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1 he Korean Journal of Microbiology, Vol. 43, No. 1, March 2007, p Copyright 2007, he Microbiological Society of Korea ss w Acinetobacter koreensis sp. nov. š w Á ³ 1 Á v 2 Á 3 Á 3 Á *» w œw y w» w p y w œw l JB10 JB15 ³ ew ss ss. y» ³, µm ³. JB10, JB15 ³ t ³ Á yw p x. JB10 JB15 ³ 16S rdna» w γ-proteobacteria w, Acinetobacter tandoii 4N13 (97.3%), Acinetobacter haemolyticus ACC17906 (97.2%), Acinetobacter johnsonii DSM6963 (97.2%), Acinetobacter junii DSM6964 (96.7%), Acinetobacter schindleri LUH5832 (97.0%) Acinetobacter ursingii LUH3792 (96.6%)». š t ³ 93-96%». ³ C 18:1 ω9c C 16:1 ω7c/c 15:0 iso 2OH w wš y w, JB15 ³ C 19:1 iso I. Á yw p, 16S rdna» ³ JB10 JB15 ³ t ³ p ùkü y JB10 (=KEMC ) JB15 (=KEMC ) ³ Acinetobacter koreensis sp. nov. w. Key words ý 16S rdna sequence, Acinetobacter koreensis, fatty acid Acinetobacter ³, µm ³ xk. y» ³ Moraxellaceae ( ) w, m,,,,, e, v y» Ÿ w ùkù. Acinetobacter ³ rypticase Soy Agar (SA, BBL) l ƒ ñ š x w g x w. s x w, ³ š, catalase x, ( ) w Moraxella, Psychrobacter oxidase x. Acinetobacter spp C o w, ³ 37 o C, 41 C o 44 o C w ù, w (5). Acinetobacter spp. w ã, Juni w (10, 13). Acinetobacter Bouvet Grimont w 12 genome species, 6 Acinetobacter calcoaceticus A. baumannii, A. haemolyticus, A. johnsonii, A. lwoffii (1, 3). z, A. radioresistens ³ ƒ 1988 (16), A. ursingii A. schindleriƒ 2001 Á š(14), A. baylyi A. bouvetii, A. grimontii, A. tjernbergiae, A. towneri, A. tandoii, A. gerneri 7 Emma w (7), A. *o whom correspondence should be addressed. el: , Fax: sslee@kyonggi.ac.kr parvusƒ Memec w 2003 Á (15). ew ss ³ Acinetobacter JB10 ³ JB15 w ³ Á ww. ss ss w C o Nutrient Broth (NB, Bacto) w 13³ (JB3, JB5, JB7, JB9, JB10, JB11, JB15, JB17, JB19, JB29, JB37, JB41, JB54) w, ³ JB10 JB15 ³ t ³ w. š KCCM (Korean Culture Center of Microorganisms, Korea) KCC (Korean Collection for ype Cultures, Korea) Acinetobacter t w x w (able 1). ³ xkw p NB w ³ w, w x (BX50, Olympus, Japan; 1,000) w w. ³ q š, µm ³, NB š l ƒ ñ š, x w g x wš, NB 0.3% š y w. ƒ ³ Áyw p API 20E 20NE kit (BioMeriux, France) t (6, 11) w w. Bergey's Manual Acinetobacter w xk p (5). Acinetobacter ³ o C w, ƒ w ƒ 66
2 Vol. 43, No. 1 ss w Acinetobacter koreensis sp. nov. š 67 able 1. List of Acinetobacter type strains and isolated JB strains used in this study Acinetobacter strains Strain no. Accession no. Acinetobacter baumannii KCCM40203 G (=DSM3007 ) X81664 Acinetobacter baylyi KCC12413 G (=DSM14961 ) AF Acinetobacter bouvetii KCC12414 G (=DSM14964 ) AF Acinetobacter calcoaceticus KCCM40204 G (=DSM3006 ) X81661 Acinetobacter gerneri KCC12415 G (=DSM14967 ) AF Acinetobacter grimontii KCC12416 G (=DSM14968 ) AF Acinetobacter haemolyticus KCCM40205 G (=DSM6962 ) Z93437 Acinetobacter johnsonii KCCM40206 G (=DSM6963 ) X81663 Acinetobacter junii KCCM40207 G (=DSM6964 ) X81664 Acinetobacter lwoffii KCCM40172 G (=DSM2403 ) X81665 