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Antimicrobial Activities of Microbial Strains Isolated from Soil of Stressed Ecological Niches of Eastern Uttar Pradesh, India

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Antimicrobial Activities of Microbial Strains Isolated from Soil of Stressed Ecological Niches of Eastern Uttar Pradesh, India Indian Journal of Experimental Biology Vol. 47, April 2009, pp. 298-303 Antimicrobial activities of microbial strains isolated from soil of stressed ecological niches of Eastern Uttar Pradesh, India Vineeta Singh, Vandana Praveen, Jaspreet Banga & C K M Tripathi* Division of Fermentation Technology, Central Drug Research Institute, Chattar Manzil Palace, PO Box 173, Lucknow 226 001, India Received 1 September 2008; revised 12 January 2009 Antimicrobial activities of twenty bacterial strains isolated from ten different stressed agro-ecological niches of Eastern Uttar Pradesh, India were evaluated against bacteria, yeasts and molds. Eleven isolates showing strong antimicrobial activities were characterized. Eight antifungal compounds were purified and partially characterized by Ultra-Violet (UV) absorption spectra and grouped into polyenes and non-polyenes. Antibacterial metabolites produced by four isolates were purified and chemically characterized, of which one isolate (AB) produced a new form of olivanic acid, and other three isolates (C5, Py and M4) produced antibacterial compounds having phenoxazone nucleus. Keywords: Antibiotic production, Antimicrobial activity, Olivanic acid, Polyenes. Frequent clinical and veterinary use of antibiotics plating to eliminate very common microbes. One promotes the development of antibiotic resistances in gram of each soil sample was suspended in 10 ml of infectious microbial strains in hosts and eventually in normal saline and distributed in aliquots, one aliquot the environment which necessitate search for novel was treated by heat (1hr at 120ºC) and the other was antibiotics through natural, synthetic or semi- treated with 1·5% phenol (30ºC, 30 min) as described synthetic sources. The microorganisms, especially by Hayakawa et al.5. The pretreated soil samples were actinobacteria, are the most potential source for plated by serial dilution method on actinomycetes production of natural therapeutic agents. Traditional isolates agar (AIA), nutrient agar (NA), sabouraud screening methods lead to isolation of common dextrose agar (SDA), potato dextrose agar (PDA), microbes producing known compounds1,2. Selective czepecdox agar (Himedia) and yeast glucose malt isolation of rare microorganisms may allow the agar (glucose, 4; yeast extract, 4; malt extract, 10; 3,4 -1 discovery of new bioactive metabolites . CaCO3, 2; and agar, 20 gl ). Plates were incubated at In this investigation an attempt has been made to 28ºC for 1-2 weeks. The colonies selected on the basis screen bacterial strains isolated from different of morphological features were purified and subjected unexplored agro-ecological niches of Eastern Uttar to screening for antimicrobial activity. Pradesh, India by subjecting the soil sample to Antimicrobial activities of the axenic cultures were physico-chemical treatments. Potential antibiotic determined by perpendicular streak method against producing strains were characterized and