Islamic Azad University, Qom Branch, Iran

Corresponding author Email: Fatima.alzameli@yahoo.com

Article Publishing History

Introduction

Enterococcus can be resistant to different antibiotics. One of the main causes that distributes drug-resistant bacteria is routed in antibiotic misuse by communities. This new phenomenon causes severe infections and death of patients. Challenging problems of increasing drug-resistant bacteria urges the need to find more effective antibiotics for treatment (Yehuda et al, 2002). Antimicrobial producing microorganisms are distributed 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 fishing industry (Usha, 2010 and Mohitosh Biswas et al,2011).

Figure 1a

 

Figure 1b

 

Material and Methods

One hundred and twenty five 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 five 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 further study on isolates. Visual observation of both morphological and microscopic characteristics of isolates were done by colony investigation color and shape and using light microscopy.Those colonies that were identified as Streptomyces in previous stage were transferred to ISP2 agar incubated in 28 degree centigrade for up to seven days to obtain purified isolates for further study (Kavya, 2012).The standard isolate of VRE ATCC 51299 were brought from Reference Laboratory of Tehran.

Figure 1c

 

Figure 1d: A:The inhibition zone on the right is made by the E.P1 isolate B: Gram staining of E.P1 isolate under the light microscopy C: Colony morphology of E.P1 isolate on ISP4 culture medium

 

Figure 2: The inhibition zone of the isolate E.P1 was produced more in the seventh day of the incubation

 

Figure 3: Biochemical structure of antimicrobial agent produced by E.P1

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 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 solvent was centrifuged at 5000 rpm for 15 min to remove traces of fermentation broth. The extracted antimicrobial substance was identified by Gas-Chromatography-Mass Spectrometry (GC-MS) analysis (Naggar et al., 2006).

Table 1: Thickness of the inhibition zone made by E.P1 isolate against VRE
Inhibition zone size(mm)	Streptomycetes isolates
26	E.P1

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

Table 2: Biochemical test results of Streptomyces E.P1 isolate
E.P1 isolate	Biochemical 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

The 16 s rRNA sequence of Streptomyces E.P1 isolated showed 100% similarity to Streptomyces tendae. Moreover, antimicrobial agent obtained from the target isolate is a quinoline compound.

Figure 4: GC-MS analysis result of antimicrobial agent

There were similar studies done in this area and different 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 potential Actinomycete strain KH-614 against VRE that produced 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 purified with Sephadex LH-20 column. Finally HPLC was done to prove the purification of the antimicrobial agent (El-Naggar., 2007).

Acknowledgement

The author is thankful to professor Ahmad Ali Pourbabaei (Lecturer at Tehran University, Iran) and Dr Seyyed Soheil Aghaei (Lecturer at Azad University of Qom, Iran).

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