Advances in Environmental Biology Streptomyces sp. nov Isolated From Hot-Springs

Advances in Environmental Biology, 8(7) May 2014, Pages: 2008-2011
AENSI Journals
Advances in Environmental Biology
ISSN-1995-0756
EISSN-1998-1066
Journal home page: http://www.aensiweb.com/aeb.html
Anti-Bacterial Potential of New Streptomyces sp. nov Isolated From Hot-Springs
North of Iran
1
Akhgari Zahra, 2Heshmatipour Zoheir, 3Mahtabipour Mohammad Reza
1
MS.c in Microbiology, Tonekabon Branch, Islamic Azad University, Tonekabon, IRAN
Department of Microbiology, Tonekabon Branch, Islamic Azad University, Tonekabon, IRAN
3
MS.c in Clinical Biochemistry, Department, School of Medicine, Tabriz University of medical Sciences, Tabriz, IRAN
2
ARTICLE INFO
Article history:
Received 28 February 2014
Received in revised form 19
April 2014
Accepted 23 April 2014
Available online 25 June 2014
Key words:
Antibacterial activity, Hot-springs,
Streptomyces, AIA, SCA.
ABSTRACT
Actinomycetes are useful as producing organisms of antibiotics, enzymes and other
bioactive metabolites because of their rapid growth rate and rapid autolysis of
mycelium. They are useful for studying the evolution and adaptation mechanism of
organisms to extreme environments such as hot spring .They are also an important
resource for development of industrial products. Thus, the aim of this study is isolation
and study characterization new isolated from hot spring. In this study new isolated
from other species have shown different characteristics and this isolated strain can be
grows on Actinomycetes conventional media such AIA and SCA.
© 2014 AENSI Publisher All rights reserved.
To Cite This Article: Akhgari Zahra, Heshmatipour Zoheir, Mahtabipour Mohammad Reza., Anti-Bacterial Potential of New Streptomyces
sp. nov Isolated From Hot-Springs North of Iran. Adv. Environ. Biol., 8(7), 2008-2011, 2014
INTRODUCTION
The most abundant Actinomycetes in nature are Streptomyces. Streptomyces have more than 140 species.
Streptomyces are naturally found in some inches below soil, in organic materials and hot springs [1]. Their most
distinctive feature is that they form aerial branched mycelium and substrate filaments. Streptomyces are
naturally occurring below the surface of the soil, water and decomposed organic matter in Hot-springs. Many
strains produce one or more antibiotics. They are Gram-positive but not acid-alcohol fast [2]. Their optimum
growth temperature is 25-35°C; and also some species grow at temperatures within Psychrophilic and
thermophilic range. Optimum pH range for growth is 6.5 to 8. They have a relatively slow growth as their
bacterial colony can be seen after a few days. Some Streptomyces species are pathogenic for animals and
humans and other species are phytopathogenic. The isolation and identification of species of this genus is very
difficult [3]. Antibiotics produces by Streptomyces are stable against many infections such as, Streptomycin
producing by S.griseus, Nystatin producing by S.noursei, Neomycin and Tylosin producing by S.fradiae [2].
The numbers of antibiotics produced by Actinomycet up to 2002 are far more than other bacteria and fungi
around 8700, 39 % of produce components have antifungal activity, 32% of components are from active
microorganisms which are gram positive and 10 % of combinations are gram negative [1]. To classify
Streptomyces genus phenotypic and genotypic methods are used. Phenotypic methods such as fatty acids,
serumology, phagtyping, protein profiles. Genotypic methods like hybridization of DNA- DNA, fingerprinting
techniques and comparison of 16srRNA and other genes.
MATERIALS AND METHODS
Water samples were collected from Hot-springs in North of Iran. Samples inoculation at 37 ° C for 3-7
dayes in 180 rpm. Streptomyces were isolated and purified from these samples by selective methods and SCA
(Starch Casein Agar) [4, 5] and AIA (Actinomycete Isolation Agar) media [6] and Bilayer culture method. After
isolation single colony of Streptomyces base on Bergys manual microbiology we have a few tests of phenotypic
and biochemical on these colonies. Such as Gram dying, catalase, oxidase, Indol, motility, SH2, Starch
hydrolysis, casein hydrolysis. Then on isolated colony we have effecte environmental important factors such ee
and pH (Table.1and 2). They were classified based on nature and morphology. Cross-streak method was used
for 7 pathogen species [7]. The Anti-bacterial active substance was extractable in ethyl acetate [8, 9]. Bacterial
supernatant was collected and were screened by agar diffusion method include agar well diffusion method and
Corresponding Author: Akhgari Zahra, MS.c in Microbiology, Tonekabon Branch, Islamic Azad University, Tonekabon,
Iran.
E-mail: [email protected]
2009
Akhgari Zahra et al, 2014
Advances in Environmental Biology, 8(7) May 2014, Pages: 2008-2011
disk diffusion method [10] (fig1). Detection and final approval of the isolates was carried out by 16srDNA
analysis.
RESULTS AND DISCUSSION
In this study, the different bacterial colonies were isolated from the Ramsar spas. After enrichment of
samples in SCB, medium and their culture on agar containing SCA and AIA, seven colonies similar to
Actinomycets colonies were identified and isolated. Then, Cross-streak method test was conducted and
reference pathogens screened to determine the anti-microbial potential of colonies. At last two colonies were
chosen and disk diffusion method and well diffusion method were conducted on the metabolits produce from