active different strains of Bacillus subtilis, Staphylococcus metabolites produced by them were purified and aureus, Escherichia coli and Candida albicans. partially characterized. Strains showing moderate to good activity were selected for secondary screening, which was Materials and Methods performed by agar well method using 100 μl of their Isolation and screening of cultures−Soil samples fermented broth against different test organisms. collected from different geographical areas were Activities of the strains were compared with that of pretreated with chemical and physical methods before known antibiotics, erythromycin (E15), ampicillin ___________________ (A10), amphotericin B (AmB100) and fluconazole *Correspondent author (Fu10), (Table 1). Telephone: +91 522-2624198, Fax: +91 522-2623938, 2623405 Characterization and identification of selected E-mail: [email protected] strains−The producer strains selected from secondary SINGH et al.: ANTIMICROBIAL ACTIVITY OF MICROBIAL ISOLATES 299 Table 1—Activity profile zone of inhibition (zoi) of the producer strains zoi (in mm) Known Test strains Soil Isolates antibiotics [A] BACTERIA Sb Sd SM3 Md T1 C5 Py CR M4 B4 B5 B8 B14 B17 B27 C8 AB 5A 5W A17 E 15 A10 Bacillus subtilis ATCC 6633 28 24 25 16 31 35 40 26 38 21 20 30 21 23 30 16 35 21 26 20 30 28 Bacillus subtilis ATCC 11774 - 14 27 19 29 36 38 28 29 14 22 25 - - 13 - 24 15 16 25 30 27 Bacillus coagulans MTCC 2302 12 24 34 22 28 32 36 28 35 24 30 26 - 25 - - 36 - - 28 - 25 Escherichia coli MTCC 64 14 - 25 - 22 27 28 26 30 16 23 24 - 16 14 - - - 17 20 15 15 Escherichia - 15 17 19 25 25 28 26 - 17 10 27 - 15 14 - - - - - - 15 coli MTCC 739 Escherichia coli ATCC 1303 14 - 30 - 24 22 30 26 26 16 25 20 15 - 15 - 23 15 15 22 20 - Escherichia coli ATCC 1304 12 17 25 19 22 - - - 30 16 20 25 - 13 - - 20 16 15 20 20 12 Klebsiella - - - - 20 21 24 18 - - - - - - - - - - - - - - pneumoniae ATCC13884 Micrococcus - - 17 - 26 28 30 24 28 16 14 27 - 17 - 12 26 15 18 - 33 20 flavus ATCC 10240 Proteus 16 16 - 20 25 35 38 27 39 - - - - - - 14 32 - 23 - 12 25 vulgaris ATCC 6380 Pseudomonas - - - - - - - - - 27 - 31 - - 21 - 21 15 17 - 15 22 aeruginosa MTCC 424 Salmonella - - - - 18 30 30 22 28 - - - - - - - 35 22 25 - - 25 typhi ATCC 6539 Staphylococcus 21 - 30 - 23 28 28 24 35 - 28 - 14 - - 21 32 16 19 24 35 30 aureus ATCC 12598 Staphylococcus - - 17 - 24 25 28 22 35 - 10 - - - - - 42 14 19 - 32 30 aureus MTCC 96 (-) – Not active 300 INDIAN J EXP BIOL, APRIL 2009 Table 1—Activity profile zone of inhibition (zoi) of the producer strains—Contd zoi (in mm) Known Test strains Soil Isolates antibiotics [B] FUNGI Sb Sd SM3 Md T1 C5 Py CR M4 B4 B5 B8 B14 B17 B27 C8 AB 5A 5W A17 AmB Fu Alternaria - - - - 32 - - 20 - - - - - - - - - - - 28 28 20 alternata MTCC 1362 Beauveria 12 - - - - - - - - - - - - - - - - 25 40 - - - nivea MTCC 557 Candida albicans ATCC 24433 27 22 25 - 35 - - 28 24 - 25 21 - - - - - 27 29 23 28 21 Candida - - 20 - 26 - - 25 28 - 20 17 - - - - - 25 40 20 25 18 albicans ATCC 10231 Candida - - 30 - 26 - - 28 30 - 26 20 - - - - - - 15 26 25 15 albicans ATCC 90028 Candida - - 18 - 22 - - 22 26 - 17 - - - - - - 24 27 24 25 15 albicans ATCC 24433 Candida - - 25 - 28 - - 26 23 - 26 - - - - - - 24 24 26 25 12 albicans MTCC 183 Candida - - - - 22 - - 21 22 - - - - - - - - 17 19 - 20 12 tropicalis