5 : 1085 (V. fluvialis, KCCM40827) (Escherichia coli, ATCC29532) (SAT) SAT [13] mol/l NaH 2 PO 4 (ph 6.8) 0.2 mol/l, mol/l San

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1 2011 9, 18(5): Journal of Fishery Sciences of China 研究论文 DOI: /SP.J 李华, 王扬, 李强, 乔帼, 摘要 : (Vibrio splendidus)ap622,,, AP622, 0.5% LB, 24 h AP622 Ⅳ AP622, (MIC) 32, AP622, : ; ; ; ; 中图分类号 : S94 文献标志码 : A 文章编号 : (2011) , [1 3],,, [4],,, [1], [1,5] (Vibrio splendidus), (Apostichopus japonicus) [6 7], (Scophthalmus maximus) [8] (Ruditapes decussatus) [9] (Paralichthys olivaceus) [10], [11] [12] [8],,,, (Vibrio splendidus, AP622) (V. splendidus, KCTC12679) (V. harveyi, ATCC14126) (Shewanella marisflavi, KCCM41822) (V. anguillarum, HUFP5001) (V. alginolyticus, KCCM 40513) (V. parahaemolyticus, KCTC2471) 收稿日期 : ; 修订日期 : 基金项目 : ( ); (2011BAD13B03); ( ). 作者简介 : (1958 ),,, lihua@dlou.edu.cn

2 5 : 1085 (V. fluvialis, KCCM40827) (Escherichia coli, ATCC29532) (SAT) SAT [13] mol/l NaH 2 PO 4 (ph 6.8) 0.2 mol/l, mol/l Santos [14] SAT : 0~1.0 mol/l, 1.0~2.0 mol/l, 2.0~4.0 mol/l, >4.0 mol/l (MATH) MATH Won [15] Santos [14] (CSH %), CSH% = (D 0 D i )/D 0 100%, D 0 OD 600, D i OD 600 MATH : CSH%>50%, CSH% 20% 50%, CSH% 20% O Toole [16] AP622 LB ( 10 g/l; 5 g/l) 10 6 /ml 3 ml (PVC), h, PBS(0.01 mol/l, ph7.2, ) 3,, 1% (CV) 15 min, 3,, 95% PVC CV, 600 nm LB /ml AP622, 3, h, /ml AP622 (PVC), h, AP622 TSB( 15 g/l; 5 g/l) LB M63[68 g/l KH 2 PO 4, 10 g/l (NH 4 ) 2 SO 4, g/l MgSO 4 7H 2 O, 2.5 mg/l FeSO 4 7H 2 O, 2 g/l D- ] CM9(12.8 g/l Na 2 HPO 4, 3 g/l KH 2 PO 4, 0.5 g/l NaCl, 1.0 g/l NH 4 Cl, 2.0 g/l D-, g/l MgSO 4 7H 2 O, g/l CaCl 2 2H 2 O, 0.01 g/l, 0.01 g/l FeSO 4 7H 2 O, 2 g/l ) 10 6 /ml, 28 PVC 24 h, AP622 0% 0.05% 0.125% 0.25% 0.5% 0.75% 1% LB 10 6 /ml, 28, PVC 24 h, Deziel [17] AP622 KCTC KCCM41822 HUFP 5001 KCCM ATCC KCTC2471 KCCM ATCC (swimming) 9, NA ( 10 g/l, 3 g/l, 15 g/l), h ( 10 g/l, 3 g/l), h (swarming) 1.4.1, ( 8 g/l, 5 g/l, 5 g/l), h,

3 (twitching) 1.4.1, (3 mm)lb ( 10 g/l, 5 g/l, 10 g/l),, 28 24~48 h, 1%, (MIC) [18], 3 AP622 NB ( 10 g/l, 3 g/l) 10 6 /ml, mg/ml ( >590 μg/mg), h, (MIC) Walters [19] AP622 PBS 10 6 /ml,, 10 MIC 28, 180 r/min, 30 min 1 h 1.5 h 2 h 2.5 h 3 h 3.5 h 4 h 24 h, (MIC) AP622 TSB, h, PBS OD 600 =0.2; ( 25 mm), TSA, 5 μl, h, 12 h TSA [18] 48 h, 9 ml PBS, 2 min, , SPSS17.0 x ±SD P< , AP622 AP mol/l,, Santos [14] ; KCTC mol/l,, AP622 CSH% 54.83%, ; KCTC12679 CSH% 21.40%, 2.2 (CV), (P<0.05)( 1) AP622,, AP622 (P<0.05) AP622, ( 2A) AP622 (P<0.05) AP622 LB, M63 TSB, CM9 ( 2B) AP622 (P<0.05) 0.5%, AP622,, ( 2C)

