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 BIOL, APRIL 2009

The crude preparations obtained were passed deduce some preliminary information on structure of through Sephadex LH-20 column (Pharmacia) using the compounds. All the active metabolite contained methanol as an eluting agent at 3 ml/min flow rate carboxyl or peptide moiety in the compounds as they and 254 nm and fractions collected were bioassayed showed maximum absorbance between 205-216 for activity by disc diffusion method against B. nm8,9. Absorbance band near 253 nm indicated subtilis ATCC 6633 and C. albicans ATCC 24433. presence of benzene moiety in 5A, T1 and B5 and Purity of the active fractions was further examined on conjugated absorption pattern of UV profile indicated high pressure liquid chromatography (HPLC) using polyene nature10 in the antifungal compounds of M4, reverse phase silica column (RP18). UV spectrum of 5A, 5W, CR and A17, whereas absence of these different antibacterial and antifungal compounds were conjugated absorption suggested non-polyene nature determined in methanol with a Perkin Elmer Lambda- of compounds of T1, B5 and SM3. Presence of 25 UV spectrophotometer at 200-500 nm UV-Visible phenoxazone nucleus in the antibacterial compounds range. of M4, C5 and Py were confirmed by the absorption hump at 443 nm11. Results and Discussion Cultural, biochemical and morphological tests Antimicrobial activity−Sixty-two microbial strains, revealed that out of eleven strains, eight belong to including bacteria, actinomycetes and fungi, isolated gram positive Streptomyces genus and three to gram from ten soil samples, were subjected to primary negative bacteria. Out of eleven strains four strains screening against Bacillus subtilis ATCC 6633, AB (Streptomyces olivaceus MTCC 6820), M4 Staphylococcus aureus ATCC 12598, Escherichia (Streptomyces capoamus MTCC 8123), Py coli MTCC 1304 and Candida albicans ATCC 24433. (Streptomyces annulatus MTCC 6818) and C5 Out of these twenty cultures showing moderate to (Streptomyces halstedii MTCC 6817) were high activity subjected to secondary screening characterized through 16S rDNA homology studies at (Table 1), and eleven strains (AB, 5A, 5W, M4, T1, Microbial Type Culture Collection (MTCC) C5, Py, CR, A17, B5 and SM3) showing strong Chandigarh. The strains of S. olivaceus are known to activity were selected for further studies. Results of produce elloramycin (polyketide), kanchanamycins cultural along with biochemical and physiological (polyol macrolide), rabelomycin (anthraquinone) and characteristics of the above selected active strains beta-lactam compounds (carpetimycin or olivanic (Table 2) confirmed the diversity of the strains. acids)12-15. The compound purified from our isolate Chemical characterization of the active (S. olivaceus MTCC 6820) was identified as olivanic compounds−Antimicrobial compounds were purified acid having different molecular weight than other and partially characterized by observing UV olivanic acids as reported earlier16. S. halstedii is absorbance (λmax) and absorption pattern (Table 3) to known to produce vicenistatins (macrolactum

Table 3—UV absorbance (MeOH) of the isolated metabolites polyketide glycoside), aclidinomycins (naphthyridinomycin)17,18. S. capoamus produces ciclamycin, Strains Nature of compound UV absorbance (nm) ciclacidine and capoamycin (anthracyclin)19-21 and isolated Maximum Shoulders S. annulatus is reported to produce endophenazines 22 AB Antibacterial 211 403, 487 A~D, tubermycin B (phenazine) . Antibacterial 5A Antifungal 208 257, 314, 328, compounds produced by the above three strains 345 contains phenoxazone nucleus confirmed by 5W Antifungal 210 260, 310, 320 absorbance at 443 in UV, whereas the compounds M4 Antibacterial 205 434 reported from these strains did not contain this ring, Antifungal 210 318, 334, 352 confirming some novelty with the reported ones. T1 Antifungal 211 266 Antifungal activities are reported from S. halstedii C5 Antibacterial 210 443 and not from S. capoamus. The metabolites isolate S. Py Antibacterial 210 443 capoamus contained polyene antifungal compounds, CR Antifungal 204 339, 356, 376, and that of S. halstedii were strictly antibacterial. 399 A17 Antifungal 218 243, 314, 327 Acknowledgement B5 Antifungal 216 250, 328 This study was financially supported by Council of SM3 Antifungal 216 238, 330 Scientific and Industrial Research, India. The authors