MTCC 184 Cryptococcus - 29 - - 20 - - 22 23 - - 24 - - - - - 32 35 - 12 12 terreus MTCC 1716 Fusarium - - - - - - - - - - - - - - - - - - 28 - 21 12 moniliforme MTCC 156 Penicillium - - 25 - - - - - 22 - 20 - - - - - - - - 20 - - ochroc-hloron MTCC517 Rhizoctonia - - - - 34 - - 22 - - - - - - - - - - - - - - oryzae MTCC2162 Saccharomyces - - 24 - - - - - 25 - 20 - - - - - - 23 27 20 20 15 cerevisiae MTCC 307 Trichophyton - - 40 - 27 - - 25 28 - 30 - - - - - - 23 - 35 32 15 rubrum MTCC 296 (-) – Not active SINGH et al.: ANTIMICROBIAL ACTIVITY OF MICROBIAL ISOLATES 301 screening were characterized by morphological, A17, B5, SM3 in nutrient broth (Himedia) by biochemical, cultural and physiological tests inoculating the production medium with seed according to the methods described by Shirling and inoculums of the producer strains. Inoculated flasks Gottlieb6 and Holt et al.7. Strains were characterized were incubated at 28ºC; 180 rpm for 72 hr and growth and deposited in Microbial Type Culture Collection kinetics along with antibiotics production were (MTCC), IMTECH Chandigarh, India. studied. Extraction, purification and chemical Cell cultivation and antibiotic production− characterization of the active compounds−Fermented Submerged fermentation was carried out in culture was centrifuged at 8000 rpm for 20 min to Erlenmeyer flasks for production of active separate cells from the fermented broth. Antimicrobial metabolites. Fermentation of the active strains AB, metabolite was recovered from the fermented broth by 5A, 5W, M4, T1, C5, Py, CR were carried out in a two phase solvent extraction using ethyl acetate. Cells complex medium (Soybean meal, 10 g; CaCO3, 3 g; were extracted with methanol. The solvents MgSO4.7H2O, 0.5 g; (NH4)2HPO4, 0.5 g; NaCl 3, g; containing active compounds were concentrated in K2HPO4, 1 g; glycerol, 15 ml; DW 1l, pH 6.9-7.0) and vacuum to give dried crude materials. Table 2—Biochemical and physiological characteristics of the producer strains Characteristics AB 5A 5W M4 T1 C5 Py CR A17 B5 SM3 Assimilation Arabinose + - - 2+ - 2+ 3+ - + + + Dextrose 2+ 4+ 3+ 3+ + 4+ 3+ - + + + Fructose - - - + + + 4+ + + + + Galactose 3+ - - 2+ + 3+ 4+ + + + + Inositol + + + + + - 3+ + + + + Mannose - - - + + 4+ 3+ + + + + Raffinose + + + - 2+ 2+ 4+ + + + + Rhamnose + - - + - 4+ 4+ - + + + Salicin 4+ 4+ 2+ + + + 3+ + + + + Sucrose 4+ 4+ 2+ 2+ 2+ 2+ 2+ + + + + Xylose 3+ - - + 2+ 2+ + 2+ + + + Utilization Citrate - - - - - - - - 3+ 3+ 3+ Asparagine 3+ + + + 3+ 3+ 3+ - - - - Oat meal 2+ 2+ 2+ 3+ 5+ 2+ 3+ 2+ - - - Enzyme production Catalase + + + + + + + + + + + Protease + + + + + + + + + + + Urease 3+ 2+ 2+ + 3+ - - + + + + Amylase 3+ 3+ 3+ 3+ 4+ 3+ 2+ + + + 3+ Tyrosinase 3+ + + - 4+ - 3+ + 3+ + - Gram reaction + + + + + + + + - - - Nitrate reduction 3+ + + + + - - + 3+ 3+ 3+ pH tolerance 7 2+ 2+ 2+ 2+ 2+ 3+ + + 3+ 3+ 3+ 8 2+ 2+ 2+ 2+ 2+ 2+ 2+ + 3+ 3+ 3+ 9 2+ 2+ 2+ 2+ 2+ + + + 3+ 3+ 3+ NaCl tolerance 1% 2+ 2+ 2+ 2+ 2+ 2+ 2+ + 3+ 3+ 3+ 3% 2+ + + 2+ 2+ 2+ + + 2+ 2+ 2+ 5% 2+ - - + + + - + + - - 7% - - - - - - - - - - - Growth at 27ºC 2+ 2+ 2+ 2+ 2+ 2+ 2+ 2+ 2+ 2+ 2+ 37ºC 2+ + + 2+ 2+ + + 2+ 2+ 2+ 2+ 50ºC - - - - - - - - - - - (+) - Normal growth; (2+, 3+, 4+, 5+) - comparative dense growth; (-) - No growth 302 INDIAN J EXP
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