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Research Article
 

Characterization and In vitro Antimicrobial Activity of the Two Novel Compounds of Streptomyces Species



A. S. M. Anisuzzaman, Naoki Sugimoto , S. A. Bhuiyan , Golam Sadik and M. A. Gafur
 
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ABSTRACT

The objective of this investigation is to determine the in vitro antimicrobial activity of the two novel compounds from Streptomyces. This antagonistic micro-organism was isolated and identified on the basis of morphological and biochemical study from the soil of the Northern district of Pabna, Bangladesh. The organism yielded maximum antimicrobial principle when grown at Czapek-Dox broth medium of pH 8 for 8 days at temperature 37.5C having rhamnose as carbon source. The antimicrobial principles were extracted from the fermentation broth by choloroform. The extract on chromatographic resolution yielded two antimicrobial compounds: Streptomysone-A (I) and Streptomysone-B (II). Both the compounds were screened for antimicrobial activity against eighteen pathogenic organisms which showed moderate to strong antimicrobial activity. The minimum inhibitory concentration (MIC) of the compounds against five pathogenic organisms were found to be between 32 to 128μg ml‾1.

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  How to cite this article:

A. S. M. Anisuzzaman, Naoki Sugimoto , S. A. Bhuiyan , Golam Sadik and M. A. Gafur , 2001. Characterization and In vitro Antimicrobial Activity of the Two Novel Compounds of Streptomyces Species. Journal of Medical Sciences, 1: 220-223.

DOI: 10.3923/jms.2001.220.223

URL: https://scialert.net/abstract/?doi=jms.2001.220.223

Introduction

In the old age, millions of people were died in the epidemic form of infectious diseases like plague, cholera etc. But after the introduction of newer antibiotics and tremendous advancement of medical sciences and technology, these infectious diseases could be managed successfully (Carlson et al.,1983). Micro-organisms have historically provided a rich source of structurally diverse, biologically active metabolites (Jan et al., 1979).

In recent years owing to indiscriminate use of antibiotics the pathogenic organisms are gaining resistance to the existing antimicrobial and chemotherapeutic agents (Julia et al., 1992). Hence the search for newer antimicrobial drugs active against those strains is a pressing need. As a result, scientists have engaged themselves to isolate the newer and effective antibiotics from microbes.

As a part of our continuing search of metabolites from micro-organisms, soil samples were collected throughout Bangladesh. In this investigation, we report the antimicrobial spectra of the two compounds isolated from the culture filtrate of the Streptomyces species.

Materials and Methods
Collection of soil samples and identification of the organism:
For screening purposes, soil samples of various depth up to 1m, were collected from various places like construction sites, road sides, grave yards, food wastage, agriculture waste, drains and sewage of Bangladesh. The organism was identified on the basis of morphology and biochemical study (Holt et al.,1994).

Selection of suitable broth medium for antibiotic production:

A number of different media such as Czapek-Dox broth (acidic), Czapek-Dox broth (alkaline), Glucose broth, Yeast extract glucose broth, Jenson medium modified, Nutrient broth etc., were tried for the maximum antibiotic production from the organism. The antibiotic activity of the liquid cultures was tested against Bacillus subtilis, Pseudomonas aureginosa, Staphylococcus aureus, Shigella dysenteriae by disc diffusion method (Bauer et al., 1966).

Selection of the suitable culture conditions: The effect of different carbon sources (e.g. Sucrose, D-Glucose, D-Fructose, D-Galactose, Maltose, D-Mannose, lactose, D-mannitol, L-arabinose, D-xylose and rhamnose); incubation period (up to 14 days); temperature (30, 32.5, 37.5, 30, 40, 42.5 and 45°C); pH values (3, 4, 5, 6, 7, 8 and 9) and NaCl concentration (0.5, 1, 2, 3, 4, 5, 6, 7, 8 and 9 % respectively), on antibiotic production by the organism were studied by disc diffusion method (Bauer et al., 1966) using Shigella dysenteriae as test organism.

Production, isolation and characterization of the compounds:
The organism was allowed to grow in a number of culture flasks of 500ml capacity containing Czapek-Dox broth alkaline medium (100ml in each flask) at 37.5°C.

Image for - Characterization and In vitro Antimicrobial Activity of the Two Novel
Compounds of Streptomyces Species

After 8 days (due to maximum yield of antibiotic) the broth was separated from the mycelial mat. The culture filtrate then subjected to repeated chloroform (CHCl3) extraction (3 x 30 ml) and the extract was evaporated under reduced pressure. The crude antibiotic fraction was resolved by thin layer chromatography (TLC), preparative TLC (PTLC) and obtained on large scale by column chromatography (CC) (Beckett et al., 1986). The isolated antibiotics were characterized as streptomysone-A (I) and streptomysone-B (II) on the basis of their UV, IR and NMR data.

Antimicrobial screening: The antimicrobial activity of the compounds I and II (25 and 50μg disc-1 respectively) were determined against six gram positive bacteria, eight gram negative bacteria and four pathogenic fungus by the standard disc diffusion method (Bauer et al., 1966). Amoxycillin disc (25μg disc-1) for bacterial and Grisofulvin (20μg disc-1) for fungal test were used as standard for the comparison of antimicrobial activity. The test organisms were collected from the Department of Microbiology, Universtiy of Dhaka, Bangladesh. The MIC values of the compounds were determined against Shigella dysenteriae, Salmonella typhi, Escherichia coli, Bacillus subtilis and Staphylococcus aureus by serial dilution technique (Hammond et al., 1978).

