performance Liquid Chromatography we get five active compound

(Tetracycline, streptomycin, neomycin, vancomycin, kanamycin).

6. Our result that obtains show powerful antitumor activities against various cancerous cell

lines, especially for developing the potential antitumor drugs with precise cellular targets,

however, this requires more research to identifying its importance therapeutic properties and

determining their mechanisms on target cellular metabolisms.

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