4 5 : AP622 (P<0.05) AP622 4 h,, 24 h, 24 h ( 2D), AP622, 48 h 72 h, AP h 1 24 h, 24 h, ( 2D) 2.4, 9,,, AP622 Fig.1 1 Comparison on biofilm formation capability of different bacteria strains Fig.2 2 AP622 A: ; B: ; C: ; D. Effect of different factors on biofilm formation of Vibrio splendidus AP622 A:materials; B: media; C: concentration of glucose D: culturing time.

5 , KCTC12679, Ⅳ, Ⅳ,,, AP622 ; KCTC12679,, AP622, ( 1) 2.5 AP622 MIC 4 μg/ml 256 μg/ml, MIC 128 μg/ml μg/ml AP622 MIC MIC 32, MIC MIC 4 10 MIC, 3,, (P<0.05) AP h, 3.5 h 4 h 3, - [20 21], [1 4], AP622, AP622,,, AP h 1, bacterium 表 1 不同菌株的运动性 Tab.1 The motility of different bacteria swimming motility swarming Vibrio splendidus (AP622) + + twitching Vibrio splendidus(kctc12679) + Vibrio harveyi (ATCC14126) + + Shewanella marisflavi (KCCM41822) Vibrio, anguillarum (HUFP5001) + Vibrio alginolyticus (KCCM 40513) Vibrio parahaemolyticus (KCTC2471) Vibrio fluvialis (KCCM40827) Escherichia coli (ATCC29532) : + ; -. Note: + and - indicated positive and negative, respectively.

6 5 : AP622 KCTC Fig.3 Sensitivity of planktonic cells and resuspended biofilm cells of Vibrio splendidus AP622 or KCTC to gentamycin sulphate, AP622,, Donlan [22],,, AP622 LB, CM9, [23] (Pseudomonas aeruginosa), [24], AP %, [24],,,, ;, [1,5],, AP622 Ⅳ, AP622,, [25] [7], AP622 MIC, AP622 MIC 32, MIC MIC 4, AP622, 3 : 1) ; 2), ; 3), [26], AP622, AP622,

7 参考文献 : [1],. [J]. :, 2007, 31(6): [2],. [J]., 1995, 21(2): [3] Balebona M C, Morinigo M A, Borrego J J. Hydrophobicity and adhesion to fish cells and mucus of Vibrio strains isolated from infected fish [J]. Int Microbiol, 2001, 4 (1): [4],,,. [J]., 2003, 13(12): [5] Hossain M M, Tsuyumu S. Flagella-mediated motility is required for biofilm formation by Erwinia carotovora subsp. Carotovor [J]. J Gen Pathol, 2006, 72: [6],,. [J]., 2006, 30(1): [7] Li H, Qiao G, Gu J Q, et al. Phenotypic and genetic characterization of bacteria isolated from diseased cultured sea cucumber Apostichopus japonicus in northeastern China [J]. Dis Aquat Org, 2010, 91: [8] Gatesoupe F J, Lambert C, Nicolas J L. Pathogenicity of Vibrio splendidus strains associated with turbot larvae, Scophthalmus maximus [J]. J Appl Microbiol, 1999, 87, [9] Gomez-Leon J, Villamil L, Lemos M L, et al. Isolation of Vibrio alginolyticus and Vibrio splendidus from aquacultured carpet shell clam (Ruditapes decussatus) larvae associated with mass mortalities [J]. Appl Environ Microbiol, 2005, 71(1): [10],,,. [J]., 2003, 34(2): [11] Vazquez J A, Docasal S F, Miron J, et al. Proteases production by two Vibrio species on residuals marine media [J]. J Ind Microbiol Biotechnol, 2006, 33: [12] Farto R, Armada S P, Montes M, et al. Presence of a lethal protease in the extracellular products of Vibrio splendidus Vibrio lentus related strains [J]. J Fish Dis, 2006, 29, [13],. 2 [J]., 2006, 26(3): [14] Santos Y, Bandin I, Bruno D W, et al. Comparison of the cell surface hydrophobicity of bacterial fish pathogens by different procedures [C]//Perkins O F, Chen T C. Pathology in Marine Sciences. San Diego: Academic Press, 1990: [15] Won K M, Park S I. Pathogenicity of Vibrio harveyi to cultured marine fishes in Korea [J]. Aquaculture, 2008, 285: [16] O Toole G A, Kolter R. Initiation of biofilm formation in Pseudomonas fluorescens WCS365 proceeds via multiple, convergent signaling pathways: a genetic analysis [J]. Mol Microbiol, 1998, 28(3): [17] Deziel E, Comeau Y, Villemur R. Initiation of biofilm formation by Pseudomonas aeruginosa 57RP correlates with emergence of hyperpiliated and highly adherent phenotypic variants deficient in swimming, swarming, and twitching motilities [J]. J Bacteriol, 2001, 183(4): [18],,,. [J]., 2008, 29 (1) : [19] Walters III M C, Roe F, Bugnicourt A, et al. Contributions of antibiotic penetration, oxygen limitation, and low metabolic activity to tolerance of Pseudomonas aeruginosa biofilms to ciprofloxacin and tobramycin [J]. Antimicrob Agents CH, 2003, 47(1): [20],,,. JO 1 [J]., 2002, 25 (2): [21] Uchimura M, Yamamoto T. Production of hemagglutinins and pill by Vibrio mimicus and its adherence to human and small intestines in vitro [J]. Fems Microbio1 Lett, 1992, 91 (1): [22] Donlan R M. Biofilms: microbial life on surfaces [J]. Emerg Infect Dis, 2002, 8(9): [23],,. [J]., 2007, 11(5): [24],,,. [J]., 2005, 45(3): [25],. [J]., 2007, 47(5): [26]. [J]., 2004, 31(5):

