K-B 16S rdna Shewanella putrefaciens LD cfu / g A

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Acta Microbiologica Sinica 52 5 558-565 4 May 2012 ISSN 0001-6209 CN 11-1995 / Q http / / journals. im. ac. cn / actamicrocn Research Paper 222005 16S rdna K-B 16S rdna Shewanella putrefaciens LD 50 2. 1 10 3 cfu / g Ⅳ 16S rdna S941. 42 A 0001-6209 2012 05-0558-08 Carassius auratus gibelio S. putrefaciens Cyprinus carpio var. singuonensis Carassius auratus gibelio Bloch 1 1. 1 1 1. 1. 1 1-2 3 4 5 1. 1. 2 TSB 6 TaqDNA dntp 2008HS018 KQ07051 1978 - Tel + 86-518-85895427 E-mail qinlei1978@ 163. com 2011-10-23 2012-02-02

. / 2012 52 5 559 SS Neighbor-joining method Bootstrap 1. 2 1. 5 47 g 14 10 cm 27 ± 1 1 6 8 MS-222 5 SYY-1 9 10 8 9 10 7 9 10 6 9 10 5 9 10 4 9 10 2 cfu / 1. 3 ml 0. 2 ml 75% 28 TSB 28 24 h SYY-1 Karber TCBS SS LD 10 50 1. 6 15% - 70 1. 4 1. 4. 1 12 h 30 μg 10 μg 10 μg 0. 5% 30 μg 10 μg 30 μg 30 μg 5 μg 10 U 1. 4. 2 15μg 30 μg 15 μg 7 30 μg 5 μg 5 μg 2 μg 30 μg Ⅳ 30 μg 5 d Ⅴ 30 μg 30 μg 15 μg 300 μg 5 μg 1. 4. 3 16S rdna SYY-1 DNA 27f 1492r 8 16S rdna PCR PCR 1 PCR 2 mmol / L MgCl 2 200 μmol / L dntp 2. 1 0. 4 μmol / L 0. 5 μl TaqDNA 1 μl DNA 34. 5 μl ddh 2 O 94 3 min 94 1 min 50 1 min 72 2 min 30 72 10 min GenBank Blast 1000 K-B SYY-1 30 μg 30 μg 30 μg 2 EzTaxon server 2. 1 9 30% Mega 4

560 Lei Qin et al. / Acta Microbiologica Sinica 2012 52 5 2. 2 5 7 SYY TSB 24 h 7 1 mm - 2 mm TCBS SS 48 h α β 2. 3 7 SYY 0. 3-0. 5 μm 1. 5-3. 0 μm 1 VP 28-30 40 0-9% NaCl ph 6-8 ph 4 10 1 7 S. putrefaciens 1 Fig. 1 Electron micrograph showing negative staining of isolates. 2. 4 16S rdna SYY-1 16S rdna 1414 bp Table 1 GenBank JN555612 Blast 1 Phenotypic characteristics of isolates from diseased Carassius auratus gibelio Test SYY n = 7 S. putrefaciens * Gram - - Motility + + Gelatine liquefication + + Motility + + Pigment Red-brown Red-brown Oxidase + + Catalase + + Urease - - Ornithine decarboxylase + + Arginine dihydrolase - - Lysine decarboxylase - - Gas from glucose - nd H 2 S production + + Nitrate reduction + + Indole production - - Gelatine liquefication + d Acid production from Glucose - - Lactose - - Melibiose - nd Trehalose - - Fructose - - Arabinose - d Sorbitol - - Rhamnose - nd Sucrose - d Ribose - - Maltose - d Inositol - - Raffinose - nd Salicin - - Adonitol - nd Aesculin - - Xylose - - Mannose - - Citrate + d MR - nd VP - - Growth at 4 + + 28 + + 37 + + 40 - - Growth in 0% NaCl + + 3% NaCl + + 6% NaCl + d 9% NaCl + - 11% NaCl - - Growth in ph 4. 0 - nd ph 6. 0 + nd ph 8. 0 + nd + positive - negative nd no data d variable results * Data from Bergey's Manual of Determinative Bacteriology 11 Bacteria from Fish and Other Aquatic Animals a practical identification manual 12 Biochemical and Pathogenic of Shewanella algae and Shewanella putrefaciens 13 and Polyphasic Taxonomy of the Genus Shewanella and Description of Shewanella oneidensis sp. nov. 14.

