微生物学通报 AUG 20, 2008, 35(8): 1186~1191 Microbiology tongbao@im.ac.cn 研究报告 2008 by Institute of Microbiology, CAS 鲟源致病性嗜水气单胞菌 X1 的分离 鉴定与药敏特性研究 * 李圆圆 * 曹海鹏 ** 何珊杨先乐 ( 200090) 摘要 : 从患细菌性败血症的西伯利亚鲟 (Acipenser baerii) 的体内分离到一株致病菌株 X1, 其对西伯利亚鲟的半数致死浓度 (LC 50 ) 为 5.62 10 5 CFU/mL, 具有较强毒力 ; 菌株 X1 为革兰氏阴性杆菌, 菌体大小为 1.0 μm~1.2 μm 2.1 μm~2.4 μm, 周生侧鞭毛, 在兔血琼脂平板上能形成明显的 β- 溶血圈, 经 ATB 细菌鉴定仪鉴定和 16S rdna 序列分析, 菌株 X1 为嗜水气单胞菌 (Aeromonas hydrophila)( 登录号 :EU669667); 其系统发育分析表明, 菌株 X1 与嗜水气单胞菌 ATCC35654( 登录号 :X74676.1) 的亲缘关系最近, 其同源性为 99% 此外, 菌株 X1 对先锋必 左氟沙星等 2 种药物高度敏感, 对妥布霉素 氟哌酸 舒普深 卡那霉素 庆大霉素 复达欣 万古霉素 新霉素 多粘菌素 B 洛美沙星等 10 种药物中度敏感 关键词 : 西伯利亚鲟, 嗜水气单胞菌, 鉴定, 药敏特性 Isolation and Identification of Aeromonas hydrophila Strain X1 from Acipenser baerii and Its Antibiotic Sensitivity LI Yuan-Yuan * CAO Hai-Peng * HE Shan YANG Xian-Le ** (Aquatic Pathogen Collection Center of Ministry of Agriculture, SHOU, Shanghai 200090) Abstract: A pathogenic bacterial strain X1 was isolated from Siberian sturgeon (Acipenser baerii) suffering with bacterial septicemia. The 50% lethal concentration (LC 50 ) of strain X1 was 5.62 10 5 CFU/mL, which showed that strain X1 was rather strong virulent to Acipenser baerii. Strain X1 was gram negative and 1.0 μm~1.2 μm 2.1 μm~2.4 μm in size with peritrichous flagella, and had β-hemolytic activity on rabbit blood agar. By means of ATB expression identification and 16S rdna sequence analysis, strain X1 was identified as Aeromonas hydrophila (locus number: EU669667), which was the closest relative to Aeromonas hydrophila strain ATCC35654 (locus number: X74676.1) with 99% homology. In addition, strain X1 was highly sensitive to cefoperazone and cravit, and intermediately sensitive to ten kinds of antibiotics including tobramycin, norfloxacin, sulperazone, kanamycin, gentamycin, fortum, vancomycin, neomycina, polymyxin B and lomefloxacin. 基金项目 :(No. 2006BAD03B0506); (No. B-8101-08-0017); * (No. 6690107251) ** 通讯作者 :Tel: 021-65710870; : xlyang@shfu.edu.cn 收稿日期 :2008-01-12; 接受日期 :2008-04-25
: X1 1187 Keywords: Acipenser baerii, Aeromonas hydrophila, Identification, Antibiotic sensitivity (Acipenser baerii) (Acipenserdae)(Acipenser), 26, [1], [2] 1996, (Acipenser schrenckii), 2 [3],, 2006 6,,,, 1 X1, ;, X1, X1, X1 16S rdna, 2,,, 1 材料与方法 1.1 鲟鱼来源, 50 g~70 g, 13 cm~16 cm, 1.2 致病菌的分离与鉴定 1.2.1 致病菌的分离 :, 75,, (TSA), 28 24 h,, TSA, 4 1.2.2 致病菌的人工回归感染试验 :, 2.2 10 7 CFU/mL,, 0.2 ml 8, 19 ~22 7 d,, 1.2.3 致病菌株毒力测定 : 2.2 10 4 CFU/mL 2.2 10 5 CFU/mL 2.2 10 6 CFU/mL 2.2 10 7 CFU/mL,, 0.2 ml 8, 19 ~22 7 d, [4] (LC 50 ) 1.2.4 病原菌的鉴定 :, 28 24 h, ;,, ATB, 16S rdna, NCBI Blastn GenBank + EMBL + DDBJ + PDB 16S rdna, BioEdit 7.0 Mega 4.0 1.3 致病菌药敏特性的测定 KB [4], ( ), 28 24 h, (mm), [5], 2 结果 2.1 致病菌的分离 4, X1 X2 X3 X4,, X1,