Acinetobacter radioresistens KCC12411 G (=DSM6976 ) X81666 Acinetobacter schindleri KCC12409 G (=DSM16038 ) AJ Acinetobacter tandoii KCC12417 G (=DSM14970 ) AF Acinetobacter tjernbergiae KCC12418G (=DSM14971 ) AF Acinetobacter towneri KCC12419 G (=DSM14962 ) AF Acinetobacter ursingii KCC12410 (=DSM16037 ) AJ JB10 KEMC EF JB15 KEMC EF *KCC: Korean Collection for ype Cultures, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Korea; KCCM: Korean Culture Center of Microorganisms, Korean Federation of Culture Collections (KFCC), Korea; KEMC: Korea Environmental Microorganism Center, Korea. (5). ss JB10 JB15 ³ ƒ o C, ³ 37 C o 41 C o w ù, w w. š 44 C w o (able 2). ƒ ³ catalase oxidase x ƒƒ, gelatine ww w. JB10 JB15 ³ D-glucose L- arabinose x w. š k citrate, sucrose, phenylacetate, malonate, caprate malate Fig. 1. Phylogenetic tree for 16S rrna gene nucleotides of genus Acinetobacter and newly isolated strains analyzed by the neighbor-joining method (A) and maximum-parsimony (B). Strict consensus of five equally parsimonious tree (B). he genetic distances tree were computed by using Jukes and Cantor model (A). he 16S rrna sequences of Psychrobacter immobilis and Moraxella lacunata were included as outgroups. he scale bar indicates a genetic distance of substitutions/site (A). he number shown next to each node indicates the percentage bootstrap value of 1000 replicates.
3 68 Ha-Yan Lee et al. Kor. J. Microbiol able 2. Biochemical characteristics of isolated JB strains and Acinetobacter type strains Characteristic Motility Catalase Oxidase Growth at: 37 o C o C o C Nitrate reductase Methyl Red Voges Proskauer Urea ONPG PNPG H 2 S Acid production from: D-Mannitol D-Sorbitol D-Sucrose D-Glucose v L-Arabinose L-Rhamnose D-Melibiose Hydrolysis of: ESC Gelatine Assimilation of: L-lysine L-ryptophane L-Arginine L-Ornithine v - - Phenylalanine L-Maltose D-Mannitol NAG GN Citrate L-Arabinose D-Mannose D-Glucose Sucrose Adipate able 2. Continued Characteristic Phenylacetate Malonate Caprate Malate Strains: 1, JB10; 2, JB15 ; 3, A. junii DSM6964 ; 4, A. schindleri LUH5832 ; 5, A. towneri AB1110 ; 6, A. bouvetii 4B02 ; 7, A. radioresistens DSM6976 ; 8, A. gerneri 9A01 ; 9, A. baylyi B2 ; 10, A. calcoaceticus DSM30006 ; 11, A. haemolyticus DSM6962 ; 12, A. ursingii LUH3792 ; 13, A. lwoffii DSM2403 ; 14, A. tandoii 4N13 ; 15, A. baumannii DSM30007 ; 16, A. johnsonii DSM6963 ; 17, A. tjernbergiae 7N16. Symbols: +, positive; -, negative; V, variable. Abbreviation: 1 Esc, Esculin ferric citrate; 2 NAG, N-acetyl-gluconate; 3 GN, Potassium gluconate. w, L-arginine, L-arabinose adipate k w w. Bergey's Manual of Systematic Bacteriology Volume 2 Part B š x y w JB10 JB15 ƒƒ 94% 98% A. lwoffii DSM2403 w yw p ùkü (3, 5, 7, 10, 14, 15). ³ 16S rdna» w Proteinase K w genomic DNA y w (1), universal primer 27F 1492R (13) w PCR z,» w. JB10 JB15 ³ w 16S rdna» GenBank accession no. EF EF w.» w