大菱鲆源杀鱼爱德华氏菌的分离鉴定及其毒力和耐药基因检测分析

Isolation, identification, and analysis of virulence gene and drug resistance gene in pathogenic Edwardsiella piscicida from cultured turbot Scophthalmus maximus

为探究2019年山东省烟台市某大菱鲆Scophthalmus maximus养殖场出现的以腹水、体表及内脏出血为主要症状的疾病发病原因,通过平板划线法从患病濒死大菱鲆(体质量为350 g±5 g)肝、肾、脾和心等组织中分离获得1株优势菌,采用形态学观察,16S rRNA、gyrB基因和rpoB基因序列分析,PCR特异性引物扩增,以及人工回归感染试验等方法对优势菌进行鉴定,检测其毒力基因、耐药基因携带情况,并采用纸片扩增法(K-B)检测分离株对10类23种抗生素的敏感性。结果表明:从患病鱼分离获得的菌株,生理生化特征为革兰氏阴性杆菌,赖氨酸、鸟氨酸和葡萄糖产酸等试验呈阳性,枸橼酸、精氨酸、蔗糖、甘露醇、苦杏仁苷、尿素、硝酸盐还原和氧化酶等试验呈阴性;经16S rRNA、gyrB基因、rpoB基因序列比对,以及爱德华氏菌特异性引物扩增等鉴定,确定其为杀鱼爱德华氏菌Edwardsiella piscicida;该菌的毒力基因型为fimA+-fimB+-fimC+-fimD+-esrB+-mukF+-katB+-sodB+-citC+-gadB+,耐药基因型为TEM+-ant(3″)-Ⅰ+-tet(A)+-mcr-1+-Sul2+-Sul3--oqxA-;将分离菌株制备成7.8×1010、7.8×109、7.8×108、7.8×107CFU/mL 4个浓度的菌悬液,腹腔注射健康大菱鲆,得到菌株对受试鱼的半致死浓度(LD50)为1.2×108CFU/mL;药敏试验显示,该菌株对磺胺类2种、多肽类2种和利福霉素类1种在内的5种抗生素类药物耐药;对β-内酰胺类5种、氨基糖苷类3种、喹诺酮类5种、大环内酯类1种、氯霉素类1种、四环素类2种和呋喃类1种在内的18种抗生素敏感。研究表明,本试验首次从大菱鲆上检出携带5种耐药基因的杀鱼爱德华氏菌,本研究结果为鱼源耐药性杀鱼爱德华氏菌的分子特性、致病机理和药物防治等研究提供了科学参考。

In order to explore the causes of diseases characterized by ascites and sepsis in turbot Scophthalmus maximus with body weight of(350±5)g farmed in Yantai, Shandong Province in 2019, predominant bacterial strains were isolated from moribund individual tissues including liver, kidney, spleen, and heart, and identified by morphological observation, 16S rRNA gene sequence analysis, gyrB gene sequence analysis, rpoB gene sequence analysis, and specific PCR amplification. Genetic characteristics of the isolated strain were analyzed by virulence genes and drug resistance gene, and the susceptibility of the isolated strain to 10 categories of drugs including 23 antimicrobial dugs was determined using Kirby-Bauer diffusion method. The results showed that the strain isolated from the visceral tissues as Gram-negative bacillus named as H4-S18 was identified as Edwardsiella by the physiological features of positive lysine, ornithine and glucose acid production, and negative citrate, arginine, sucrose, mannitol, amygdalin, urea, nitrate reduction and oxidase. The strains H4-S18 was identified as Edwardsiella piscicida by 16S rRNA, gyrB and rpoB gene sequence alignment and specific amplification of Edwardsiella. The virulence genotype was found to be fimA+-fimB+-fimC+-fimD+-esrB+-mukF+-katB+-sodB+-citC+-gadB+. The drug resistance genotype was found to be TEM+-ant(3″)-Ⅰ+-tet(A)+-mcr-1+-Sul2+-Sul3--oqxA-. The strain H4-S18 had median lethal concentration( LD50)of 1.2×10~8 CFU/mL determined in the healthy turbot injected with the bacterial solution intraperitoneally at a dose of 7.8×1010, 7.8×10~9, 7.8×10~8 and 7.8×10~7 CFU/mL. Antibiotic sensitivity test showed that among 23 antibiotics tested, H4-S18 was resistant to 5 antibiotics such as sulfonamides, polypeptides, and rifamycins, but was sensitive to 18 antibiotics such as β-lactams, aminoglycosides, quinolones, tetracyclines, macrolides, chloramphenicols, and furan. It is considered that H4-S18, E.piscicida bacterium is the first pathogenic strain isolated from turbot with five resistant genes. The finding provides basic support for the research of fish-sourced E.piscicida disease surveillance, precision prevention, and control.