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1 The Korean Journal of Microbiology, Vol. 43, No. 1, March 2007, p Copyright 2007, The Microbiological Society of Korea w³y m ³ Paenibacillus polymyxa DY1 Á Á «1 Á½ z 1 Á«2 Á 3, * w w ü ³q w w w w ü ³ x w w v š. w ³y wš m l w³ w ³ w š, w. yw x 16S ribosomal DNA» Paenibacillus polymyxa³ ƒ. ³ Paenibacillus polymyxa ƒ ƒ¾. ³ ƒ w w³ 1 j Samonella enterica serovar Typhi Shigella dysentery, enterohaemorrhagic Escherichia coli, š Vibrio cholera ³ z ùkþ, ü ³ z ùkþ. ³ ƒ w w³y š, Ÿ w w³y w w z ƒ sƒ. Key words ý 16S ribosomal DNA sequence, antibacterial activity, fatty acid, Paenibacillus polymyxa w ³ w ù j w³ wù. ³ w w³ 1928 A. Fleming Penicillium notatum l penicillin w (12), 2 z e w ƒ w e wù w. ù 1950 penicillin w Staphylococcus ³ ƒ ùkù» w, penicillin ü ùkü, tetracycline erythromycin ü ùkü Staphylococcus ³ ƒ š (25). Methicillin» w 1960 z methicillin ü ùkü y s ³(methicillin-resistant Staphylococcus aureus, MRSA) š (39), vancomycin ü ùkü Enterococcus faecium %, %, % š % ƒw (3). Vancomycin x ¾ r q w ü ³ e ƒ z š ù, 1986 vancomycin ü Enteroccociƒ š (2, 8, 23), ü vancomycin ü ü ³ (VRSA; Vancomycin-resistant Staphylococcus aureus)ƒ š (1). z (36), v, yg 5 6 ƒ š (14). 3 cephalosporin ü Escherichia coli Klebsiella pneumoniaƒ ƒwš (16), *To whom correspondence should be addressed. Tel: , Fax: khyoo@mail.sangji.ac.kr fluoroquinolone ü ü ³ wš (7) l 2000 ¾ ü Shigella sonnei 122³ w w³ x trimethoprim streptomycin 100% ü ùkü, sulfamethoxazole 94%, tetracycline 93% š nalidixic acid 90% ü ùkü (37). x w ü ü ³ ƒ e ƒ ƒ, ³ e ƒ ƒ dwš (21). ü ³ w w³z ƒ w š, w w w» š (26). w yw, p rk, rk, w rk š x - w l q w³ rk ( : hemocyanin) 4ƒ š (4). ¾ w³y ùkü t ph ã ù» s m ww z ùkü» (29), ³ z ƒ (17) š m yw (38). ³ l w³w, w, z w, w, w y ü» w k š (30). w l w³ wš w 47

2 48 Eun-seok Shin et al. Kor. J. Microbiol w š. ³ Listeria monocytogens w w³z ƒ š (24), y» ³ w w wš š (20, 26). w wš w š, ³ l w wš w y w š (10, 19). p Bacillus polymyxa polymyxin B rk w w š (18, 33). ³ gatavaline, jolipeptin, gavaserin, saltavalin, substance BN 109 LI-F w w³ w š (22, 34). w w w ³ w w z x» w w w, š w j ww w k w ƒ š (15). œw» w s ü w w» w w w ƒ x w š. ú ü w w v f š z ù y w w³ y» wš (32), w w penicillin, cephalosporin, tetracycline glycopeptide» w w oxazolidinine x w w w ƒ w š (31). ƒ ³ w w³y ùkü ³ m l w, w³y» wš, ³ w xkw, yw, w wš w. ³ m w ³ w³y» w t ³ Escherichia coli ATCC Staphylococcus aureus ATCC ³ ƒ. ³ Bacillus cereus, Listeria monocytogens, Vibrio parahaemolyticus, Yersinia enterocolitica, Salmonella Typhimurium, enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC), enteroinvasive E. coli (EIEC)ƒ. w 1 j enterohaemorrhagic E. coli (EHEC), Salmonella Typhi, Shigella sonnei, Shigella flexneri, Shigella dysenteriae, Shigella boydii, Vibrio choleraeƒ 18 ³ ƒ (Table 1). Nutrient Agar ƒ š, w³y w k 4 µg/ml penicillin G, 5 µg/ml polymyxin B sulfateƒ ƒ w ƒ. ³ Nutrient Broth, Brain Heart Infusion, Mueller Table 1. Bacterial strains used in this study Items Bacterial strains Standard strains Escherichia coli ATCC25922 Staphylococcus aureus ATCC29213 Group I communicable diseases Salmonella Typhi bacteria Salmonella Paratyphi A E. coil (EHEC) a