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Identification and Characterization of Soil-Isolated Streptomyces Sje177 Producing Actinomycin

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Identification and Characterization of Soil-Isolated Streptomyces Sje177 Producing Actinomycin CHARACTERIZATION OF STREPTOMYCES SJE177 IDENTIFICATION AND CHARACTERIZATION OF SOIL-ISOLATED STREPTOMYCES SJE177 PRODUCING ACTINOMYCIN Jirayut Euanorasetr1,2, Arjaree Nilvongse1,2, Srisurang Tantimavanich3, Takuya Nihira2,4, Yusuhiro Igarashi5 and Watanalai Panbangred1,2 1Department of Biotechnology, 2Mahidol University-Osaka University Collaborative Research Center for Bioscience and Biotechnology (MU-OU:CRC), Faculty of Science, 3Department of Clinical Microbiology, Faculty of Medical Technology, Mahidol University, Thailand; 4International Center for Biotechnology, Osaka University, 5Biotechnology Research Center, Toyama Prefectural University, Japan Abstract. One hundred seventy-seven actinomycetes strains were isolated from soils collected from fruit orchards in Thailand. All were tested for antibacterial activity against seven pathogenic bacteria using co-cultivation methods. Forty strains (22.6%) were active against at least one indicator bacteria. Twenty-seven strains (15.3%) inhibited only gram-positive bacteria, four strains (2.3%) inhibited only gram-negative bacteria, and nine strains (5.1%) showed activity against both. Strain SJE177 had potent activity against all tested bacteria, and was selected for further investigation. A crude ethyl acetate extract of this strain retained inhibi- tory activity as tested by disk-diffusion method. Analysis of morphological and biochemical characteristics and the 16S rRNA gene sequence indicated this strain belonged to the genus Streptomyces. The strain formed a monophyletic line in a phylogenetic tree of 16S rRNA gene sequences with other Streptomyces reference strains. High performance liquid chromatography (HPLC) analysis showed SJE177 produced actinomycin. Since many isolates showed inhibitory activity against indicator bacteria, these results suggest Thai soil could be an interesting source to explore for antibacterial substances. Key words: Streptomyces, antibacterial activity, co-cultivation method, actinomy- cin, Thailand INTRODUCTION the genus Streptomyces are potential sources for secondary metabolites possess- Streptomyces spp are gram-positive ing a variety of biological activities, includ- soil-dwelling filamentous bacteria with a ing antibacterial activity, which is used for complex cycle of morphological differen- human and animal treatment. It is esti- tiation (Claessen et al, 2006). Members of mated this bacteria synthesizes more than Correspondence: Watanalai Panbangred, De- 7,000 metabolites (Bérdy, 2005). Antibiotic partment of Biotechnology, Faculty of Science, resistant bacteria emerge clinically within Mahidol University, 272 Rama VI Road, months to years following their use Bangkok 10400, Thailand. (Walsh, 2000; Palumbi, 2001). There is an Tel: +66 (0) 2201 5927; Fax: +66 (0) 2201 5926 urgent need to study how to overcome the E-mail: [email protected] resistance of pathogenic bacteria and fungi Vol 41 No. 5 September 2010 1177 SOUTHEAST ASIAN J TROP MED PUBLIC HEALTH to commonly used antimicrobials. Identi- on Mueller-Hinton (MH) (Becton, fication of new antimicrobials and new Dickinson, Sparks, MD) plates at 4ºC. ecological niches are important. This in- Stock cultures were kept in 20% glycerol cludes discovery of antibiotic-producing at -80ºC. bacteria, mining of uncovered natural- Isolation of actinomycetes product biosynthetic clusters in bacterial One gram of each soil sample was genomes, and inhibitors of antibiotic efflux 10-fold serially diluted in saline solution, systems (Wright and Sutherland, 2007). 0.1 ml of each solution, at dilutions of Thailand is an agricultural country 10-4-10-6, then spread on selective media: located in the tropics and contains great Pridham medium [per 1 liter: 10 g glucose, ecological diversity. Relatively few scien- 10 g starch, 2 g (NH4)2SO4, 2 g CaCO3, 1 g tific studies have been carried out search- K2HPO4, 1 g MgSO4, 1 g NaCl, and 12 g ing for new antibiotics from microorgan- agar, pH 7.0] and water proline medium isms isolated from Thai soil, where acti- (per 1 liter: 10 g L-proline, 12 g agar, pH nomycetes, in particular Streptomyces spp, 7.0), then incubated at 28ºC for 7-14 days. is found abundantly. This study aimed to Both screening media were supplemented isolate Streptomyces from Thai soil and test with 25 µg/µl nalidixic and 50 µg/µl for antibacterial activity against multiple cyclohexamide to prevent growth of other drug resistant bacteria. Strain SJE177, bacteria and fungi, respectively. Typical showing potent activity against multiple Streptomyces colonies were picked and drug resistant bacteria, was selected for purified by streaking onto an agar plate biochemical