Microbiological
Communication
Biosci. Biotech. Res. Comm. 10(2): 220-224 (2017)
Isolation and characterization of
Streptomyces
species
against vancomycin resistant enterococcus (VRE) from
agricultural soils of Qom, Iran
Fatemeh Alzamoli
Islamic Azad University, Qom Branch, Iran
ABSTRACT
Infections due to Vancomycin Resistant Enterococcus (VRE) are increasing worldwide. Resistance of VRE to glyco-
peptides antibiotics is related to van A,B,C,D,E genes which decreases the impact of this antibiotics on the cell wall
of bacteria , making these bacteria innately Tuberamycin-resistant. Therefore, Jentamicin is used to treat infections.
In recent years it has been reported that many of VRE are showing high resistance to Aminoglycosids and even
to Jentamycin. There have been reports on distribution of VRE strains in clinical environments. This research was
done to obtain novel antimicrobial agents which is effective against VRE infections. Enterococcus mainly cause
nosocomial infections especially in ICU and CCU sections of hospitals and are threat to patients. Soil samples were
collected from agricultural soils of Qom. Then isolation and primary identi cation of Streptomycetes was done on
ISP4 culture medium. Then the antimicrobial activity of all Streptomycetes obtained were examined against VRE on
Hickey-Trenser medium, Brain-heart infusion medium, Muller-Hinton agar with cross-streak method. Five out of
125 isolates showed inhibitory effect on VRE. In the next step the Streptomycete Strain that left bigger inhibition
zone against VRE on medium were selected to be characterized biochemically and morphologically and was named
as E.P1. Additionally were identi ed on the basis of its 16S rRNA sequence. Identi cation results indicated that the
obtained Streptomyces strain is 100% similar to Streptomyces tendae ATCC 19812.Finally, GC-Mass analysis was done
on the antimicrobial agent that was extracted by ethyl acetate. Results revealed that it was a quinuline compound.
KEY WORDS: STREPTOMYCES ISOLATION, VRE, SECONDARY METABOLITE
220
ARTICLE INFORMATION:
*Corresponding Author: Fatima.alzameli@yahoo.com
Received 19
th
March, 2017
Accepted after revision 22
nd
June, 2017
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Vafa, Saeedizadeh and Bostani
Table 1. Thickness of the inhibition zone made by
E.P1 isolate against VRE
Inhibition zone size(mm)Streptomycetes isolates
26 E.P1
INTRODUCTION
Enterococcus can be resistant to different antibiotics.
One of the main causes that distributes drug-resistant
bacteria is routed in antibiotic misuse by communi-
ties. This new phenomenon causes severe infections
and death of patients. Challenging problems of increas-
ing drug-resistant bacteria urges the need to nd more
effective antibiotics for treatment (Yehuda et al, 2002).
Antimicrobial producing microorganisms are distrib-
uted widely in nature and play key role in soil, water
and degradation. Actinomycetes contains one of the
largest of bacterial genera, Streptomyces known to be
the source for many antibiotics. These are saprophytic,
gram-positive that are major contributors to biological
buffering of soils. This useful bacteria group produces
secondary metabolites such as antibiotics, enzymes,
immunoregulators, immunosupresores, antitumors and
vitamins. These bacteria group play key part in organic
compounds, food processing, pharmaceutical products,
agriculture and shing industry (Usha, 2010 and Mohi-
tosh Biswas et al,2011).
FIGURE 1. A:The inhibition zone on the right is made by the E.P1 isolate B: Gram stain-
ing of E.P1 isolate under the light microscopy C: Colony morphology of E.P1 isolate on
ISP4 culture medium
A
B
C
BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS ISOLATION AND CHARACTERIZATION OF
STREPTOMYCES
SPECIES AGAINST (VRE) 221
Vafa, Saeedizadeh and Bostani
FIGURE 2. The inhibition zone of the isolate E.P1 was produced more in the seventh day of the incubation
MATERIAL AND M ETHODS
One hundred and twenty ve Streptomycetes were
obtained from different sites in Qom. Soil samples were
taken from 5-15 cm depth of soil and then were put in
bags containing 0.1 g of caco3 . Samples were incubated
in 28 degree centigrade for about ve days (Kavya et al.,
2012 and Gurung et al., 2009).
Samples were diluted and 0.1 ml of each sample were
transferred to ISP4 culture medium, after that, were
incubated for seven days in 28 degree centigrade for fur-
ther study on isolates. Visual observation of both mor-
phological and microscopic characteristics of isolates
were done by colony investigation color and shape and
using light microscopy.Those colonies that were identi-
ed as Streptomyces in previous stage were transferred
to ISP2 agar incubated in 28 degree centigrade for up to
seven days to obtain puri ed isolates for further study
(Kavya, 2012).The standard isolate of VRE ATCC 51299
were brought from Reference Laboratory of Tehran.