Food poisoning bacteria a EHEC: Enterohemorrhagic E. coli b EIEC: Enteroinvasive E. coli c EPEC: Enteropathogenic E. coli d ETEC: Enterotoxigenic E. coli Shigella sonnei Shigella dysenteriae Shigella boydii Vibrio cholerae Bacillus cereus E.Gcoli (EIEC) b E.Gcoli (EPEC) c E.Gcoli (ETEC) d Listeria monocytogens Vibrio parahaemolyticus Yersinia enterocolitica Salmonella Typhimurium Hinton Broth (Difco, Maryland, USA) ƒ, ³ ³ TSA (Difco, Maryland, USA) ƒ. w³y ³ ew y t 2-5 cm ¾ m g w ³ š ice box w x 4 o C þ š w. m 1 g ³ 9 ml 80 C 10 z d o 1,000 w. k (4 µg/ml penicillin G, 5 µg/ ml polymyxin B sulfate w ) wš 28 o C w» 48 w. k ƒƒ w Nutrient Broth (NB) w 30 C 24 o k z, 13,000 g 2 wš, w 0.22 µm vl w g. w³y ³ Escherichia coli ATCC Staphylococcus aureus ATCC ³ Brain Heart Infusion (BHI) 18 w z, ³ ³ w Nutrient Agar sq t š w t jš, ³ 25 µl k z w³ y w (Fig. 1).

Vol. 43, No. 1 Paenibacillus polymyxa DY1 w³y 49 Fig. 1. Antibacterial activities of DY1 strain on the TSA medium indicated by clear inhibition zone formed around the drop.

3 Vol. 43, No. 1 Paenibacillus polymyxa DY1 w³y 49 Fig. 1. Antibacterial activities of DY1 strain on the TSA medium indicated by clear inhibition zone formed around the drop. D1: Bacillus thuringiensis, E. coli; Escherichia coli ATCC 25922, Sal; Salmonella Typhi, Shigella sonnei: Shigella sonnei. ³ w w³y x ³ l w w w³y x disk diffusion w ww. ³ disk œ» w 8 w 60 µl disk z w x³ ƒ sq 30 o C k z j» d w. w³y ³ xkw, yw w³y ³ w» w k w ³ NA 24 w z w ³ j», parasporal crystal x, hemolysis, motility, catalase oxidase test, VP test, glucose l acid, β-galactosidase, w. w API 50 CHB kit (Bio Merieux, France) w 50 w (27). w Bergy's Manual of Systematic Bacteriology (9) The Genus Bacillus (13) šw. 16S ribosomal DNA» ³ 16S rdna Polymerase Chain Reaction (PCR) s g. 16S rdna s Universal primer 16S Forward primer; 5'-AGA GTT TGA TCC TGG CTC AG-3' 16S Reverse primer; 5'-GTT TAC CTT GTT ACG ACT T-3' w. PCR 95 o C 5 k z, 95 o C 30 sec, 55 o C 30 sec, 72 o C 2 min» 30 cycles ww, 72 o C 3 e z 4 o C w. PCR DNA purification kit (Qiagen Inc., Valencia, CA, USA) w w z, automatic sequencer mw» w. NIH w NCBI yr œw nucleotide blast search program w w w ( Trypticase Soy Agar (Difco, Maryland, USA) ³ 3 w ³ v w 1.5 ml Effendorf tube š, methanol NaOHƒ 1 ml 100 C ƒ w ƒ o ww z, methanol HCl 2 ml ƒwš, 80 C 10 ew o fatty acid methyl ester (FAME)y g. n-hexane 1.25 ml ƒw ywwš, w n-hexane d w ƒ k z, NaOH methanol w w w GC vial w. GCFID (Hewlett-Packard 6890, USA) Gas liquid chromatography (GLC) w š, Sherlock software (Version 4.5) w w. Column capillary column w, detector Flame ionization detector (FID) w. w³y ³ ³ w w ³ DY1 l DY11 ³ ¾ 11 ³ w ( ). w 11³ Escherichia coli ATCC ³ ƒ j z ùkü DY1 ³ ³ w. DY1 ³ w z w d E. coli, Sligella sonnei Salmonella Typhiƒ sq z 37 o C 18 k sq w ù kü (Fig. 1). ³ w w³y x DY1 ³ x 18 ü ³ 15 ³ w w³y (Table 2). 1 ³ E. coli (EHEC), Salmonellia Typhi, Shigella sonnei, Shigella flexneri, Shigella dysenteriae, Shigella boydii, Vibrio cholerae ³ Vibrio parahaemolyticus, Yersinia enterocolitica, Salmonella Typhimurium, E. coli (ETEC, EPEC, EIEC) j z ùkþ. ù ³ Staphylococcus aureus, Bacillus cereus Listeria monocytogens w z ùkü (Table 2).

