Screening of antagonistic bacteria against visceral white-spots disease of Larimichthys crocea and preliminary study on its biological characteristics
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摘要: 为实现大黄鱼 (Larimichthys crocea) 内脏白点病的生物防控，推进水产养殖用药减量，从健康大黄鱼肠道中筛选出对其内脏白点病病原菌——杀香鱼假单胞菌 (Pseudomonas plecoglossicid) 有拮抗作用的益生菌。采用琼脂扩散法筛选菌株，通过生理生化特征以及分子生物学分析，对菌株进行鉴定，并评价其溶血性、药敏性、安全性、产酶能力及抗菌广谱性。从健康大黄鱼肠道中初步分离出37株潜在益生菌，通过拮抗试验筛选出3株具有拮抗效果的菌株，分别命名为P1-17、P2-33和P3-11。通过生理生化特性及16S rRNA 测序分析，菌株P1-17和P2-33鉴定为贝莱斯芽孢杆菌 (Bacillus velezensis)，菌株P3-11鉴定为粪肠球菌 (Enterococcus faecalis)；溶血性试验和药敏试验结果表明3株菌均无显著溶血圈，且所含耐药因子较少，不具备潜在致病性；人工回感试验证实，3株拮抗菌对健康大黄鱼无致病性；抗菌谱测定结果表明，2株芽孢杆菌对溶藻弧菌 (Vibrio alginolyticus)、哈维氏弧菌 (V. harveyi)、美人鱼发光杆菌 (Photobacterium damselae) 等多种水产病原菌具有拮抗效果，同时2株芽孢杆菌具有产淀粉酶和蛋白酶的能力；而该株粪肠球菌只对杀香鱼假单胞菌有拮抗作用。研究结果可为后续大黄鱼肠道益生菌的筛选和应用提供科学依据。Abstract: In order to achieve the biological prevention and control of visceral white-spots disease of Larimichthys crocea, and promote the reduction of drug use in aquaculture, we isolated and screened the probiotics with an antagonistic effect on Pseudomonas plecoglossicid, which is a pathogen of visceral white spot disease in L. crocea, from the intestine of healthy L. crocea. The strains were screened by agar diffusion method, identified by physiological and biochemical characteristics and molecular biology analysis, and evaluated for hemolysis, drug sensitivity, safety, enzyme production ability and broad-spectrum antibacterial activity. Thirty-seven strains of potential probiotics were isolated and the three most potent strains were further characterized for their probiotic potential, named as P1-17, P2-33 and P3-11. The three most promising isolates were identified by sequencing the 16S rRNA gene and physiological and biochemical characteristics. Strains P1-17 and P2-33 were identified as Bacillus velezensis and P3-11 was identified as Enterococcus faecalis. According to the hemolytic test and disk diffusion method, none of the three strains had sigificant hemolytic rings, containing few drug resistance factors, so they had no potential pathogenicity. The result from the artificial infection safety test confirmed that these three strains of antagonistic bacteria had no pathogenicity to healthy L. crocea. The results of the antimicrobial spectrum show that two strains of Bacillus had an antagonistic effect on common aquatic pathogens such as Vibrio alginolyticus, V. harveyi and Photobacterium damselae. Moreover, two strains of Bacillus could produce amylase and protease, while the strain of E. faecalis only had an antagonistic effect on P. plecoglossicida. The study provides a scientific basis for the subsequent screening and application of intestinal probiotics in L. crocea.
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图 1 拮抗菌P1-17、P2-33、P3-11菌落形态
Figure 1. Colony morphology diagram of antagonistic bacteria P1-17, P2-33 and P3-11
图 2 拮抗菌P1-17、P2-33、P3-11革兰氏染色图
Figure 2. Gram staining of antagonistic bacteria P1-17, P2-33 and P3-11
图 3 基于菌株P1-17、P2-33及相关菌株16S rRNA基因序列的系统发育树
注：括号中的序号代表菌株的GenBank登录号；分支点上的数字代表bootstrap值；标尺刻度代表碱基替代率。图4同此。
Figure 3. Phylogenetic tree based on 16S rRNA sequences of Strains P1-17, P2-33 and other related strains
Note: The sequence number in the brackets is the GenBank accession number of the strain; the numbers at the node are the bootstrap values; the scale bar indicates nucleotide substitution ratio. The same case in Fig. 4.