The target Streptomycetes isolates were cultured on
the center of Hicky-Trenser agar and then were incubated
in 28 degree centigrade for 10 days. Next the Brain-Heart
Infusion broth containing VRE was added to this medium
to observe antimicrobial activity of every streptomycete
isolate. The results were investigated after 24-48 hours
(Alireza Dehnad et al., 2010). Biochemical test done on
the target isolate of E.P1 includes: Melanin pigment
production, Starch hydrolysis, Tween 80 hydrolysis,
lecithinase test, Nitrate Broth, Oxidase test, Catalase test,
DNase, Tryptophan hydrolysis, Acid Fast Staining and
Lysozyme Resistance (Houssam, 2009). Antimicrobial
producing Streptomyces E.P1 was cultured on ISP4 salnt
medium in tube and incubated at 28 degree Centigrade
for 10 days. Then spore suspension was provided. Firstly,
10 ml of sterile water was added to slant medium and
Table 2. Biochemical test results of
Streptomyces E.P1 isolate
E.P1 isolateBiochemical tests
+Melanin Pigmentation
+Starch hydrolysis
- Tween 80 hydrolysis
+Licethinase test
-Nitrat test
-Oxidase test
+Catalase test
-DNase test
+Tryptophan hydrolysis
-Acid fast staining
-Lysosyme resistance
E.P1 colonies scratched. Secondly, 1ml of the suspension
was transferred to fermentation erlen containing 50 ml
of culture medium. This erlen was incubated in shaker
at 150,170,200 rpm in 28 degree centigrade for 14 days.
Then 1 ml of fermentation medium was centrifuged at
5000 rpm for 15 min to remove traces of fermentation
broth. Finally 20 microliter from supernatant was added
to the center of Muller-Hinton agar in which VRE was
cultured on previously. This plate was incubated at 30
degree centigrade and the inhibition zone was observed
and measured (Osman et al.,2011).
Ethyl acetate solvent was used for the extraction of
the antimicrobial from culture supernatant. The sol-
vent was centrifuged at 5000 rpm for 15 min to remove
traces of fermentation broth. The extracted antimicro-
bial substance was identi ed by Gas-Chromatography-
Mass Spectrometry (GC-MS) analysis (Naggar et al.,
2006).
222 ISOLATION AND CHARACTERIZATION OF
STREPTOMYCES
SPECIES AGAINST (VRE) BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS
Vafa, Saeedizadeh and Bostani
BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS ISOLATION AND CHARACTERIZATION OF
STREPTOMYCES
SPECIES AGAINST (VRE) 223
RESULTS AND DISCUSSION
Five Streptomycetes isolates could inhibited the growth
of VRE . The isolate that showed the strongest inhibitory
effect (E.P1) was chosen to be characterized .
After careful investigation of the result of GC-MS
analysis of the antimicrobial compound it was named
as: 3-Quinoline Carboxylic acid,1-hexyl-1,2-dihydro-
2-oxo
The 16 s rRNA sequence of Streptomyces E.P1 iso-
lated showed 100% similarity to Streptomyces tendae.
Moreover, antimicrobial agent obtained from the target
isolate is a quinoline compound.
There were similar studies done in this area and dif-
ferent results obtained. Rhee et al., attempted to isolate
an Actinomycete that inhibited the growth of VRE in
2002 in Korea. They were successful to isolate a poten-
tial Actinomycete strain KH-614 against VRE that pro-
duced antitumor metabolite, too (Rhee et al., 2002).
El-Naggar could isolate Streptomyces HAL64 from
Eygept soils that produced antimicrobial agent against
gram-positive pathogens but not effective against
gram-negative pathogens. He could extract the obtained
antimicrobial substance by silicagel and then was puri-
ed with Sephadex LH-20 column. Finally HPLC was
done to prove the puri cation of the antimicrobial agent
(El-Naggar., 2007).
FIGURE 3. Biochemical structure of antimicrobial
agent produced by E.P1
FIGURE 4. GC-MS analysis result of antimicrobial agent
Vafa, Saeedizadeh and Bostani
224 ISOLATION AND CHARACTERIZATION OF
STREPTOMYCES
SPECIES AGAINST (VRE) BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS
ACKNOWLEDGEMENT
The author is thankful to professor Ahmad Ali Pourba-
baei (Lecturer at Tehran University, Iran) and Dr Seyyed
Soheil Aghaei (Lecturer at Azad University of Qom, Iran).
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