4 50 Eun-seok Shin et al. Kor. J. Microbiol Table 2. Antimicrobial activities of DY1 against various pathogenic enteric bacteria Group 1 communicable diseases bacteria Food poisoning bacteria Pathogenic bacterial strains Inhibition zones (mm) Salmonella Typhi 13 Salmonella Paratyphi A 9 Shigella sonnei 14 Shigella flexneri 14 Shigella dysenteriae 12 Shigella boydii 12 E. coli (EHEC) 14 Vibrio cholerae 14 Salmonella Typhimurium 10 Salmonella Enteritidis 9 E. coli (EIEC) 12 E. coli (ETEC) 12 E. coli (EPEC) 15 Staphylococcus aureus 0 Yersinia enterocolitica 12 Bacillus cereus 0 Listeria monocytogens 0 Vibrio parahaemolyticus 12 xkw yw p DY1 ³ xkw yw p w s j» 1 µm j» ùkþ, catalase ùkþš, L-arabionose l acid w L-arabinose ùkþ (Table 3). ³ ù kû, xk ³ xk ( ). Sporangium swollen parasporal crystal, x», VP test, gelatin ƒ w, Egg-yolk lecithinase, indol,, oxidase, β-galactosidase ùkû (Table 3). API 50 CHB kit w x DY1 ³ glycerol 26 ùkþ ù, erithritol 23 ùkü (Table 4). Biomerieux ( industry.biomerieux-usa.com/industry/watertesting/api/ apiweb.htm,hazelwood, MO, USA)z yr œw Apiweb ( mw Paenibacillus polymyxa ³ 84% ùkü. 16S ribosomal DNA» DY1 ³ DNA w PCR 16S rdna s w» w 1,416 bp w» (GeneBank Accession number EF ).» NCBI œw Nucleotide BLAST search ww, DY1 ³ Peanibacillus polymyxa ³ Table 3. Morphological and biochemical characteristics of DY1 strain Items Characteristic Items Characteristic Cell diameter>1.0 µm + Hydrolysis gelatin - Gram stain + Egg-yolk lecithinase - Catalase + Formation of indole - Anaerobic growth Hemolysis of blood - agar plate - Voges-Proskauer test - Sporangium swollen - Acid from Parasporal crystal - D-Glucose - Motility - L-Arabinose + Oxidase - D-Xylose - Dihydrolase of grainine - D-Mannitol - β-galactosidase - Table 4. The biochemical characteristics of strain DY1 by API 50 CHB test Carbohydrate source DY1 Carbohydrate source DY1 Glycerol + Salicin + Erythritol - Celibiose + D-Arabinose - Maltose + L-Arabinose + Lactose + Ribose + Melibiose + D-Xylose + Sucrose + L-Xylose - Trehalose + Adonitol - Inulin + β-methyl-d-xylose + Melezitose - Galactose + Raffinose + Glucose + Starch + Fructose + Glycogen + Mannose + Xylitol - Sorbose - Gentiobiose + Rhamnose - D-Turanose - Dulcitol - D-Lyxose - Inositol - D-Tagatose - Mannitol + D-Fucose - Sorbitol - L-Fucose - α-methyl-d-mannoside - D-Arabitol - α-methyl-d-glucoside - L-Arabitol - N-Acetyl-Glucosamine - Gluconate - Amygdalin + 2-Keto-Gluconate - Arbutin + 5-Keto-Gluconate - Esculin + pairwise identity Paenibacillus polymyxa strain GBR-603 (Accession number AY ) 99.79% (1,413/1,416) ƒ.

