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Research Journal of Microbiology

Year: 2007  |  Volume: 2  |  Issue: 3  |  Page No.: 272 - 277

Isolation and in vitro Antimicrobial Activities of Ethyl acetate Extract from Streptomyces bangladeshiensis

M. Abdul Alim Al-Bari, Alam Khan, M. Robiul Islam, M. Kudrat-E-Zahan, M.M. Sazedur Rahman, M. Anwar Ul Islam and M. Ashik Mosaddik

Abstract

The aim of the present study was to investigate the antimicrobial activities of ethyl acetate extracts against Gram-positive and Gram-negative bacteria and fungi. An attempt has been made to investigate the indigenous drugs of choice in infectious diseases for mitigation of suffering of the vast masses of humanity. The organism, Streptomyces bangladeshiensis was isolated from a soil sample collected from Natore, Bangladesh at the depth of 0.75 m using crowded plate technique. The maximum secretion of metabolites from the strain was found at the 7th day of incubation in Czapek Dox broth (alkaline pH 8.5) medium at 32.5°C by maintaining the physicochemical factors in optimum level for the culture. In vitro antimicrobial susceptibility was determined as per National Committee for Clinical Laboratory Standards guidelines and serial dilution technique for the determination of Minimum Inhibitory Concentration (MIC) of extracts. Ethyl acetate extract from a new actinomycetes, Streptomyces bangladeshiensis, showed good antibacterial and antifungal activities against a total of 14 bacteria (5 gram positive plus 9 gram negative) and 8 fungi. The Minimum Inhibitory Concentrations (MIC) were determined and found to be 16 μg mL-1 against Bacillus subtilis, Staphylococcus aureus, Shigella dysenteriae and Aspergillus flavus while 32 μg mL-1 against Salmonella typhi, Candida albicans and Aspergillus niger. Present data shows that all the pathogenic microorganisms (Gram positive and negative bacteria and fungi) showed a substantial sensitivity towards the crude extract. But further work is necessary in order to establish the individual antibiotics present of this isolated compound.

Table 1) and eight pathogenic fungi (Table 2) were used in this antimicrobial screening. Briefly in disc diffusion method, the molten nutrient agar medium (Difco) for bacteria and molten potato dextrose agar medium for fungi respectively was poured in 15 mL quantity in each in the clean test tubes and plugged with cotton.


Table 1: Antibacterial activity of the ethyl acetate extract

Table 2: Antifungal activity of the ethyl acetate extract

The test tubes and a number of Petridishes were sterilized in an autoclave at 121°C and 15 Ibs-1 sq. inch pressure for 30 min and were transferred into laminar airflow unit and then allowed to cool to about 45 to 50°C. The test organisms (both bacteria and fungi) were transferred from the fresh subculture to the test tube containing 15 mL autoclaved medium with the help of an inoculating loop in an aseptic condition. Then the test tube was shaken by rotation to get a uniform suspension of these organisms. These suspensions were immediately transferred to the sterile Petridishes in an aseptic area. The Petridishes were rotated several times, first clockwise and then anticlockwise to assure homogenous distribution of the test organisms. The media were poured into Petridishes in such a way as to give a uniform depth of approximately 4 mm. For the placement of the discs, by means of a pair of sterile forceps, the sample impregnated discs and standard disc were placed gently on the solidified agar plates seeded with the test organisms to ensure contact with the medium. The plates were then kept in a refrigerator at 4°C for 24 h in order to provide sufficient time to diffuse the extract into the medium.

The petridishes were then incubated at 37°C for overnight to allow the bacterial growth and 72 h for fungal growth. The antibacterial and antifungal activities of the extract were then determined by measuring the respective zones of inhibition in mm.

For demonstrating the antibacterial activity and subculture of the test organisms nutrient agar medium (Difco) was used. Nutrient broth (Difco) was used as liquid culture of all the tested bacteria and is used in the minimum inhibitory concentration determination experiments. Potato Dextrose Agar (PDA) media was prepared in the lab to maintain the fungal growth. Antifungal activity of the complexes was done on PDA media spreading with fungal spores and kept at 28°C for about 72 h. For PDA preparation 20 g potato was extracted with distilled water 100 mL at 100°C for 1 h and it was then filtered off by cotton filter. The potato juice (100 mL) was then mixed with 2 g Dextrose and 1.5 g agar and finally the pH of the prepared media (PDA) was adjusted at 7.00.