1188 微生物学通报 2008, Vol.35, No.8 7 d, 100 ( 1),,,, X1 ( ) [ log(cfu/ml) ] y =36.25x 158.58( 1), X1 (LC 50 ) 5.62 10 5 CFU/mL, Devesa [6], X1 Strain Concentration(CFU/mL) Table 1 表 1 人工回归感染结果 Results of artificial infection test of strain X1 Death number( ) Number ( ) 1 d 2 d 3 d 4 d 5 d 6 d 7 d Mortality(%) X1 2.2 10 7 8 2 4 6 6 7 8 8 100% X2 2.2 10 7 8 0 0 0 0 0 0 0 0 X3 2.2 10 7 8 0 0 0 0 0 0 0 0 X4 2.2 10 7 8 0 0 0 0 0 0 0 0 图 1 鲟死亡率 (%) 与菌液浓度对数 ( log (CFU/mL)) 的关系曲线 Fig. 1 The relation curve of sturgeon mortality with logarithm of bacterial concentration 2.2 致病菌的鉴定 X1, 1.0 μm~ 1.2 μm 2.1 μm~2.4 μm, ( 2), β- ( 3) ATB X1 ( 2), X1 (Aeromonas hydrophila), 99NCBI X1 16S rdna 16S rdna, ( 3), X1 16S rdna GenBank (Aeromonas), ( 4), X1( EU669667) ATCC35654( X74676.1), 99 图 2 电镜下菌株 X1 的形态 Fig. 2 The morphological characteristics of strain X1 by electro microscope 图 3 菌株 X1 在兔血琼脂平板上产生的 β- 溶血圈 Fig. 3 The beta hemolysis zone of strain X1 on rabbit-blood agar plate
: X1 1189 Table 2 表 2 ATB 细菌鉴定仪对菌株 X1 的鉴定结果 The identification of strain X1 by ATB bacteria identification instrument Test item Result Test item Result Test item Result OX + CIT SAC + D- GLU + His + MAL + SAL + 2-2 kg ITA D- MEL 3-- 30BU SUB L- FUC 5-- 5 kg MNT D- SOR 3-- 30BU ACE L- ARA + RHA + DL- LAT + PROP N- NAG + L- Ala + CAP L- Pro + GLYG + VALT + INO MAN + L- Ser + Note: +: Denotes positivity; : Denotes negativity Table 3 表 3 菌株 X1 的 16S rdna 序列同基因库中细菌菌株同源性的比较 The comparison of 16S rdna sequence of strain X1 with high homological strains in GenBank Species Strain number Locus number Score Identity( ) Aeromonas hydrophila ATCC49140 AY987754.1 2579 99 Aeromonas hydrophila subsp. decolorations AY686711.1 2573 99 Aeromonas hydrophila CCM7232 DQ207728.2 2567 99 Aeromonas hydrophila WAB1968 AM184287.1 2567 99 Aeromonas hydrophila T20 AF099022.1 2567 99 Aeromonas hydrophila WAB1877 AM184219.1 2566 99 Aeromonas hydrophila ATCC35654 X74676.1 2560 99 Aeromonas hydrophila WAB1943 AM184282.1 2560 99 Aeromonas veronii 211c AY987746.1 2556 99 Aeromonas hydrophila WAB1920 AM184260.1 2553 99 Aeromonas hydrophila ATCC7966T X74677.1 2549 99 Aeromonas media CDC AY987773.1 2545 99 Aeromonas media ATCC33907 X60410.1 2543 99 Aeromonas veronii HQ010516C DQ029351.1 2532 99 Aeromonas media ATCC37907T X74679.1 2532 99 Aeromonas punctata RK27611 AY987760.1 2518 98 Aeromonas punctata WAB1928 AM184267.1 2503 98 16S rdna, X1 (Aeromonas hydrophila) 2.3 致病菌的药敏特性 X1 30 ( 4), X1 2, B 10 3 讨论,, (Aeromonas hydrophila) (Aeromonas caviae) (Plesiomonas shigelloides) [7 9], 4, X1, LC 50 5.62 10 5 CFU/mL,, X1
1190 微生物学通报 2008, Vol.35, No.8 Fig. 4 图 4 基于 16S rdna 序列的系统发育树 The phylogenetic tree based on 16S rdna sequence Antibiotics Sensitivity 表 4 菌株 X1 的药物敏感性 Table 4 The antibiotic sensitivity of strain X1 Antibiotics Sensitivity Antibiotics Tobramycin I Novobiocin R Cefoxitin R CXM R Augmentin R Lomefloxacin I Norfloxacin I Fortum I Ofloxacin R Sulperazone I Avocin R Cefradine R Kanamycin I Penicillin R Cefaran R Cefoperazone S Ampicillin R Cefalotin R Gentamycin I Vancomycin I Bactocill R Spectinomycin R Neomycina I Carbenicillin R Furacilin R V Cefalin V R Cefamandole R Cravit S BPolymyxin B I Timentin R : S: ( d >15 mm); I: (10 mm d 15 mm); R: (0 mm d <10 mm) Sensitivity Note: S: Denotes high sensitivity (d >15 mm); I: Denotes moderate sensitivity (10 mm d 15 mm); R: Denotes low or no sensitivity (0 mm d<10 mm), X1 X1 16S rdna (Aeromonas hydrophila),, [10], X1, [7,8],, flha lafk flij L flgn flge maf-5 [11] Gavin,, [12], X1,
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