. / 2012 52 5 561 EzTaxon server 2. 1 SYY-1 S. putrefaciens LMG26268 T 98. 7% 2 SYY-1 bootstrap 2 16S rdna SYY-1 Fig. 2 Phylogenetic tree of strain SYY-1 based on the result of 16S rdna sequence analysis. Numbers at branching points indicate bootstrap values 1000 replicates Numbers in brackets indicate GenBank accession number The sequence of Pseudomonas fluorescens ATCC13525 T AF094725 was used as outgroup. 2. 5 SYY-1 Ⅳ 3 9 10 8 cfu / ml 5 100% 9 10 2 cfu / ml 28 d SYY-1 28 d LD 50 2. 1 10 3 cfu / g 11-14 16S rdna SYY-1 2. 6 Shewanella 2 SYY-1 3 xiamenensis Shewanella oneidensis 16 S rdna SYY -1

562 Lei Qin et al. / Acta Microbiologica Sinica 2012 52 5 Pipemidic Acid 30 μg Nalidixic acid 30 μg S Fleroxacin 5 μg S Albamycin 30 μg R Enrofloxacin 5 μg S Fluperacid 10 μg S Jiemycin 2 μg R Enoxacin 10 μg S Ceftriaxone 30 μg S Penicillin 10 U I Cefalexin 30 μg S Streptomycin 10 μg I Cephazolin 30 μg I Neomycin 30 μg I Ceftazidine 30 μg S Florfenicol 30 μg S Roxithromycin 15 μg S Rifampicin 5 μg S Sulfamethoxazole 300 μg R Acetylspiramycin 30 μg I Levofloxacin 5 μg S Minocycline 30 μg S Midecamycin 30 μg I Azithromycin 15 μg I Kitasamycin 15 μg I S susceptible I intermediate susceptible R resistant. 16S rdna Achromobacter spp. 17 Pseudomonas spp. 18 99. 9% Alteromonas spp. 19 1985 MacDonell Colwell 5S rrna 16S rdna Shewanella spp. 20 Gilardi 21 14-15 S. SS 6% NaCl xiamenensis 3 3 6% NaCl 16 SYY-1 6% NaCl S. xiamenensis SYY-1 S. oneidensis 12 14 SYY-1 S. xiamenensis S. oneidensis Venkateswaran 14 16S rdna S. oneidensis S. putrefaciens 16S rdna S. oneidensis 3 SYY-1 S. oneidensis AF005251 Fig. 3 Mortality of Carassius auratus gibelio after experimental infection with strain SYY-1 at selected doses. S. xiamenensis 2 SYY-1 16S rdna Table 2 Drug sensitivity of strain SYY-1 Antibiotics Sensitivity Antibiotics Sensitivity Venkateswaran 14 S. putrefaciens ATCC 8071 16S rdna S Vancomycin 30 μg R 97% SYY-1 16S rdna SYY-1 S. putrefaciens Koch S. putrefaciens Derby Hammer

