Journal of Microbiology and Biotechnology Research

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J. Microbiol. Biotech. Res., 2011, 1 (2): 74-79 (http://scholarsresearchlibrary.com/archive.html) ISSN : 2231 3168 CODEN (USA) : JMBRB4

Antagonistic activity of actinomycetes isolates against human pathogen

Usha Rakshanya. J* Hema Shenpagam .N** and D. Kanchana Devi ** ** Department of Microbiology, Hindusthan College of Arts and Science, Coimbatore ______________________________________________________________________________ ABSTRACT The present study was designed to isolate actinomycetes and evaluate their antimicrobial activity against different pathogenic bacteria. Actinomycetes isolates were identified as Streptomyces sp, Micromonosporasp, Nocadia sp. The cell free supernatant of the selected isolate were able to inhibit the growth of all human pathogens ( S.aureus, Proteus vulgaris, P.aeroginosa, E.coli, B.subtilis, B.megaterium, K.pneumoniae, C.albicans, A.niger, S.cervisiae) used in this study. Out of the 5 actinomycetes isolates Streptomyces sp( S1) exhibited high antibacterial activity against Staphylococcusaureus. The remaining isolates also showed very promising activity against human pathogens Key words: Actinomycetes , screening, antibacterial activity, pathogens ______________________________________________________________________________ INTRODUCTION Actinomycetes are gram-positive bacteria frequently filamentous and sporulating with DNA rich in G+C from 55-75%P [1].Actinomycetes are prokaryotes which have a hyphal morphology. Most of the actinomycetes are unparalleled sources of bio-active metabolites including antibiotics, plant growth factors and other substances[2].They are soil microorganisms and are active in the decomposition of plant tissues and thereby in the recycling of carbon and nitrogen [3] Streptomyces and other actinomycetes are major contributes to biological buffering of soils and have role in organic matter decomposition conductive to crop production [4].Actinomycetes are known to produce bioactive substances, especially antibiotics that are effective against phytopathogenic fungi [5]. During the last 40 years, more than 1000 substances and preparations which posses antibiotic properties, i.e., have the capacity to inhibit the growth of and even to destroy various microorganisms, in dilute solutions, have been isolated from culture of an actinomycetes may, therefore, be said to have been isolated in 1940. It was designated as actinomycin by [6] 74
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Usha Rakshanya. J et al J. Microbiol. Biotech. Res., 2011, 1 (2):74-79 ______________________________________________________________________________ Among all the known microbes ,members of the actinomycetes genus especially Streptomyces sp. have been recognised as prolific producer of useful bioactive metabolite with broad spectrum of activities which has antibacterial, antifungal, antibiotic ,antiphrastic antitumor, antiviral, insecticide, herbicide, immunomodulators, antithrombatic agents [7-10]. Thus screening and isolation of promising strains of actinomycetes with potential antibiotics is a thrust area of search since many years [11-14] The aim of this present study was to isolate, identify and characterize the actinomycete isolates from different soil samples and to determine their antagonistic activity against selective human pathogenic micro-organism. MATERIALS AND METHODS Sample collection: Soil samples were collected from different areas of Coimbatore and stored in the sterilized glass bottles and refrigerate at 4C during 2010 -2011. Isolation of actinomycetes from soil samples: The medium used for the isolation and cultivation of actinomycets was starch casein agar medium contains starch 1.0%, casein 0.1%, agar 2.0. After autoclaving, the medium was supplemented with 50 and 20 g ml-1 of tetracycline and nystatin respectively as antibacterial and antifungal agents to inhibit the bacterial and fungal contamination. Soil samples serially diluted and from each dilution 0.1 ml was taken and spread evenly with sterile L -shaped glass rod over the surface of starch casein agar and kept for incubation at 36C Repeated streaking method was used to purify the soil actinomycetes colonies. Morphology and biochemical characterization: Actinomycetes isolates were incubated on starch casein agar