INTRODUCTION:

The discovery of novel antibiotic and other potential compound lead molecules of pharmaceutical interest through microbial secondary metabolite screening is becoming increasingly fruitful. There is wide acceptance that microorganisms are virtually unlimited sources of novel substances with many therapeutic applications. Among them actinomycetes hold a predominant position due to their diversity and had proven their ability to provide new and novel substances. Actinomycetes are aerobic, spore forming and gram positive bacteria that have DNA with a high GC content (69-73%).They form extensive branching substrate, aerial mycelia and widely distributed in soil [Oskay et al.,2004].

Actinomycetes have provided many important bioactive compounds of high commercial and continue to be routinely screened for new bioactive substances .Most of the actinomycetes have clinical applications on the basis of their activity against different kinds of microorganisms viz., antibacterial, antiparasite and antiviral [Campbell et al.,1984]. Keeping this point of view, the present study is focused on the diversity of actinomycetes in salt pan region of Kodiakarai and to detect their antibacterial activity against common bacterial pathogen. The selected actinomycetes has subjected to molecular characterization.

MATERIALS AND METHODS:

SAMPLE COLLECTIONS:

The soil samples were collected from salt pan environment of Kodiakarai, Vedaranyam, Nagapattinam [District], Tamilnadu, India. The soil samples were collected at radom, brought to the laboratory in sterile polythene bags and used for the analysis.

ISOLATION OF ACTINOMYCETES:

The 10 fold serial dilutions of the samples were prepared, using filtered and sterilized 50% sea water. One ml of filtered and serially diluted sample was plated in the stared casein agar [Kuster and Williams, 1964]. The plates were incubated at 28°C for seven days. After incubation, the selected colonies of actinomycetes were transferred from mixed culture of the plates and incubated at 28°C for 7 days. Plates containing pure cultures were stored at 4°C until further examination.

TEST ORGANISMS:

Antibacterial activities of selected actinomycetes were tested for in vitro against human bacterial pathogens that included Staphylococcus aureus, Escherchia coli and Klebsiella pheumoniae.

ANTIBACTERIAL ACTIVITIES OF ISOLATES:

The selected bacterial strains were pre-grown in nutrient broth at 37°C for 24hrs.Sterilized nutrient agar medium was poured into petridishes and after solidification the bacterial culture were evenly spread over appropriate media by using sterile cotton. Then the wells were punched in the bacterial spread medium with 3mm diameter gel puncher. A 100µl of actinomycetes broth cultures were pipetted into separate wells. Inoculated plates were incubated at 54°C for 24-48 hr. After incubation, the results were observed and measured the diameter of inhibition zone around the each well [Madigan et al.,1997].

MOLECULAR CHARACTERISATION:

ISOLATION OF DNA:

DNA of the actinomycete was isolated from cells grown in yeast malt extract broth from cells (YMB) with 0.2% of glycine [Yamada and Komagata 1970]. The isolated DNA was amplified by thermalcycler [PCR].

16S rDNA AND PHYLOGENETIC ANALYSIS:

The amplified DNA fragments were purified by gel electrophoresis and sequenced directly by using a Tag Dyedeoxy terminator cycle sequencing kit [applied biosystems] and previously described oligonucleotide primers. The 16s rDNA sequence of the test strain was aligned manually with available actinomycete nucleotide sequence retrieved from GENE /EBML/DDBJ database by CLUSTAL W version 1.81 program. Evolutionary tree was constructed using neighbor joining method [Saito and Nei,1987].

SECONDARY STRUCTURE PREDICTION:

The secondary structure of actinomycete was predicted using the bioinformatics tools available online www.genebee.msu.sulservices/rna2 reduced-html

RESTRICTION SITE ANALYSIS IN 16S rDNA:

The restriction sites in 16s rDNA of actinomycete were analysed using NEB culture program version 2.0 tools in online www.neb.com/NEB cutter2/index.php

RESULTS:

PHYSICO-CHEMICAL PARAMETERS:

The physico-chemical features of the test soil were given in Table-1

Table.1-Physico-chemical analysis of soil

S. No	Name of the parameter	Analytical values
1.	pH	7.82
2.	Electrical conductivity (dsm^-1)	0.18
3.	Organic carbon (%)	0.36
4.	Organic matter (%)	0.72
5.	Available Nitrogen (Kg/ac)	97.9
6.	Available Phosphorus (Kg/ac)	4.98
7.	Available Potassium (Kg/ac)	125
8.	Available Zinc (ppm)	0.98
9.	Available Copper (ppm)	0.59
10.	Available Iron (ppm)	4.53
11.	Available Manganese (ppm)	2.63
12.	Cat ion exchange capacity (c. Mole. Proton^- /kg)	18.9
Exchangeable bases (c. Mole. Proton^- /kg)
13.	Calcium	8.9
14.	Magnesium	7.2
15	Sodium	1.26
16	Potassium	0.22

ISOLATION OF ACTINOMYCETES:

A total of 20 different actinomycetes were recovered from salt pan region of Kodiakarai, Nagapattinam [District] using starch casein agar medium [Table-2]. From 20 isolated actinomycetes 10 were dominant in their growth. Among the 10 actinomycetes Streptoverticillium album was highly dominant from their isolates [Table-3].