. / 2012 52 5 563 2 SS 6% NaCl 2 CDC 2 Khashe 13 30% 2 42 β 42 β 16S rdna Saeed 31 2 Kozi ska 35 22-23 24-27 1. 28 29 30. Journal of Fisheries of China 1991 15 2 130-139. 2.. Siganus rivulatus 31 Dicentrarchus Journal of Hydroecology 2010 3 4 labrax 32 Pseudosciaena crocea 33 102-107. Scophthalmus maximus 34 3.. Freshwater Fisheries 2009 39 2 48-53. Kozi ska 35 Cyprinus carpio L. 4. Oncorhynchus mykiss. Chinese Veterinary Science 2009 39 4 303- Eriocheir scinensis 36 309. 5.. Aquaculture 2008 5 40-42. 6 Lu HD Zhu GZ Fan LP Zhang LY. Etiology and pathology of epidermal papillomas in allogynogenetic crucian carp Carassius auratus gibelio x Cyprinus carpio var. singuonensis. Diseases of Aquatic Organisms 2009 83 1 77-84. 7.. 2001.

564 Lei Qin et al. / Acta Microbiologica Sinica 2012 52 5 8 Lane D. 16S /23S rrna sequencing / / Stackebrandt E Goodfellow M. Nucleic Acid Techniques in Bacterial Systematics. Chichester John Wiley & Sons Ltd. 1991 115-147. 9 Chun J Lee JH Jung Y Kim M Kim S Kim BK Lim YW. EzTaxon a web-based tool for the identification of prokaryotes based on 16S ribosomal RNA gene sequences. International Journal Systematic and Evolutionary Microbiology 2007 57 2259-2261. 10.. rods / / Gilardi GL. Nonfermentative gram-negative rods. 1982 587. 11 Holt JG Krieg NR Sneath PHA Staley JT Williams ST. Bergey's Manual of Determinative Bacteriology. 9 th eds. Baltimore Williams & Wilkins Press 1994. New York Marcel Dekker Inc 1985 17-84. 22. Alteromonas putrefaciens. Chinese Journal of Applied & Environmental Biology 2002 8 3 294-297. 12 Nicky BB. Bacteria from Fish and Other Aquatic Animals a practical identification manual. Cambridge CABI Publishing 2004. 13 Khashe S Janda JM. Biochemical and Pathogenic of Shewanella algae and Shewanella putrefaciens. Journal of Clinical Microbiology 1998 36 3 783-787. 14 Venkateswaran K Moser DP Dollhopf ME Lies DP Saffarini DA MacGregor BJ Ringelberg DB White DC Nishijima M Sano H Burghardt J Stackebrandt E Nealson KH. Polyphasic taxonomy of the genus Shewanella and description of Shewanella oneidensis sp. nov.. International Journal of Systematic Bacteriology 1999 49 705-724. 15 Wayne LG Brenner DJ Colwell RR Grimont PAD Kandler O Krichevsky MI Moore LH Moore WEC Murray RGE Stackebrandt E Starr MP Truper HG. International Committee on Systematic Bacteriology Report of the ad hoc committee on reconciliation of approaches to bacterial systematics. International Journal of Systematic Bacteriology 1987 37 463-464. 16 Huang JX Sun BL Zhang XB. Shewanella xiamenensis sp. nov. isolated from coastal sea sediment. International Journal of Systematic and Evolutionary Microbiology 2010 60 1585-1589. 17 Derby HA Hammer BW. Bacteriology of butter. IV. Bacteriological studies on surface taint butter. Iowa Agricultural Experiment Station Research Bulletin 1931 145 385-416. 18 Long HF Hammer BW. Classification of organisms important in dairy products III. Pseudomonas putrefaciens. Iowa Agricultural Experimental Station Research Bulletin 1941 285 176-195. 19 Baumann L Baumann P Mandel M Allen RD. Taxonomy of aerobic marine eubacteria. Journal of Bacteriology 1972 110 402-429. 20 MacDonell MT Colwell RR. Phylogeny of the Vibrionaceae and recommendation for 2 new genera Listonella and Shewanella. Systematic and Applied Microbiology 1985 6 171-182. 21 Gilardi GL. Cultural and biochemical aspects for identification of glucose-nonfermenting gram-negative 23.. Environmental Science & Technology 2004 27 3 14-15. 24 J rgensen BR Huss HH. Growth and activity of Shewanella putrefaciens isolated from spoiling fish. International Journal of Food Microbiology 1989 9 51-62. 25 Gram L Huss HH. Microbiological spoilage of fish and fish products. International Journal of Food Microbiology 1996 33 121-137. 26.. Journal of Fisheries of China 2006 30 6 824-830. 27.. Food Science 2011 32 13 184-187. 28 Bulut C Ertem GT G kcek C Tulek N Bayar MA Karakoc E. A rare cause of wound infection Shewanella putrefaciens. Scandinavian Journal of Infectious Diseases 2004 36 692-694. 29 Leonardo P Adolf L Claudio V Oswald M Giovanni R Raffaele P Peter M. Soft tissue infection and bacteremia caused by Shewanella putrefaciens. Journal of Clinical Microbiology 2003 41 2240-2241. 30 Benu D Rama C Baijayanti MM. Isolation of Shewanella putrefaciens from a rheumatic heart disease patient with infective endocarditis. Journal of Clinical Microbiology 1998 36 2394. 31 Saeed MO Alamoudi MM Al-Harbi AH. A Pseudomonas associated with disease in cultured rabbitfish Siganus rivulatus in the Red Sea. Diseases of