GenBank database ww. w ³ y w z, Acinetobacter ü ƒ t ³ ³ w, Acinetobacter tandoii 4N13 (97.3%), Acinetobacter haemolyticus ACC17906 (97.2%), Acinetobacter johnsonii DSM6963 (97.2%), Acinetobacter junii DSM6964 (96.7%), Acinetobacter schindleri LUH5832 (97.0%)» š, t ³ 94-96%». m t ³ ³ 16S rdna» MacClade 4.06 OS v PAUP 4.0 b10 v w, Neighbor-joining (NJ)(18), Maximum-likelihood (ML) Maximum-parsimony (MP) w m w (1, 19). Jukes and Cantor Kimura's twoparameter w w, outgroup ( ) w Psychrobacter immobilis ACC Moraxella lacunata ACC17967 w (3, 4). Branch (bootstrap ) 1,000z l m w w (3, 4, 8, 9, 19). JB10 JB15 ³ NJ MP 100% w cluster x w š, 16S rdna ƒ A.
4 Vol. 43, No. 1 ss w Acinetobacter koreensis sp. nov. š 69 able 3. 16S rdna sequence homology (%) between isolated JB strains and the Acinetobacter type strains Strains: 1, JB10; 2, JB15 ; 3, A. baumannii DSM30007 ; 4, A. baylyi B2 ; 5, A. bouvetii 4B02 ; 6, A. calcoaceticus DSM30006 ;7, A. gerneri 9A01 ; 8, A. haemolyticus DSM6962 ; 9, A. johnsonii DSM6963 ; 10, A. junii DSM6964 ; 11, A. lwoffii DSM2403 ; 12, A. radioresistens DSM6976 ; 13, A. schindleri LUH5832 ; 14, A. tandoii 4N13 ; 15, A. tjernbergiae 7N16 ; 16, A. towneri AB1110 ; 17, A. ursingii LUH3792. tandoii 4N13 NJ MP ƒƒ 95% 90% cluster. ƒ ³ rypticase Soy Agar (SA, BBL) plate 24 y» w ³ y w, Makula and Finnerty (11) w w. Sigma t w. w Gas Chromatography Frame Ionization Detector (GC-FID)(6890 N, Agilent, USA) w, peak Sherlock MIS version 4.5 w w (MIDI, Inc., NewYork, USA)(17). x ³ C 18:1 ω9c C 16:1 ω7c/c 15:0 iso 2OHƒ ùkû,, 3OH, C 14:0 ³ (able 4). A. tandoii 4N13 16S rdna m cluster JB10, JB15 ³ w A. tandoii 4N13 C 15:1 iso G C 14:0 3OH/C 16:1 iso I, A. tandoii 4N13 C 15:0 isoƒ. p, C 19:1 iso I w JB15 ³ p. yw p x, 16S rdna mw, m ww JB10 JB15 ³ y w, Acinetobacter koreensis w. Acinetobacter koreensis sp. nov.» Acinetobacter koreensis (ko.re.en' sis. N.L. fem. adj. koreensis pertaining to Korea, from where to novel micro-organism was isolated.) ³ ³, µm j» ³. Spore x w,. 37 C o 41 C o w, 44 C w o w o C Nutrient Agar rypticase Soy Agar y» y ù, l ƒ ñ š, x w g x w. Gelatine ww w, D- glucose L-arabinose x w. š k citrate, sucrose, phenylacetate, malonate, caprate malate w, L-lysine, L-tryptophane, L-ornithine, phenylalanine, L-maltose, D-mannitol, D-mannose, L-arginine, L- arabinose adipate k w w. š C 18:1 ω9c C 16:1 ω7c/c 15:0 iso 2OHƒ, w C 19:1 iso I ƒ. Acinetobacter koreensis sp. JB15 (=KEMC ) ew ss. y w y» w»
5 70 Ha-Yan Lee et al. Kor. J. Microbiol able 4. Cellular fatty acid composition (% of total fatty acids) of isolated JB strains and related Acinetobacter species Fatty acid C 10: C 10:0 2OH C 10:0 3OH OH OH C 14:1 ω5c C 14: C 13:0 2OH C 15: C 15:1 iso G C 16:1 ω7c alcohol N alcohol ω9c ω5c OH iso C 17:1 ω8c methyl C 18:3 ω6c(6,9,12) C 18:1 ω9c C 18:1 ω7c C 18:1 ω5c C 13: C 19:1 iso I Summed feature C 14:0 3OH/C 16:1 iso I C 15:1 iso I/C 13:0 3OH C 16:1 