图 4 基于菌株P3-11及相关菌株16S rRNA基因序列的系统发育树
Figure 4. Phylogenetic tree based on 16S rRNA sequences of Strain P3-11 and other related strains
表 1 菌株P1-17、P2-33、P3-11的生理生化特征
Table 1. Physiological and biochemical characteristics of Strains P1-17, P2-33 and P3-11
鉴定项目
Tested item菌株 Strain P1-17 P2-33 P3-11 蛋白胨水 peptone water + + + 蔗糖 Sucrose − − + 甘露糖 Mannose − − − 阿拉伯糖 Pectinose + + + 乳糖 Lactose − − − 葡萄糖氧化发酵试验 OF − − − 邻硝基苯-半乳糖苷酶
o-nitrophenyl-galactosidase− − − 柠檬酸盐 Citrate − − − 硫化氢 H2S − − − 尿素酶 Urease − − − VP试验 Voges-Proskauer test + + + 明胶液化 Gelatin Liquefaction + + + 吲哚试验 Indole test − − + 葡萄糖 Glucose − − + 甘露醇 Mannitol − − + 肌醇 Inositol − − + 山梨醇 Sorbitol − − + 鼠李糖 Rhamnose − − − 密二糖 Melibiose − − − 苦杏仁苷 Amygdalin − − + 氧化酶试验 OX − − − 注：+. 阳性；−. 阴性。 Note: +. Positive; –. Negative. 
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表 2 菌株P1-17、P2-33 和 P3-11的药敏特性
Table 2. Antibiotic sensitivity of Strains P1-17, P2-33 and P3-11
抗菌药物种类
Type of Antibacterial药物名称
Drug纸片含药量
Drug content抑菌圈直径/敏感性
Diameter of inhibition zone (mm)/SusceptibilityP1-17 P2-33 P3-11 青霉素类
Penicillins氨苄西林 Ampicillin 10 μg·片−1 27.41/S 22.34/S 14.56/R 青霉素 Penicillin 10 μg·片−1 32.65/S 27.54/S 21.37/S 阿莫西林 Amoxicillin 20 μg·片−1 28.57/S 18.26/S 16.22/I 新生霉素 Neomycin 30 μg·片−1 28.57/S 28.57/S 17.30/S 头孢菌素类
Cephalosporins头孢噻肟 Cefotaxime 30 μg·片−1 37.58/S 31.48/S 0/R 头孢曲松 Ceftriaxone 30 μg·片−1 37.45/S 33.56/S 0/R 喹诺酮类
Quinolones诺氟沙星 Norfloxacin 10 μg·片−1 32.36/S 31.43/S 19/S 依诺沙星 Enoxacin 10 μg·片−1 32.54/S 31.64/S 22/S 环丙沙星 Ciprofloxacin 5 μg·片−1 41.05/S 36.55/S 24/S 氧氟沙星 Ofloxacin 5 μg·片−1 41.24/S 39.15/S 25/S 左氧氟沙 Levofloxacin 5 μg·片−1 39.04/S 38.86/S 19/S 恩诺沙星 Enrofloxacine 10 μg·片−1 40.32/S 36.58/S 24/I 洛美沙星 Lomefloxacin 10 μg·片−1 40.27/S 38.36/S 21/S 磺胺类
Sulfonamides磺胺异恶唑 Sulfisoxazole 300 μg·片−1 20.34/S 18.87/S 0/R 复方新诺明 Cotrimoxazole 25 μg·片−1 34.66/S 32.78/S 0/R 四环素类
Tetracyclines四环素 Tetracycline 30 μg·片−1 25.16/S 23.36/S 15/I 多西环素 Doxycycline 30 μg·片−1 33.27/S 33.27/S 26/S 大环内酯类
Macrolides阿奇霉素 Azithromycin 15 μg·片−1 30.26/S 27.67/S 13/R 罗红霉素 Roxithromycin 15 μg·片−1 37.57/S 33.35/S 14/R 氨基苷类
