The Korean Journal of Microbiology, Vol. 43, No. 1, March 2007, p. 47-53 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 1999 29%, 2000 32%, 2001 28% š 2002 33% 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: 82-33-730-0433, Fax: 82-33-730-0430 E-mail: khyoo@mail.sangji.ac.kr fluoroquinolone ü ü ³ wš (7). 1991 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
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 25922 Staphylococcus aureus ATCC 29213 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 50-100 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 25922 Staphylococcus aureus ATCC 29213 ³ Brain Heart Infusion (BHI) 18 w z, ³ ³ w Nutrient Agar sq t š w t jš, ³ 25 µl 18-24 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. 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 18-24 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 (http://www.ncbi.nlm.nih.gov/blast). 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 25922 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).
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 (http:// industry.biomerieux-usa.com/industry/watertesting/api/ apiweb.htm,hazelwood, MO, USA)z yr œw Apiweb (https://apiweb.biomerieux.com) 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 108320).» 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 359632) 99.79% (1,413/1,416) ƒ.
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 18 15 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. 2005 w ü. š x 1. «. 1996. ü ³ w w w. w z 7, 97-102. 2. ½, kx,, Ÿ. 1995. Benzimidazole N-phenylcarbamate ³ w q ³. w wz 11, 146-150. 3. ½ ³, ½, ½yz, x,, k. 2003.
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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 220-702, 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.