ω7c /C 15:0 iso 2OH C 18:2 ω6,9c /C 18:0 ANE uuknown Strains: 1, JB10; 2, JB15 ; 3, A. baumannii DSM30007 ; 4, A. baylyi B2 ; 5, A. bouvetii 4B02 ; 6, A. calcoaceticus DSM30006 ;7, A. gerneri 9A01; 8, A. haemolyticus DSM6962 ; 9, A. johnsonii DSM6963 ; 10, A. junii DSM6964 ; 11, A. lwoffii DSM2403 ; 12, A. radioresistens DSM6976 ; 13, A. schindleri LUH5832 ; 14, A. tandoii 4N13 ; 15, A. tjernbergiae 7N16 ; 16, A. towneri AB1110 ; 17, A. ursingii LUH3792.» w w,. š x 1. Ashraf, I., G.S. Peter, and L. Werner Phylogenetic relationship of the twenty-one DNA groups of the genus Acinetobacter as revealed by 16S ribosomal DNA sequence analysis. Int. J. Syst. Evol. Microbiol. 47, Ausubel, F.M. R. Brent, R.E. Kingston, D.D. Moore, J. G. Seidman, J.A. Smith, and K. Struhl Short Protocols in Molecular Biology 5th ed., p. 2-11, Wiley, USA. 3. Bouvet, P.J.M. and P.A.D. Grimont axonomy of the genus Acinetobacter with the recognition of Acinetobacter baumannii
6 Vol. 43, No. 1 ss w Acinetobacter koreensis sp. nov. š 71 sp. nov., Acinetobacter haemolyticus sp. nov., Acinetobacter johnsonii sp. nov., and Acinetobacter junii sp. nov. and emended description of Acinetobacter calcoaceticus and Acinetobacter lwoffii. Int. J. Syst. Bacteriol. 36, Bouvet, P.J.M. and S. Jeanjean Delineation of new proteolytic genomic species in the genus Acinetobacter. Res. Microbiol. 140, Brenner, D.J., N.R. Krieg, and J.. Staley Bergey's manual of systematic bacteriology 2nd ed. he Williams & Wilkins Co., Baltimore, Maryland, USA 6. Cappuccino, J.G. and N. Sherman Microbiology: A Laboratory Manual, 6th ed., Benjamin-Cummings Publishing Company 7. Emma, L.C., K. Peter, K.C.P. Bharat, G. Volker, and R.J. Seviour Seven novel species of Acinetobacter isolated from activated sludge. Int. J. Syst. Evol. Microbiol. 53, Felsenstein, J Confidence limits on phylogenies, an approach using the bootstrap. Evolution. 39, Juni, E Interspecies transformation of Acinetobacter: genetic evidence for a ubiquitous genus. J. Bacteriol. 47, Loubinoux, J., L. Mihaila-Amrouche, A.L. Fleche, E. Pigne, G. Huchon, P.A.D. Grimont, and A. Bouvet Bacteremia caused by Acinetobacter ursingii. J. Clin. Microbiol. 41, Makula, R.A. and W.R. Finnerty Microbial assimilation of hydrocarbone : cellular distribution of fatty acids. J. Bacteriol. 112, MacFaddin, J.F., L. McGrew, A. Heubeck, D. Hartman, and K. Ruppert Biochemical tests for identification of medical bacteria. 3rd edition. Lippincott Williams & Wilkins 13. Misbah, S., H. Hassan, M.Y. Yusof, Y.A. Hanifah, and S. Abubakar Genomic species identification of Acinetobacter of clinical isolates by 16S rdna sequencing. Singapore. Med. J. 46, Nemec, A., D.. Baere, I. jernberg, M. Vaneechoutte,.J.K. Reijden, and L. Dijkshoorn Acinetobacter ursingii sp. nov. and Acinetobacter schindleri sp. nov., isolated from human clinical specimens. Int. J. Syst. Evol. Microbiol. 