5 Vol. 43, No. 1 Paenibacillus polymyxa DY1 w³y 51 Fig. 2. Phylogenetic dendrogram derived by the 16S rdna sequences of DYI strain and various Paenibacillus spp. in genus Paenibacillus. The tree shows DY1 is the closest to Paenibacillus polymyxa of the genus Paenibacillus. The tree was rooted from a Jotun-Hein method of 16S rdna sequences from DY1-related species available in GenBank. GenBank accession numbers were shown in front of species name. GenBank ³ 16S rdna» DNASTAR MegAlign v w Jotun-Hein method (28) w» molecular phylogenetic tree (Fig. 2). x w gas chromatography w ( ), ³ DY1 ³ isobranched fatty acid anteiso-branched fatty acid, C 15:0 anteiso-fatty acidƒ 63.55% ƒ w, C 16:0 iso-fatty acidƒ 9.3%, C 17:0 anteiso-fatty acidƒ 9.25% w. ³ iso-type anteiso-type, anteiso-type j wš. w DY1 ³ w Paenibacillus w Paenibavillus polymyxa ƒ ƒ¾. Paenibacillus polymyxa Paenbacillus terrae w ùkþ (35). š m l w³y w ³ wš, ƒ w³y DY1 ³ w. DY1 ³ w yw x, x, 16S rdna» Paenibacillus polymyxaƒ ƒ ù. w Gram p, xkw ¾ ww Paenibacillus polymyxaƒ DY1 ƒ ƒ¾. w DY1 ³ Paenibacillus polymyxa DY1 w. Paenibacillus polymyxa rdna w w 3 Bacillus genus š m (24), w³y w ³. Paenibacillus polymyxa DY1 x ü ³ ³ ww z ùkþ. Paenibacillus polymyxa ³ ƒ w polyxin w Bacillus cereus sww 17 ³ w w³z d w, m ³ w³ ù, Pseudomonas Salmonella w³ (38). ù w Paenibacillus polymyxa DY1 ³ ƒ w w³ Salmonella sww ³ 1 ³ w w³ ùkü ù, ³ w³. Paenibacillus polymyxa DY1 w w³ š w³ q. w ƒ ü ³ ù w³y (Table 2) w z ƒ. w³ w ³ š w ƒ ƒ š. ù ü ³ w ü wš. p Paenibacillus polymyxa l w w³y w w y. Paenibacillus z, olive-mill s, m, m (5, 6, 11, 40). 1,300 m š m Paenibacillus polymyxa DY1 w. Paenibacillus polymyxa ³ w k y ³ k y w ƒ q. Paenibacillus polymyxa DY1 w w³y» w w ù w z ƒe ƒ q w» w x ƒ š ƒ ü ³ w w w³ sƒƒ w, x mw sƒ w. w³y w³p Ÿ w w³ wz w³ z ƒ š ƒ w ü. š x 1. « ü ³ w w w. w z 7, ½, kx,, Ÿ Benzimidazole N-phenylcarbamate ³ w q ³. w wz 11, ½ ³, ½, ½yz, x,, k

6 52 Eun-seok Shin et al. Kor. J. Microbiol w³ ü 11, , w w³rk y w. 31, Aguilera, M., M. Monteoliva-Sanchez, A. Suarez, V. Guerra, C. Lizama, A. Bennaser, and A. R. Cormenzana Paenibacillus jamilae sp. nov., an exopolysaccharide-producing bacterium able to grow in olive-mill waste water. Int. J. Syst. Evol. Microbiol. 51, Ash, C., F.G. Priest, and M.D. Collins Molecular identification of rrna group 3 bacilli using a PCR probe test. Proposal for the creation of a new genus Paenibacillus. Antonie van Leeuwenhoek 64, Bush, K., G.A. Jacoby, and A.A. Mederios A functional classification scheme for β-lactamases and its correlation with molecularg structure. Antimicrob. Agents Chemother. 39, Cheong, H.J Vancomycin resistant Enterococci. J. Korean Soc. Chemother. 15, David, R.B. and R. N. Castenhol Bergey's manual systematic bacteriolgy. p Springer, press 2nd ed. New York, USA 10. Duffy, B.K. and G. Defago Environmental factors modulating antibiotic and siderophore biosynthesis by Pseudomonas fluorescens biocontrol srains. Appl. Environ. Microbiol. 65, Elo, S., I. Suominen, P. Kampfer, J. Juhanoja, M. Salkinoja-Salsonen, and K. Haahtela Paenibacillus borealis sp. nov., a nitrogen fixing species isolated from spruce forest humus in Finland. Int. J. Syst. Evol. Microbiol. 