Table.2-Actinomycetes isolated from salt pan

S. No	Name of the actinomycetes
1.	Actinobispora yunnanensis
2.	Actinosynnema pretiosum
3.	Actinoplanes brasiliensis
4.	Agromyces ramosus
5.	Actinomadura citrea
6.	Catellatospora citrea
7.	Jonesia denitrificans
8.	Micromonospora echinospora
9.	Microtetrospora fastidiosa
10.	Nocardia amarae
11.	Pseudonocardia thermophila
12.	Saccharothrix australiensis
13.	Saccharomonospora viridis
14.	Saccharopolyspora hirsute
15.	Streptoverticillium album
16.	Streptomyces albus
17.	S. rochei
18.	S. anulatus
19.	S. cyaneus
20.	S. microflavus

Table.3-Dominant Actinomycetes in salt pan

S. No	Actinomycetes	Number of isolates
1.	Streptoverticillium album	25
2.	Streptomyces albus	20
3.	Saccharomonospora viridis	19
4.	Actinobispora yunnanensis	17
5.	Nocardia amarae	15
6.	Streptomyces rochei	15
7.	Saccharopolyspora hirsute	13
8.	Microtetrospora fastidiosa	12
9.	Actinomadura citrea	10
10.	Micromonospora echinospora	10

ANTIBACTERIAL ACTIVITY:

Antibacterial activity of 10 dominant actinomycetes were tested against 3 human pathogens. The highest antibacterial activity showed by Streptoverticillium album against Staphylococcus aureus. The maximum zone of inhibition 20mm was observed in S. aureus against S. album ,and minimum zone of inhibition 10mm was observed against K. pneumoniae and 6mm zone of inhibition in E. coli against [Table-4][Fig-1].

Table.4-Antibacterial activity of Actinomycetes

S. No	Name of the organism	Zone of inhibition (mm)
S. No	Name of the organism	*Staphylococcus aureus*	*Klebsiella* *pneumoniae*	*Escherichia coli*
1.	A. yunnanensis	5	7	-
2.	A.citrea	-	6	-
3.	M. echinospora	3	-	-
4.	M. fastidiosa	9	4	3.5
5.	N. amarae	-	-	-
6.	S. hirsuta	-	8.5	-
7.	S. albus	3.5	-	-
8.	S. album	20	10	6
9.	S. rochei	-	-	4.5
10.	S. viridis	7	-	-

MOLECULAR CHARACTERISATION OF S. ALBUM:

The molecular characterization of S. album was evaluated by PCR amplification of 16S rDNA gene. The genomic DNA and amplified products were separated in agarose gel. The 16S rDNA genes of S. album from the marine soil was partially sequenced using 16S rDNA sequence primer. The sequence of S. album was deposited in NCBI to get the accession number. The sequence comparisons with sequences in the EMBL database, the phylogenetic analysis (neigbhour joining tree) revealed that the sequence of the marine isolate is similar (98%) to the existing uncultured actinomycetes clone.

The secondary structure of 16S rDNA of S. album showed 33 loops, 33 stems and 24 hairpin loops. Totally 53 restriction enzyme sites were observed. The GC and AT content of S. album was found to be 53% and 44% respectively, using NEB cutter programme.

DISCUSSION:

Diversity of actinomycetes isolates was high, perhaps due to the nutritive status of the soil .The first report on marine actinomycetes was made by Nadson [1903] from the salt molds of St. Pedenburg. Other reports on the isolation of actinomycetes from marine soil were by Dhanasekaran et al. [2005]. Streptoverticillium album having antibacterial activity reported by Ting et al [2004] to isolation and characterization of actinobacteria with antibacterial activity from soil and rhizosphere soil. Our study also established the antibacterial activity of actinomycetes isolated from salt pan region. Totally 20 actinomycetes was isolated and determined the antibacterial activity against the three human pathogens. Streptoverticillium album showed the high antibacterial activity against the S. aureus. To conclude, results from our present study has shown that there is potential in sourcing for inhibitory compounds produced from actinomycetes and to identify the antibacterial components for further use.

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Received on 31.07.2011 Accepted on 24.08.2011

Asian J. Pharm. Tech. 1(3): July-Sept. 2011; Page 79-81