51, Nemec, A., L. Dijkshoorn, I. Cleenwerck,. Baere, D. Janssens,.J.K. Reijden, P. Jezek, and M. Vaneechoutte Acinetobacter parvus sp. nov., a small-colony-forming species isolated from human clinical specimens. Int. J. Syst. Evol. Microbiol. 53, Nishimura, Y.,. Ino, and H. Iizuka Acinetobacter radioresistens sp. nov. isolated from cotton and soil. Int. J. Syst. Bacteriol. 38, Osterhout, G.J., V.H. Shull, and J.D. Dick Identification of clinical isolates of Gram-negative nonfermentative bacteria by an automated cellular fatty acid identification system. J. Clin. Microbiol. 29, Saitou, N. and M. Nei he neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4, Satoshi, Y. and H. Shigeaki Phylogenetic analysis of Acinetobacter strains based on the nucleotide sequences of gyrb genes and on the amino acid sequences of their products. Int. J. Syst. Evol. Microbiol. 46, (Received November 20, 2006/Accepted December 22, 2006) ABSRAC : Isolation and Identification of Acinetobacter koreensis sp. nov. from Jang-Baek Waterfall Ha-Yan Lee, Yong-Kyu Yoo 1, Pil-Soo Seo 2, Jung-Sook Lee 3, Keun-Chul Lee 3, and Sang- Seob Lee* (Department of Biological Engineering, Kyonggi University, Suwon , Korea, 1 Korea Environmental Microorganism Center, Kyonggi University, Suwon , Korea, 2 Depart-ment of Korea Biological Resourse Center, Kyonggi University, Suwon , Korea, 3 Korean Collection for ype Cultures, Korea Research Institute of Bioscience and Biotechnology, 52 Oeundong, Yusong, Daejeon , Korea) wo isolates of genus Acinetobacter were obtained from Jang-Baek waterfall in North Korea. Morphological characteristics of the isolated 2 strains were Gram-negative, aerobic and rod shape bacteria. Physiological and biochemical characterization of the isolated 2 strains were some different aspect from those of type strains. 16S rdna sequence analysis showed that the two isolates shared 99.9% sequence similarity. Strains JB10 and JB15 were shown to belong to the Gammaproteobacteria and showed the highest levels of sequence similarity to Acinetobacter tandoii 4N13 (97.3%), Acinetobacter haemolyticus ACC17906 (97.2%), Acinetobacter johnsonii DSM6963 (97.2%), Acinetobacter junii DSM6964 (96.7%), Acinetobacter schindleri LUH5832 (97.0%) and Acinetobacter ursingii LUH3792 (96.6%). he major cellular fatty acid in Acinetobacter type strains and isolated strains included C 18:1 ω9c and C16:1 ω7c/c 15:0 iso 2OH. Eventhough it was ascertained that the isolated strains were closely related to genus Acinetobacter, physiological and biochemical characteristics and the result of the isolated strains 16S rdna analysis indicate some different aspects from those of type strains of genus Acinetobacter. It is considered that the isolated JB10 (= KEMC ) and JB15 (KEMC ) strains be new species of genus Acinetobacter. We name it as Acinetobacter koreensis sp. nov.
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