Result and Discussion
Identification of the organism:
The organism was identified as Streptomyces species, on the basis of the following characteristics (Holt et al.,1994):

Spore chain morphology.The spores were ornamented.
The mature spores were colored.
It was able to produce melanoied pigment.
It was able to utilize under standardized conditions particular carbon containing compounds for growth.
It was able to produce antibacterial factors.
Vegetative mycelia were colored.

Suitable culture condition: The effect of different physical parameters on antibiotic production was observed. It was found that the production of antibiotic from the organism in Czapek-Dox broth alkaline medium after 8 days of incubation (Fig. 1), at pH 8 (Fig. 2) at temperature 37.5°C and rhamnose as carbon sources (Fig. 3) was found to be the most suitable.

Isolation and characterization of the compounds: The CHCl3 extract of the culture filtrate of Streptomyces sp. on chromatographic analysis yielded two novel antimicrobial compounds designated as streptomysone-A (I) and Streptomysone-B (II) on the basis of UV, IR, 1H-NMR, 13C-NMR (Anisuzzaman 2000). The structure of the compounds are as follows:

Image for - Characterization and In vitro Antimicrobial Activity of the Two Novel
Compounds of Streptomyces Species

Image for - Characterization and In vitro Antimicrobial Activity of the Two Novel
Compounds of Streptomyces Species
Fig. 1: Effect of incubation period on the production of antibiotic

Image for - Characterization and In vitro Antimicrobial Activity of the Two Novel
Compounds of Streptomyces Species
Fig. 2: Effect of pH on the production of antibiotic

Image for - Characterization and In vitro Antimicrobial Activity of the Two Novel
Compounds of Streptomyces Species
Fig. 3: Effect of Carbon sources on the production of antibiotic

Antimicrobial activity of the compounds: Both the compounds showed significant antimicrobial activity against the test pathogens. The results are shown in the Table 1 and Table 2. However, the compound (I) exhibited the strong activity against Bacillus subtilis, Bacillus megaterium, Escherichia coli and Klebselia species and comparatively weak activity was observed against Pseudomonas aureginosa. Salmonella typhi-A. Shigella dysenteriae. While the compound (II) exhibited strong activity against gram negative bacteria than gram positive bacteria.

Table 1: Antibacterial activity of the compound I and II.
Image for - Characterization and In vitro Antimicrobial Activity of the Two Novel
Compounds of Streptomyces Species

Table 2: Antifungal activity of the compound I and II.
Image for - Characterization and In vitro Antimicrobial Activity of the Two Novel
Compounds of Streptomyces Species

Table 3: MIC value of the compound (I).
Image for - Characterization and In vitro Antimicrobial Activity of the Two Novel
Compounds of Streptomyces Species

Table 4: MIC value of the compound (II).
Image for - Characterization and In vitro Antimicrobial Activity of the Two Novel
Compounds of Streptomyces Species
+ = Growth, - = No growth

The compounds were also active against pathogenic fungus i.e. Tinea pedis, Tinea corporis, Candida albicans and Rhizoctonia solani.

Minimum Inhibitory concentrations of the compounds: The minimum inhibitory concentration of the compound (I) and (II) were shown in the Table 3 and Table 4, respectively. The MIC values of the compound (I) against Bacillus subtilis, Streptococcus-β-hemolyticus, Escherichia Coli, Psuedomons aureginosa and Salmonella typhi - A were 16, 32, 16, 32 and 128μg ml-1 respectively, and that for compound (II) were 16, 32, 16, 32 and 128μg ml-1 respectively. From the MIC values, it was found that both the compounds were more potent against Bacillus subtilis and Escherichia Coli.

REFERENCES

1:  Hammond, S.M. and P.A. Lambert, 1978. Antimicrobial Actions. Edward Arnold Ltd., London, pp: 8-9

2:  Reiner, R., 1982. Antibiotics: An Introduction. Roche Scientific Co., Switzerland, pp: 21-25

3:  Anisurzaman, A.S.M., 2000. Characterization and biological studies on the bioactive metabolites of the Aspergillus fumigatus and Streptomyces sp. M.Sc. Thesis, University of Rajshahi, Bangladesh.

4:  Backett, A.H. and J.B. Stenlake, 1986. Chromatography. 3rd Edn., CBS Publishers and Distributors, New Delhi, India, pp: 75-76

5:  Julia, A., E. Hernandez and J. Primo, 1992. Isolation and identification of ethisolide as an antibiotic product from Penicillium capsulatum. Applied Microbiol. Biotechnol., 37: 298-300.
CrossRef  |  Direct Link  |  

6:  Holt, J.G., N.R. Kreig, P.H.A. Sneath, J.T. Staley and S.T. Williams, 1994. Bergey's Manual of Determinative Bacteriology. 9th Edn., Lippincott Williams and Wilkins, Baltimore, USA., ISBN-13: 9780683006032, Pages: 787
Direct Link  |  

7:  Bauer, A.W., W.M.M. Kirby, J.C. Sherris and M. Turck, 1966. Antibiotic susceptibility testing by a standardized single disk method. Am. J. Clin. Pathol., 45: 493-496.
CrossRef  |  PubMed  |  Direct Link  |  

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