. / 2012 52 5 565 Aquatic Organisms 1987 3 177-180. 32 Korun J Akgün-dar K Yazici M. Isolation of Shewanella putrefaciens from cultured european sea bass Dicentrarchus labrax in Turkey. Revue de Médecine Vétérinaire 2009 160 532-536. 33.. Marine Science 1999 4 58-62. 34.. 2008. 35 Kozi ska A Pekala A. First isolation of Shewanella putrefaciens from freshwater fish a potential new pathogen of fish. Bulletin European Association of Fish Pathologists 2004 24 189-193. 36.. Fisheries Science 2002 2 24-27. A new pathogen of gibel carp Carassius auratus gibelio- Shewanella putrefaciens Lei Qin * Xiaojun Zhang Keran Bi College of Marine Science Huaihai Institute of Technology Lianyungang 222005 China Abstract Objective We studied a novel disease occurred among cultured Carassius auratus gibelio at a farm located in Yancheng City Jiangsu Province. Methods The dominant bacteria were isolated from diseased fish. The pure culture of the isolated strain was analyzed using conventional physiological and biochemical tests together with 16S rdna gene sequencing. An experimental infection of Carassius auratus gibelio with the isolated strain was performed to fulfill the Koch postulates. K-B method was used for antibiotic susceptibility testing. Results The causal agent of the disease was finally proved to be one species of bacteria that was identified as Shewanella putrefaciens. Experimental infection with S. putrefaciens resulted in the same gross signs as naturally infected fish and the same bacteria were recovered in a pure culture from freshly dead fish. The LD 50 of S. putrefacien was calculated as 2. 1 10 3 cfu / g. The result of drug sensitivity test showed that S. putrefaciens was sensitive to Pipemidic acid Nalidixic acid Fluperacid Enoxacin Florfenicol Rifampicin Minocycline Fleroxacin Enrofloxacin Ceftriaxone Cefalexin Ceftazidine Roxithromycin and Levofloxacin. Conclusion This is the first report on a new pathogen of Carassius auratus gibelio revealing that S. putrefaciens as a potential new pathogen may pose a threat to the culture of Carassius auratus gibelio. Keywords gibel carp Carassius auratus gibelio Shewanella putrefaciens 16S rdna antibiotic susceptibility testing Supported by the Open Fund of Key Laboratory of Oceanic Biotechnology of Jiangsu 2008HS018 and by the Doctoral Fund of Huaihai Institute of Technology KQ07051 * Corresponding author. Tel + 86-518-85895427 E-mail qinlei1978@ 163. com Received 23 October 2011 / Revised 2 February 2012