Isolation, Characterization, Screening and Antibiotic Sensitivity of Actinomycetes from Locally (Near MCAS) Collected Soil Samples

MATERIALS AND METHODS

Isolation of Actinomycetes from soil sample: The soil samples were collected from near my institute (2009), Muthayammal College of Arts and Science (MCAS) in corn and Soya fields and air dried and used. A total soil sample was collected from two areas, collected in sterile plastic bags and brought into sterile condition. Actinomycetes were isolated by soil dilution plate technique using Arginine-Glycerol-Salt (AGS) medium, (El-Nakeeb and Lechevalier, 1962). The organism was screened by purification method, streaking on AGS medium and incubated at 30°C for 4 days.

Biochemical characterization of Actinomycetes: The potent Actinomycetes were characterized by morphological methods consist of macroscopic and microscopic methods. The mycelium structure, color and arrangement of conidiophores and arthrospore on the mycelium were observed through the oil immersion (100X). The observed structure was compared with Berbey’s manual of Determinative Bacteriology and the organism was identified. Various biochemical tests performed for the identification of potent isolates are as follows: Starch hydrolysis, fermentation of citrate, nitrate reduction and IMVIC tests.

Fermentation process: The Actinomycetes were cultured at 30°C for 120 h in a jar fermentor containing 1 L of a medium containing of maltose 4%, sodium glutamate 1.2%, K₂HPO₄ 0.01%, MgSO₄ 0.05%, CaCl₂ 0.01% and FeSO₄ 0.005% with or without sodium alginate beads by the ionotropic method. At the end of fermentation cycle, the sodium alginate beads were aseptically separated from the fermentation broth by filtration using a sterile Bunchner funnel (Pandey et al., 2001).

Isolation of antibacterial metabolites: Antibacterial compound was purified from the filtrate by solvent extraction method; Ethyl acetate was added to the filtrate in ratio of 1:1 (v/v) and shaken vigorously for 1 h complete extraction. The ethyl acetate phase contains antibiotic substances separated from the aqueous phase. It was evaporated to dryness in water bath and the residue obtained was weighed (Westley et al., 1979). The obtained compound thus used to determine the antibacterial activity.

Determination of antibacterial activity: The actinomycete isolates often encounted show antibiotic activity on agar but not in liquid culture. The results of screening method were that most of the active isolates were active against Gram-positive and Gram-negative pathogen. Antibacterial activity was tested in vitro against pathogenic bacteria: Escherichia coli MTCC 50, Pseudomonas aeruginosa MTCC 424 and Bacillus subtilis MTCC 441. Antibacterial activities were performed by disc-diffusion assay and effectiveness was measured by zone of inhibition on bacterial culture plates.

Determination of protein profile from Actinomycetes
Protein profile: For the determination of protein profile from Actinomycetes using SDS-PAGE method as described by Laemmli (1970).

Preparation of bacterial sample: The actinomycete was grown in their respective broth at 30°C for 54 h. The cells were harvested by centrifugation at 8000 rpm for 1 min. The pellet was washed with 1 M Tris HCl buffer (pH = 6.8) and resuspended in 10 mL of the same buffer and vortexed. Then 80 mL of the sample buffer (1 M Tris HCl, pH = 6.8, 20% SDS, 20% glycerol, 10% b-mercaptoethanol and 0.005% Bromophenol blue) and 6.5 mL of b-mercaptoethanol were added to the preparations and boiled immediately in water bath at 100°C for 5 min. After boiling, the samples were placed on ice for 5 min and centrifuged at 8000 rpm for 1 min.

Preparation of SDS-PAGE: For SDS-PAGE 12.5%, separating gel and 6.5% resolving gel were prepared. A volume of 30 μL of each sample was loaded on gel and was run on mini gel electrophoresis at 100 V for 2 h and stained in a solution containing 0.1% (by mass per volume) Coomassie blue, 10% (by volume) acetic acid and 40% (by volume) methanol. De staining was performed in a solution containing 10% (by volume) acetic acid and 45% (by volume) methanol. The protein molecular mass marker (100 kDa) was used as standard.

RESULTS

Out of three Actinomycetes subjected for primary screening process and subjected for purification method by streak plate method (Fig. 1a-c). The identification of the potent antibiotic producing strains reveal strain belongs to the genus streptomyces. The biochemical tests were performed in positive isolates of Actinomycetes. The isolated microorganism is Gram-positive, branching and filamentous bacteria, it has also shown positive results in methyl red, indole test, starch hydrolysis, vogus-proskauer test, catalase test and triple sugar iron test and shown negative result in fermentation of citrate, the results were represented at Table 1.

Out of three isolates, the one isolate was showed in positive result. The selected isolate was performed in whole cell immobilization and free cells. The isolates were selected for fermentation based on their broad spectrum of activity and largest zone of inhibition.