51, Fleming, A On the antibacterial action of cultures of a penicillium, with special reference to their use in the isolation of B. influenzae. Bull. World Health Organ. 79, Gordon, R.E., W.C. Haynes, and C.H. Pany The Genus Bacillus. Agriculture Handbook No Agriculture Research Service, U. S. Department of Agriculture, Washington D.C., USA 14. Hiramatsu K., N. Aritaka, H. Hanaki, S. Kawasaki, Y. Hosoda, S. Hori, Y. Fukuchi, and I. Kobayashi Dissemination in Japanese hospitals of strains of Staphylococcus aureus heterogeneously resistant to vancomycin. Lancet 350, Hutchinson, C.R. and I. Fujji Polyketide synthase gene manipulation; A structure function approach in engineering novel antibiotics. Annu. Rev. Microbiol. 49, Jacoby, G.A More extended spectrum β-lactamases. Antimicrob. Agents Chemother. 35, Kabara, J.J Medium chain fatty acids and esters, p Antimicrobial in Foods. Marcel Dekker Inc., New York, USA 18. Kajimura, Y. and M. Kaneda Fusaricidins B, C and D new depsipeptide antibiotics produced by Bacillus polymyxa KT-8 isolation, structure elucidation and biological activity. Japan J. Antibiotics 50, Kimer, S., P.E. Hammer, D.S. Hil, A. Altmann, I. Fischer. L.J. Weislo, M. Lanahan, K.H. van Pee, and J.M. Ligon Functions encoded by pyrrolnitrin biosynthetic genes from Pseudomonas fluorescens. J. Bacteriol. 180, Koo, B.S., J.C. Ryu, T.Y. Chung, and K.C. Kim Purification and characterization of natural antifungal protein from astragal seeds (Astragalus membranaceus). Kor. J. Appl. Microbiol. Biotechnol. 26, Kunin, C.M Resistance to antimicrobial drugs-a worldwide calamity. Ann. Intern. Med. 118, Kurusu, K., K. Ohba, T. Arai, and K. Fukushima New peptide antibiotics LI-F03, F04, F05, F07 and F08, produced by B. polymyxa. I. Isolation and chatacterization. J. Antibiot. 40, Leclereq, R., E. Derlot, J. Duval, and P. Courbalin Plasmid mediated resistance to vancomyhcin and teicoplanin. N. Engl. J. Med. 319, Lee, S.H. and Y. S. Lim Antimicrobial effects of Shizandra chinensis extract against Listeria monocytogenes. Kor. J. Appl. Microbiol. 25, Lepper, M.H., H.F. Dowling, G.G. Jackson, B. Moulton, and H.W. Spies Effect of antibiotic usage in the hospital on the incidence of antibiotic-resistant strains among personnal carring Staphylococci. J. Lab. Clin. Med. 42, Lim, T.H., J.M. Lee, T.H. Chang, and B.J. Cha Antifungal activity and identification of an Actinomycetes strain isolated from mummified peaches. Kor. J. Appl. Microbiol. Biotechnol. 28, Logan, N.A. and R.C. Berkeley Identification of Bacillus strains using the API system. J. Gen. Microbiol. 130, Lunter, G., I. Miklos, A. Drummond, J.L. Jensen, and J. Hein Bayesian estimation of phylogeny and sequence alignment. BMC Bioinformatics 1, Mark, A.D Antimicrobial substances from lactic acid bacteria for use as food preservatives. Oregon State University, Corvailis, OR Okami, Y. and K. Hotta Search and discovery of new antibiotics. p In M. Goodfellow (ed.). Actinomycetes in biotechnology. Academic Press. London, UK 31. Omura, S. and R. Oiwa Studies on bioactive compounds from microorganisms. Kitasato Arch. Exp. Med. 57, Park, Y.J., E.J. Oh, M.K. Kang, B.K. Kim, S.M. Kim, and S.I. Sin Emergence of teicoplanin resistant Staphylococci. J. Korean Soc. Chemother. 