Aminoglycosides链霉素 Streptomycin 10 μg·片−1 26.26/S 25.86/S 0/R 卡那霉素 Kanamycin 30 μg·片−1 35.06/S 34.89/S 0/R 丁胺卡那 Amikacin 30 μg·片−1 37.34/S 31.57/S 0/R 庆大霉素 Gentamycin 10 μg·片−1 34.36/S 31.68/S 0/R 妥布霉素 Tobramycin 10 μg·片−1 36.02/S 34.08/S 0/R 氯霉素类
Chloramphenicol氯霉素 Chloramphenicol 30 μg·片−1 35.24/S 35.24/S 18/I 氟苯尼考 Florfenicol 30 μg·片−1 38.31/S 33.65/S 22/S 肽类抗生素
Peptides万古霉素 Vancomycin 30 μg·片−1 30.10/S 28.35/S 20/S 多黏菌素 B Polymyxin B 300 IU 17.28/S 14.07/S 0/R 多烯类抗真菌抗生素
Polyeneantifungal制霉菌素 Nystatin 100 μg·片−1 10.57/R 11.21/R 0/R 注：S. 高度敏感；I. 中度敏感；R. 耐药。 Note: S. Sensitive; I. Intermediate sensitive; R. Resistance.
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表 3 拮抗菌株产酶能力
Table 3. Enzyme production ability of antagonistic strains
菌株名称
Strain水解圈直径/菌落直径
Dh/Dc蛋白酶
Protease脂肪酶
Lipase淀粉酶
AmylaseP1-17 4.23 0 4.20 P2-33 3.82 0 4.46 P3-11 0 0 0
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表 4 分离菌株对杀香鱼假单胞菌的拮抗试验
Table 4. Antagonism test of isolated strains against P. plecoglossicida
菌株
Strain抑菌圈直径 Diameter of inhibition zone/mm P1-17 P2-33 P3-11 杀香鱼假单胞菌 1303001 P. plecoglossicida 1303001 10.24±0.41 9.86±0.35 12.57±0.64 杀香鱼假单胞菌 1303002 P. plecoglossicida 1303002 10.53±0.72 10.18±1.14 12.6±0.83 杀香鱼假单胞菌 1306002 P. plecoglossicida 1306002 9.76±0.47 9.58±0.34 11.47±0.54 杀香鱼假单胞菌 202010001 P. plecoglossicida 202010001 10.43±1.40 9.76±0.65 11.85±1.21 杀香鱼假单胞菌 202010002 P. plecoglossicida 202010002 9.45±1.52 9.25±0.74 10.68±0.36 杀香鱼假单胞菌 202105001 P. plecoglossicida 202105001 9.18±0.64 8.74±1.32 10.46±0.54 杀香鱼假单胞菌 202105002 P. plecoglossicida 202105002 10.65±0.22 9.65±0.83 12.34±1.12 杀香鱼假单胞菌 202105003 P. plecoglossicida 202105003 9.34±0.72 9.05±0.63 11.38±1.43
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表 5 拮抗菌对其他致病菌的拮抗试验
Table 5. Antagonism test of antagonistic bacteria against other pathogenic bacteria
菌株
Strain抑菌圈直径 Diameter of inhibition zone/mm P1-17 P2-33 P3-11 溶藻弧菌 Vibrio alginolyticus 10.39±0.67 7.78±0.56 0 哈维氏弧菌 V. harveyi 10.65±1.54 10.34±1.56 0 副溶血弧菌 V. parahaemolyticus 11.57±0.34 11.25±0.42 0 美人鱼发光杆菌 Photobacterium damselae 15.13±0.24 14.96±0.53 0 嗜水气单胞菌 Aeromonas hydrophila 19.64±1.56 16.85±0.37 0 迟缓爱德华氏菌 Edwardsiella tarda 21.85±0.24 20.83±0.46 0 注：“0”表示无拮抗作用。 Note: "0" indicates no antagonism.
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