Fig. 1:	(a-c) Isolation and screening of microorganism

Table 1:	Bio chemical characterization of Actinomycetes strains from soil sample

Fig. 2:	Antibacterial activity for free and immobilized cell extract

Fig. 3:	Isolation of crude protein from Actinomycetes. L1: Marker (100 kDa); L2 to L7: Crude protein from Actinomycetes

The isolates were cultivated in specific fermentation liquid medium for 120 h. After fermentation process, the Antibacterial compound was purified from the filtrate by solvent extraction method. The isolate showed the activity against tested organisms. The determination of antibiotic activity of selected strain was determined using Escherichia coli MTCC 50, Pseudomonas aeruginosa MTCC 424 and Bacillus subtilis MTCC 441. The comparative studies on the total antibiotic production with free and immobilized cells are shown in Fig. 2. It was found that, the immobilized cells in sodium alginate were more efficient for the production of antibiotics, which was conformed by zone of inhibition on bacterial culture plates.

The positive Actinomycetes strains were prepared in crude protein and the protein profile were determined by SDS-PAGE method. The results were identical to that of the same genous from various species. The crude protein separated has different molecular weight from lane 2 to 7 (Fig. 3).

DISCUSSION

The composition of an arginine-glycerol-salt medium (AGS), Suitable for the selective isolation of aerobic Actinomycetes, was given. When soil samples were treated with calcium carbonate and plated on the AGS medium, higher total and relative plate counts of Actinomycetes were obtained than when other media and methods were used (El-Nakeeb and Lechevalier et al., 1962). In this present study, we are using AGS medium for isolation of Actinomycetes from soil sample. The results were observed as filamentous, branching bacteria with a fungal type of morphology. Various biochemical tests were performed, to identify but it was unable to identify the Actinomycetes up to species level due to the lack of other tests. Apart from proper identification of genera and species of Actinomycetes, besides morphological and physiological properties (Kuster, 1972). In this study, we use various biochemical tests such as Starch hydrolysis, Triple sugar iron test, Fermentation of citrate were performed for the identification of the potent isolates (Abbas, 2006). Some of the Actinomycetes are characterized by the production of various pigments on natural or synthetic media. Then, the isolates were cultivated in fermentation liquid medium.

To demonstrate use of immobilized cells of S. rimosus for the continuous production of oxytetracycline it would be improved with calcium alginate immobilization in submerged fermentation compared with free cells. The results showed that in 1 mL culture broth, free cells produced 121 to 124 μg of oxytetracycline; where as immobilized cells produced 153 to 252 μg (Yang and Yueh, 2001). In this study, we are using the whole cell immobilization technique in an efficient way to overcome the difficulty in producing antibiotics by continuous fermentations with free-cells, since the growth and metabolite yields, have been immobilized various microbial species on different support matrices for antibiotic production. The Actinomycetes isolates, which are often encounted, show antibiotic activity on agar but not in liquid culture.

In this present study, six Actinomycetes strains were isolated from soil available in the local area and screened with regard to their potential against Gram-positive and negative bacteria. Among the six isolated strains (A1, A2, A3, A4, A5 and A6) three isolates A2, A3 and A5 were found to be effective against tested organisms. These three strains were further cultivated in fermentation liquid medium for 120 h and immobilized using sodium alginate (Manjula et al., 2009). A comparative profile on the total antibiotic sensitivity of the free cells and immobilized cells showed that the immobilized strains were found to be effective against the tested microorganism then the free cells. Further, the most potent of the producer strain was selected and identified, based on the cultural and physiological characteristics. From the results, it was concluded that the three isolates showed very promising activities against tested multi-drug resistant bacteria.

The immobilized cells of Actinomycetes were found to be more efficient for the production of antibiotics with batch fermentation. The possible routes of antibiotic synthesis and its biological efficiency are currently under investigation (Pandey et al., 2001). In the present study also we are using the same methods, the results of screening method was that most of the active isolates were active against Gram-positive and Gram- negative pathogen (Escherichia coli MTCC 50, Pseudomonas aeruginosa MTCC 424 and Bacillus subtilis MTCC 441). The morphological differences between these microorganisms show that different sensitivity occurred. The reason is that Gram-negative bacteria have an outer polysaccharide membrane carrying the structural lipo polysaccharide compounds. This makes the cell wall impermeable to lipophilic solutes.

The Gram-positive should be more susceptible and only an outer peptide glycon layer which is not an effective permeability barrier. The Actinomycetes isolates are often encounted and show antibiotic activity on agar but not in liquid culture. The results of screening method was that most of the active isolates were active against Gram-positive and Gram-negative pathogen (Escherichia coli MTCC 50, Pseudomonas aeruginosa MTCC 424 and Bacillus subtilis MTCC 441).

Due to the morphological differences between these microorganisms’s different sensitivity occurred. The reason was that a Gram-negative bacterium has an outer polysaccharide membrane carrying the structural lipopolysaccharide compounds. This makes the cell wall impermeable to lipophilic solutes. The Gram-positive should be more susceptible having only an outer peptide glycon layer which is not an effective permeability barrier. From the results, it is concluded that the immobilized cells of Actinomycetes are more efficient for the production of antibiotics. The possible routes of antibiotic synthesis and its biological efficiency are currently under investigation. The crude protein samples were determined by using SDS-PAGE method (Laemmli 1970).

ACKNOWLEDGMENTS

I express my grateful and my heartfelt gratitude to Dr. N.Selvan, M.Sc., Ph.D., Head, Centre for Biotechnology, Muthayammal College of Arts and Science for his immense help for the completion of my project.