and incubated at 30C for 7 days The strain was carried out for gram staining, shape, size by under light microscope. Various biochemical tests were performed for the identification of various actinomycetes isolates . The mycelium structure , colour and arrangement of spore on the mycelium were examined under oil immersion . Different biochemical tests like starch hydrolysis ,triple sugar iron , Citrate utilization test, Indole test, methyl red test, Vogus- Proskauer test , catalase test were performed to characterize actinomycetes[15]. Test organism: The selective human pathogenic microorganism used for antagonistic activity were S.aureus, Proteus vulgaris, P.aeroginosa, E.coli, B.subtilis, B.megaterium, K.pneumoniae, C.albicans, A.niger, S.cervisiae. Antagonistic activity: Actinomycetes isolates were grown in 50 ml of starch casein broth by submerged culture containing in 250 ml flasks by incubating at 32C for 7 days and centrifuged at 10,000 rpm for 15 min and the clear supernatant broth samples were tested for their antagonistic activity against the selected pathogens by agar well diffusion method [16] wells of 6mm diameter were prepared in the nutrient agar plates 7 the test pathogenic bacterial and fungal cultures were swabbed on to the nutrient agar surface [17]and the wells were filled with the 50l of culture 75
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Usha Rakshanya. J et al J. Microbiol. Biotech. Res., 2011, 1 (2):74-79 ______________________________________________________________________________ supernatant and the diameter of inhibition zones were measured after incubation for 24 h at 37c for the bacterial species and 48h at 28C in the case of fungal species. RESULT AND DISCUSSION Isolation and screening of actinomycetes : The soil samples collected from different areas of Coimbatore, actinomycetes isolates were obtained in pure form and analysed for their antimicrobial activities .They were preserved at 4C. All the isolates were found to be gram positive and showed branched mycelium in their cell morphology [18].5 isolates which exhibited high activity against all the pathogens were selected for the further studies. According to Bergeys manual of determinative bacteriology and the laboratory manual for identification of actinomycetes, the isolates were identified as Nocardia, Micromonospora, Streptomyces sp. The colony was white to grey in colour in Micromonospora (Table 1,2,3). Biochemical characterisation of actinomycetes: The 5 isolates obtained were subjected to study morphological, physiological, biochemical characteristics (table 1,2,3). Based on the above results purified isolates of actinomycetes were identified as Micromonosporasp, Streptomyces sp (S1), Streptomyces sp (S2), Streptomyces sp(S3),Nocardiasp.Most of the isolates were efficient in hydrolysing starch except Nocardia. Indole production was strictly negative, Methyl red was strictly positive, Vogus- Proskauernegative ,Citrate positive, nitrate positive, catalase was positive in all the isolates . Determination of Antimicrobial activity: With the increasing use of antibiotics, the serious problem of antibiotic resistance is gradually increasing. All the strains were able to produce antibiotic against gram-positive and gramnegative organism.The formation of inhibition zone around the pathogenic strains is due to the production of secondary metabolites by actinomycetes isolates. Thus the result of present investigation revealed that soil actinomycetes are the potent source of novel antibiotics and were found to be of potential antagonistic against test organisms which can control variety of pathogenic organisms.
Table :1 Morphology characters Organism Micromonosporasp Mycelium and nature of colony Septate, branched, Coloured aerial mycelium Extensively branched,floccae Aerial substrate Mycelium Smooth, granular Aerial &substrate Mycelium Smooth, spirally twister Colour of mycelium White to grey Ash Type of spore Monosporophore Pigmentation Yellow Gram stain + Cell wall sugar Galactose, xylose galactose

Streptomyces sp (S1)

Short chain of spores

Brown

Streptomyces sp(S2) Streptomyces sp(S3) Nocadiasp

White pink

Long chain spore

Wine red

arabinose

Smooth, hairy, Long chain spore Yellow + galactose raised wrinkled Aerial mycelium The different actinomycetes colony showed varied colour and morphology which is given in the above table.