these isolates. With regard to active bacteria producing isolated and purified antibacterial metabolites,
Streptomyces sp, showed broad spectrum of antibacterial activities on gram positive bacteria. The extract was
active against bacteria including Bacillus subtilis PTCC1023, Staphylococcus epidermithis PTCC1436,
Staphylococcus aureus PTCC1431 3 and MRSA ATCC25923. The effect of temperature and pH on the growth
and production of metabolits were also studied. Finally, the isolated bacteria were sequenced by 16srRNA.
After analyzing 16srRNA and recording in NCBI, isolated bacteria were named Streptomyces sp.Akhgar.zh
toneka and Streptomyce sp. Akhgar.z toneka as new strains. The colonies are white and chalky form. A Few
species are pathogenic for animals and man and others are phytopathogens. It’s very difficult to isolate and
identify species of this genus. Also, the Biochemical characteristics of isolated strains are show in these isolated
show relationship to Streptomyces sp. That 99% have relationship with Streptomyces sulphureus (fig 2). Of
these two cases, i.e. 28.5% of them had strong antimicrobial activity. Both samples contain pigments that have
antimicrobial activity. In this study, the antimicrobial effect was assessed in two different ways.
a. Antimicrobial effects of the first to cross the line was cultured. The two examples of which have
antimicrobial activity showed 100% inhibition of growth by inhibiting the growth of B.subtilis and
Staphylococcus epidermidis 87.5% and 87.5% growth inhibition of Staphylococcus aureus 50% inhibition of
growth of methicillin-resistant S.aureus and 75% growth inhibition of E. coli and B.cereus showed 75% growth
inhibition, whereas it was resistant Pseudomonas aeruginosa.
b. The second method of measuring the microbial drive and well is done. In this method, bacterial extracts
were extracted with ethyl acetate and the extracting was performed. Phosphate buffer for dissolving the crude
extract and its impregnating Blank discs are used. In this method, the results were largely similar to the first
method. From two strains of E.coli and the inhibition of growth in any of the cases there was B. cereus.
Staphylococcus epidermidis, but in all cases (100%) were inhibited and inhibiting the growth of Staphylococcus
aureus in 75% growth inhibition of Bacillus subtilis and MRSA (50%) were methicillin-resistant. This has
greatly inhibited the gram positive and gram negative effect. The results of these two methods showed that this
method has an inhibitory effect on gram positive and gram negative and they were slightly inhibited. This could
be due to differences in cell wall structure of gram positive and gram negative bacteria. They have a thin layer
Peptide glycan periplasmic space between the cell wall and cell membrane are located.
In a study conducted in 2005 by Zheng et al., [11] 341 strains from water, sediments and aquatic organisms
were isolated from three different regions of China. Inhibitory effect on 42 strains tested, had at least one of the
organisms that about 12% of them had inhibitory effects. This result was consistent with results obtained in our
study.
The research was carried out by Ripa and colleagues in 2010. They were extracted and purified from
Streptomyces. The compound has good activity against the pathogenic bacteria. In this study it was shown that
the pure compound isolated against gram-positive than gram-negative bacteria are more effective. This was
clearly seen in our study. Interesting result obtained was extracted with ethyl acetate, the combined cytotoxic
effect of purified.
Fig. 1: Well diffusion Test of isolated VII extract on S.aureus.
2010
Akhgari Zahra et al, 2014
Advances in Environmental Biology, 8(7) May 2014, Pages: 2008-2011
Fig. 2: phylogeny tree isolated bacteria (akhgar.z toneka and akhgar.zh toneka).
Table 1: Effect different temperature on isolated III and VII
Day
Third
Second
First
Temperature° C
VII
VII
VII



4






25
_





30






37






42






50






Results of isolates grown in the temperature range from 4 ° C to 50 ° C (Weak growth: +, moderate growth: + +, Good growth: + + +,
and lack of growth:-)
Table 2: Effect different pH on isolated III and VII.
Day
Third
Second
PH
VII
VII


0.5




1




1.5




2




3




4




5




6




8




9




10




11




12




13




14




Growth result of isolated species (slow growth+, Medium growth++, good growth+++, no growth-)
First
VII































Conclusion
The study showed that Hot-spring exhibit diverse bacteria and it served as potential reservoirs for bacteria
of antimicrobial activity. We could use this important reservoir for isolation and identification of new strains
which produce antibacterial. The demand for new antibiotics due to the spread of antibiotic-resistant pathogens
is increasing. We found that the Ramsar spas have high potential for producing Streptomyces which are
antimicrobial substance. In contrast, it appears that this is a promising development for the treatment of
infections. In Iran we had few studies about hot springs producing anti microbial materials. Conciduring
ecological verity of Iran there is a possibility of finding new species producing antimicrobial materials.
2011
Akhgari Zahra et al, 2014
Advances in Environmental Biology, 8(7) May 2014, Pages: 2008-2011
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