and molecular characteriza- of Waksman medium (per 1 liter: 10 g glu- tion. cose, 5 g meat extract, 5 g peptone, 3 g NaCl, and 12 g agar, pH 7.0) and incubated MATERIALS AND METHODS at 28ºC for 7-14 days. The obtained isolates Soil sample collection and tested micro- were maintained on a preservation me- organisms dium: Seino’s agar slant (per 1 liter: 10 g Soil samples were collected from 4 starch, 3 g N-Z amine type A, 1 g meat provinces in 2006: Chanthaburi, Bangkok, extract, 1 g yeast extract, 3 g CaCO3, and Phetchaburi, and Nong Bua Lamphu. The 12 g agar). samples were taken at a depth of 5 centi- Antibacterial bioassay by co-culture meters from the surface in shaded areas method of fruit orchards. The seven indicator bacteria were Two standard strains (Staphylococcus grown in MH broth for 5 hours. A sterile aureus ATCC25923 and Escherichia coli cotton swab was dipped into a solution of ATCC 25922) and five multidrug resistant properly adjusted cell density and strains (methicillin-resistant S. aureus-2, swabbed on the MH agar surface. Isolated methicillin-resistant S. aureus-1302, E. coli- actinomycetes strains were previously 7, Acinetobacter-1275, and Pseudomonas grown on 301 seed medium (1 liter: 24 g aeruginosa-6) were used for the antibacte- starch, 10 g glucose, 3 g peptone, 3 g meat rial tests. Drug resistant strains were ob- extract, 5 g yeast extract, 4 g CaCO3, and tained from Siriraj Hospital (Bangkok, 12 g agar) for 7 days. Colonies of actino- Thailand). Their resistance profiles are mycetes were cut using a cork borer (8 mm shown in Table 1. They were maintained in diameter) and placed on the MH plate 1178 Vol 41 No. 5 September 2010 CHARACTERIZATION OF STREPTOMYCES SJE177 Table 1 Drug resistance profiles of indicator bacteria. Indicator bacteria Resistance profile Gram-positive bacteria S. aureus ATCC 25923 - MRSA-1302 Cefoxitin, Oxacillin Gentamicin Erythromycin,Ofloxacin, Levofloxacin Clindamycin, Cotrimoxazole MRSA-2 Cefoxitin, Oxacillin, Gentamicin Erythromycin, Ciprofloxacin Clindamycin, Cotrimoxazole Gram-negative bacteria E. coli ATCC 25922 - P. aeruginosa-6 Cefepime, Cefoperazone/sulbactam, Cefoxitin, Ceftazidime, Ceftriaxone, Imipenem, Meropenem, Piperacillin/tazobactam Amikacin, Gentamicin, Netilmicin Ciprofloxacin E. coli-7 Amoxicillin/clavulanic acid, Cefepime, Cefoperazone/sulbactam, Ceftriaxone, Ceftazidime, Piperacillin/tazobactam Gentamicin Ciprofloxacin Acinetobacter-1275 Cefepime, Cefoperazone/sulbactam, Ceftriaxone, Ceftazidime, Piperacillin/Tazobactam Imipenem, Meropenem, Amikacin, Gentamicin Ciprofloxacin, Levofloxacin previously swabbed with indicator bacte- seed medium for 2 days. The 5 ml inocu- ria. After overnight incubation at 37ºC, lum was transferred to a 250 ml baffled growth inhibition of the indicator bacte- flask containing 100 ml of production me- rium was observed by measuring the di- dium, and cultivated at 28ºC in a rotating ameters of the inhibition zones (including shaker at 150 rpm for 6 days. The whole the diameter of the cork borer) and re- culture was mixed with an equal volume corded (in mm). of ethanol and further incubated at 28ºC with rotation at 150 rpm for 30 minutes. Organic solvent extraction and chemical The cell debris was removed by centrifu- characterization gation at 3,000 rpm at 4ºC for 10 minutes. SJE177 was precultured in 5 ml of 301 The collected supernatant was evaporated Vol 41 No. 5 September 2010 1179 SOUTHEAST ASIAN J TROP MED PUBLIC HEALTH at 40ºC to half the initial volume. The re- and glycerol-asparagine agar (ISP5). Mela- sulting extract was mixed with an equal nin production was determined on pep- volume of ethyl acetate and shaken vigor- tone-yeast extract-iron agar (ISP6), ty- ously. After centrifugation at 3,000 rpm at rosine agar (ISP7), and tryptone-yeast ex- 4ºC for 10 minutes, the ethyl acetate ex- tract broth (ISP1), while the carbon utili- tract (upper layer) was collected and zation test was carried out in carbon uti- evaporated at 40ºC until dry, then the resi- lizing medium (ISP9) with the addition of due was redissolved in ethanol (at 1 ml/ one of the following sugars: D-glucose 100 ml culture). (positive control), L-arabinose, sucrose, D- The crude extract was analyzed with xylose, I-inositol, D-mannitol, D-fructose, high pressure liquid chromatography D-sorbitol, cellubiose, and in the absence (HPLC) using a Rainin Microsorb C18 col- of a carbon source (negative control) as umn (4.6 x 75 mm) on an Agilent Hp1100 described in the ISP project (Shirling and system with a photodiode array detector Gottlieb, 1966). For other biological prop- (200-600 nm) using a flow rate of 1.2 ml/ erties, milk coagulation and peptonization, minute, and additional UV detection at 254 hydrolysis of starch, and gelatin liquefac- nm. The mobile phase used was a stepwise tion were performed using the methods adapted from Cappuccino and Sherman gradient
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