8 5 : 1091 Cell surface hydrophobicity and biofilm formation ability of Vibrio splendidus LI Hua, WANG Yang, LI Qiang, QIAO Guo Dalian Ocean University, Key Laboratory of Mariculture, Ministry of Agriculture, Dalian , China Abstract: Vibrio splendidus infects a range of hosts, including fish, shellfish, and echinoderms. Infection with this pathogen has led to significant economic loss in several cultured species, including the sea cucumber (Apostichopus japonicus). Thus, there is an urgent need to understand the pathogenesis of V. splendidus. The virulence of a pathogen is partly a function of its adhesion properties. Adhesion depends on cell surface hydrophobicity and biofilm formation. The hydrophobic cell surface provides an advantage to the bacteria in vivo by increasing resistance to phagocytosis or by favoring colonization of mucosal or connective tissue in wounds and endocarditis in bacteria. We evaluated the hydrophobicity and biofilm formation in V. splendidus AP622, isolated from diseased A. japonicus. We also evaluated the effect of culture constituents, culture time, growth medium, and the concentration of glucose on biofilm formation. We compared the sensitivity of planktonic cells and resuspended V. splendidus AP622 biofilm cells to gentamycin sulphate. Last, we quantified the motility of the flagellum and measured pill and cell surface hydrophobicity using tests for salt-aggregation (SAT) and microbial adherence to hydrocarbons (MATH). V. splendidus AP622 possesses a high ability to form a biofilm. The biofilm formation cycle was 24 h in length and the optimal formation conditions consisted of polyvinyl chloride (PVC) and LB medium with 0.5% glucose. Swarming and twitching motilities played an important role in biofilm formation. The resistance of resuspended biofilm bacteria to antibiotics was significantly higher than in planktonic bacteria (P<0.05). Furthermore, the minimum inhibitory concentration (MIC) was 32 times higher than in planktonic bacteria. V. splendidus AP622, exhibiting strong hydrophobicity, could agglutinate at a concentration of 0.6 mol/l ammonium persulfate, and the adhesion partition to liquid hydrocarbons was >50%. In conclusion, the hydrophobicity and biofilm formation properties suggest that V. splendidus AP622 has strong adhesion properties. Resistance to antibiotics as a result of biofilm formation will reduce the effectiveness of treatments in aquaculture facilities. Thus, cell surface hydrophobicity and biofilm formation play an important role in the pathogenesis of V. splendidus. Key words: Apostichopus japonicus; Vibrio splendidus; biofilm; motility; hydrophobicity

5期xin

5期xin 中国农学通报 2014,30(5):23-27 Chinese Agricultural Science Bulletin 大肠杆菌生物膜的筛选及生长曲线测定喻华英, 李启峰 ( 塔里木大学动物科学学院, 新疆阿拉尔 843300) 摘要 : 为探究犊牛腹泻病中大肠杆菌生物膜的形成