White yellowish Brown White

Short chain spore

White

galactose

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Usha Rakshanya. J et al J. Microbiol. Biotech. Res., 2011, 1 (2):74-79 ______________________________________________________________________________

TABLE : 2 Biochemical characters Isolates Micromonospora sp Streptomyces sp (S1) Streptomyces sp (S2) Streptomyces sp (S3) Nocardiasp Indole MR + + + + + VP Citrate + + + + + catalase + + + + + oxidase + + + + + nitrate + + + + Starch Weakly + + + -

+ positive , - negative Based on the above biochemical characteristics the strains were identified as Micromonosporasp, Streptomyces sp (S1), Streptomyces sp (S2), Streptomyces sp(S3),Nocardiasp Table :3Utilization of sugar and acid production Human pathogens Micromonospora sp 23 10 12 17 23 20 18 20 16 15 Measurement of zone of inhibition in cm Streptomyces Streptomyces Streptomyces sp (S1) sp (S2) sp (S3) 24 14 19 9 7 8 14 10 12 16 10 14 20 12 16 22 12 15 20 18 16 19 6 14 10 16 5 12 8 15

S.aureus P.vulgaris P.aeruginosa E.coli B.subtilis B.megaterium K.pneumoniae C.albicans A.niger S.cerevisae

Nocardia sp 7 8 18 20 13 15 15 14 12 10

Table: 4 Antagonistic activity of actinomycetes isolates against human pathogens Human pathogens Micromonospora sp 23 10 12 17 23 20 18 20 16 15 Measurement of zone of inhibition in cm Streptomyces Streptomyces Streptomyces sp (S1) sp (S2) sp (S3) 24 14 19 9 7 8 14 10 12 16 10 14 20 12 16 22 12 15 20 18 16 19 6 14 10 5 8 16 12 15

S.aureus P.vulgaris P.aeruginosa E.coli B.subtilis B.megaterium K.pneumoniae C.albicans A.niger S.cerevisae

Nocardia sp 7 8 18 20 13 15 15 14 12 10

Values are the average of three replicates. Out of the 5 actinomycets isolates Streptomycessp (S1) exhibited high antibacterial activity against Staphylococcusaureus(24mm) whereas in Micromonosporasp showed more inhibition towards Staphlococcusaureus(23mm)and Bacillus subtilis(23mm). Nocardiasp exhibit high antimicrobial activity towards E.coli(20mm). 77
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Usha Rakshanya. J et al J. Microbiol. Biotech. Res. 2011, 1 (2):74-79 Res., ______________________________________________________________________________ The crude extract of the antimicrobial substances recovered from the cells of the actinomycetes isolates were tested for their inhibitory activity against the test organisms and the results are activity presented in the table 3. The crude extract of Streptomyces S1 strain showed strong antib antibacterial against gram positive andgram negative bacteria [19,20] than other isolates. gram Streptomycessp were highly susceptible to gram positive (P.aeruginosa andP.fluroscence) and aeruginosa showed moderate activity against gram negative bacteria ( (S.aureus)[21 Different types of [21]. Streptomyces strains identified [22]were S.lydicus, S.lavendulae, S.albus, S.antibioticus, S.diastaticus and S.phaeochromogenes reported that has been showed various degree of S.phaeochromogenes antibacterial activity .Thus in our present investigation, the result indicates that soil is a good source for isolation of actinomycetes and found that Nocadia,Micromonospora Micromonosporaand Streptomyces were present but predominant of actinomycetes actinomycetespopulation in Streptomyces sp. The antibacterial substances present in the actinomycetes isolate could be used to inhibit the growth of human pathogen. Antagonistic activity of actinomycetes isolates against human pathogens
25

20

15 Micromonospora sp Streptomyces sp (S1) 10 Streptomyces sp (S2) Streptomyces sp (S3) Nocardia sp 5

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