A current definition of the Minimum Inhibitory Concentration, MIC, is the lowest concentration which resulted in maintenance or reduction of inoculum viability (Carson et al., 1995). The determination of the MIC involves a semi quantitative test procedure, which gives an approximation to the least concentration of an antimicrobial needed to prevent microbial growth. The method displays tubes of growth broth containing a test level of preservative, into which an inoculum of microbes was added. The end result of the test was the minimum concentration of antimicrobial (test materials) which gave a clear solution, i.e., no visual growth (Collins, 1964; Davidson and Parish, 1989). Serial dilution technique (Reiner, 1982) was applied for the determination of minimum inhibitory concentration of the extracts. The minimum inhibitory concentrations (MICs) of the crude extract against Bacillus subtilis, Staphylococcus aureus, Salmonella typhi, Shigella dysenteriae, Candida albicans, Aspergillus flavus and Aspergillus niger were determined by serial dilution technique (Reiner, 1982; Noble and Sykes, 1977) because of maximum antimicrobial activities against these organisms. In serial dilution technique, Bacterial suspensions and fungal suspensions were prepared in sterile nutrient broth medium (Difco) and potato dextrose broth medium respectively in such a manner so that the suspension contains 107 cells mL-1. These suspensions were used as inoculums. Thirteen autoclaved test tubes were taken, ten of which marked as 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 and the rest three were assigned as Cm (medium), CS (medium + compound) and Ci (Medium + inoculum). One milliliter of sterile nutrient broth medium or potato dextrose broth medium was added to each of the twelve test tubes. One milliliter of the sample solution was added to the first test tube and mixed well. One milliliter content from the first test tube was transferred by the sterile pipette to the second test tube and mixed uniformly. Then 1 mL of this mixture was transferred to the third test tube. This process of serial dilution was continued up to the tenth test tube. One drop (10 μL) of properly diluted inoculum was added to each of the ten test tubes and mixed well. For the control test tube, CS 1 mL of the sample solution was added, mixed well and 1 mL of this mixed content was discarded. This was to check the clarity of the medium in presence of diluted solution of the compound. Ten microliter of the inoculum was added to the control test tube, Ci to observe the growth of the organism in the medium used. The control test tube, Cm containing medium only was used to confirm the sterility of the medium. All the test tubes were incubated at 37.5°C for 18-24 h.

RESULTS AND DISCUSSION

The ethyl acetate extract showed significant antimicrobial activities (both antibacterial and antifungal activities) against the selected test pathogens in comparison with that of standard kanamycin and nystatin (Table 1 and 2). However, the activity profile of the extract was investigated that all the Gram positive and Gram negative bacteria showed a potent sensitivity towards the crude extract. Moreover, better antibacterial activities were observed against Staphylococcus aureu, Streptococcus-β-haemolyticus, Salmonella typhi, Shigella dysenteriae, Shigella flexneri, Pseudomonas aeruginosa and Klebsiella species in comparison with other pathogens.

During the antifungal testing, the crude ethyl acetate extract showed better antifungal activities against Aspergillus niger, Aspergillus flavus, Candida albicans and Trichoderma species than the other fungi ranging from 19-22 mm in diameter in zone of inhibition in comparison with that of standard nystatin ranging from 19-30 mm in diameter.

The Minimum Inhibitory Concentrations (MICs), observed by serial dilution technique (Reiner, 1982) were found to be 4 μg mL-1 against Shigella dysenteriae and Aspergillus niger; 8 μg mL-1 against Bacillus subtilis, Bacillus megaterium, Salmonella typhi, Klebsiella species and Bipolavis species; 16 μg mL-1 against Staphylococcus aureus, Sarcina lutea, Shigella sonnei, Shigella boydii, Escherichia coli, Aspergillus flavus, Candida albicans, Aspergillus fumigatus, Trichoderma species and Fusarium species while 32 μg mL-1 against Streptococcus-β-haemolyticus, Shigella shiga.


Table 3: The results of MIC values of ethyl acetate extract (μg mL-1)

The MIC values of the extract against the tested organisms indicated their noticeable antibacterial and antifungal potencies compared with standard antibiotic, kanamycin and nyststin respectively (Table 3). However, further work is necessary in order to isolate active antimicrobial compounds from this organism.

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