15, Paulus, H. and E. Gray The biosynthesis of polymyxin B by growing cultures of B. polymyxa. J. Biol. Chem. 239, Pichard, B., J.P. Larue, and D. Thouvenot Gavaserin and saltavalin, new peptide antibiotics produced by B. polymyxa. FEMS Microbiol. Lett. 133, Piuri, M., C. Sanchez-Rivas, and S.M. Ruzai A novel antimicrobial activity of a Paenibacillus polymyxa strain isolated from regional fermented sausages. Lett. Appl. Microbiol. 27, Quirk, M First VRSA isolate identified in USA. Lancet Infect. Dis. 2, Seol, SY., Y.S. Kim, Y.S. Jeong, J.Y. Oh, H.Y. Kang, and D.C. Moon Molecular characterization of antimicrobial resistance in Shigella sonnei isolates in Korea. J. Med. Microbiol. 55, Somaatmadia, D., J.J. Powers, and M.K. Hamdy Anthocyanins. VI. Chelation studies on anthocyanins and other related compound. J. Food Sci. 29, Waxman, D.J. and J.L. Strominger Penicillin-binding proteins and the mechanism of action of β-lactam antibiotics. Annu. Rev. Biochem. 52, Yoon, J.H., H.M. Oh, B.D. Yoon, K.H. Kang, and Y.H. Park Paenibacillus kribbensis sp. nov. and Paenibacillus terrae sp. nov., bioflocculants for efficient harvesting of algal cells. Int. J. Syst. Evol. Microbiol. 53, (Received November 14, 2006/Accepted December 26, 2006)

7 Vol. 43, No. 1 Paenibacillus polymyxa DY1 w³y 53 ABSTRACT : Identification and Characterization of Paenibacillus polymyxa DY1 Isolated from Korean Soil with New Antibacterial Activity Eun-seok Shin, Hee-Moo Lee, Bok-Kwon Lee 1, Sung-Hoon Kim 1, Sun-il Kwon 2, and Kwan-hee Yoo 3, * (Department of Biology, Andong University, 1 Division of Enteric Bacterial Infections, Center for Infectious Diseases, Korean NIH, 2 Department of Clinical Pathology, Daegu Health College, 3 Department of biology, Sangji University, Wonju , Korea) The DY1 strain of Gram-positive, rod-shaped bacteria was isolated from the soil sample collected from Daeam mountain, Korea. The culture filtrate of DY1 strain showed a broad spectrum of antimicrobial activity on various pathogenic and food poisoning enteric bacterial species tested in vitro. It showed significant growth-inhibitory effect on Salmonella enterica sp., Shigella sp., pathogenic Escherichia coli, Vibrio cholerae, Vibrio parahemolyticus, and Yersinia enterocolitica. For the identification of the DY1 strain, morphological, biochemical and molecular phylogenetic approaches were performed. The DY1 strain was found to be a member of the genus Paenibacillus on the basis of morphological and biochemical analyses. The 16S rdna of DY1 showed the highest pairwise identity with Paenibacillus polymyxa with 99.79% (1,413 bp/1,416 bp). The antimicrobial entity from DY1 looked different from preciously reported ones and seems to have a great potential to be further studied as a candidate of new antibiotics to control multi-drug resistant pathogens.

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16(2)-7(p ).fm w wz 16«2y Kor. J. Clin. Pharm., Vol. 16, No. 2. 2006 ü t xy y w tœ ½ BÁ x BC B y w w w C y w w w Current Status and Expectations of Orphan Drugs in Korea -In point of supplying medicines for the rare