SINGH et al.: ANTIMICROBIAL ACTIVITY OF MICROBIAL ISOLATES 303

would like to thank Mr. D N Vishwakarma, (STO), 4018 I. Taxonomy, fermentation, isolation and biological Medicinal and Process Chemistry Division and Mr. R activities, J Antibiot, 49 (1996) 758. 13 Hood J D, Box S J & Verrall M S, Olivanic acids, a family of K Purushottam, SAIF, CDRI, Lucknow for HPLC β-lactam antibiotics with β-lactamase inhibitory properties purification. produced by Streptomyces species. II. Isolation and characterization of the olivanic acids Mm 4550, MM 13902 References and MM 17880 from Streptomyces olivaceus, J Antibiot, 32 1 Kurtboke D I, Chen C F & William S T, Use of polyvalent (1979) 295. phage for reduction of streptomycetes on soil dilution plates, 14 Liu W C, Parker W L, Slusarchyk D S, Greenwood G L, J Appl Bacteriol, 72 (1992) 103. Graham S F & Meyers E, Isolation, characterization, and 2 Okami Y & Hotta K, Search and discovery of new structure of rabelomycin, a new antibiotic, J Antibiot, 23 antibiotics, in Actinomycetes in Biotechnology, edited by M (1970) 437. Goodfellow, S T Williams and M Mordarski (Academic 15 Patallo E P, Blanco G, Fischer C, Brana A F, Rohr J, Mendez Press, London) 1988, 33. C & Salas J A, Deoxysugar methylation during biosynthesis 3 Bull A, Ward A C & Goodfellow M, Search and discovery of the antitumor polyketide elloramycin by streptomyces strategies for biotechnology: The paradigm shift, Microbiol olivaceus, J Biol Chem, 276 (2001) 18765. Mol Biol Rev, 64 (2000) 573. 16 Basker M J, Boon, R J & Hunter P A, Comparative 4 Cragg G M, Newman D J & Snader K M, Natural products in antibacterial properties in vitro of seven olivanic acid drug discovery and development, J Nat Prod, 60 (1997) 52. derivatives : mm 4550, mm 13902, mm 17880, mm 22380, 5 Hayakawa M, Yoshida Y & Iimura Y, Selective isolation of mm 22381, mm 22382 and mm 22383, J Antibiot, 33 (1980) bioactive soil actinomycetes belonging to the Streptomyces 878. violaceusniger phenotypic cluster, J Appl Microbiol, 96 17 Cang S, Ohta S, Chiba H, Johdo O, Nomura H, Nagamatsu Y (2004) 973. & Yoshimoto A, New Naphthyridinomycin antibiotics, 6 Shirling E B & Gottlieb D, Methods of characterization of aclidinomycin A and B from S. halstedii, J Antibiot, 54 Streptomyces species, Int J Syst Bacteriol, 61 (1966) 313. (2001) 304. 7 Holt J G, Krieg N R, Sneath P H A, Staley J T & Williams S 18 Shindo K, Kamishohara M, Odagawa A, Matsuoka M & T, Bergey’s mannual of determinative bacteriology (William Kawai H, Vicenistatin; a novel 20-membered macrocyclic and Wilkins, Baltimore) 1994 lactum antitumor antibiotic, J Antibiot, 46 (1993) 1076. 8 Bikova T & Treimanis A, UV-absorbance of oxidized xylan 19 Gonclaves D L, Delle M F, D’Albuquerque I L & Marini and monocarboxyl cellulose in alkaline solutions, Bettolo G B, Identification of ciclacidine antibiotic from Carbohydr Polym, 55 (2004) 315. Streptomyces capoamus, Tetrahedr Lett, 4 (1968) 471. 9 Becklin R R & Desiderio D M, The amount of ultraviolet 20 Hayakawa Y, Iwakiri T, Imamura K, Seto H & Otake N, absorbance in a synthetic peptide is directly proportional to Studies on the isotetracenone antibiotics I. Capoamycin, a its number of peptide bonds, Anal Lett, 28 (1995) 2175. new antitumor antibiotic, J Antibiot, 38 (1985) 957. 10 Sharma Y R, Elementary organic spectroscopy: Principles 21 Martins C S & Maior A M S, Anthracycline production by and chemical applications (S Chand Press, New Delhi) 2001. Streptomyces capoamus in batch fermentation, Braz Arch 11 Tai J C, Craig W S & Gaber B P, On the electronic spectrum Boil Technol, 46 (2003) 483. of actinomycin D, J Am Chem Soc, 98 (1976) 7925. 22 Gebhardt K, Schimana J, Krastel P, Dettner K, Rheinheimer 12 Fiedler H P, Nega M, Pfefferle C, Groth I, Kempter C, J, Zeeck A & Fiedler H P, Endophenazenes A-D, new Stephan H & Metzger J W, Kanchanamycins, new polyol phenezine antibiotics from the arthropod associated macrolide antibiotics produced by Streptomyces olivaceus Tu endosymbiont S. annulatus, J Antibiot, 55 (2002) 794.