循环水养殖系统中高毒力嗜水气单胞菌的鉴定与药敏试验

Identification and Antibiotic Susceptibility Test of Hypervirulent Aeromonas hydrophila in a Recirculating Aquaculture System

为探究中华鳖(Pelodiscus sinensis)循环水养殖过程中出现体表穿孔和溃疡的病因,对患病中华鳖的肝、脾、肺和肾等部位取样并进行病原菌分离及革兰氏染色,利用16S rRNA测序和生化鉴定相结合进行细菌鉴定,通过毒力基因PCR检测和人工回感试验评估细菌的致病性,以纸片扩散法评估几种抗生素的抑菌效果,利用琼脂打孔法评估中药提取物的抑菌效果。结果显示,从病鳖的脏器中分离到一株优势菌AH0421,该菌为革兰氏阴性菌,菌体呈短杆状;菌株AH0421与嗜水气单胞菌(Aeromonas hydrophila)模式菌株ATCC 7966的16S r RNA序列相似性高达99.66%。菌株AH0421能利用葡萄糖、蔗糖和麦芽糖,具有O/129耐受性,其携带act、aerA、aha、ahh、ahp、ahpA、alt、ast、hlyA、lip、ompA毒力基因。回感试验出现与自然发病相似病症,菌株AH0421半致死浓度(LC_(50))为1.7×10^(6)CFU/mL。中药五味子提取物、诺氟沙星、多西环素、恩诺沙星、新霉素以及头孢克肟、头孢曲松等头孢类抗生素对该菌有较强的抑制作用。研究表明,五味子和多数头孢类抗生素可作为治疗菌株AH0421感染的参考药物。

In May 2021,serious mortality of cultured Pelodiscus sinensis(Chinese softshell turtle)occurred in a recirculating aquaculture farm in Huzhou,Zhejiang Province and the infected turtles presented symptoms of perforations and ulcers.To identify the pathogen infecting the turtles and find effective inspection and prevention strategies for the disease,the dominant pathogenic strain was isolated from the liver,spleen,lung and kidney of diseased P.sinensis and labeled the AH0421 strain.Bacterial identification of the AH0421 strain was accomplished through gram staining,16S rRNA sequencing and determination of the virulence factor,as well as morphological,physiological and biochemical characteristics.The AH0421 strain is rod-shaped and gram-negative,and phylogenetic tree analysis,based on 16S rRNA gene sequences,shows that the AH0421 strain clusters with a strain of Aeromonas hydrophila ATCC 7966 with homology of 99.66%.The AH0421 strain can utilize glucose,sucrose,and maltose and has O/129 tolerance.According to PCR amplification,the AH0421 strain possesses 11 virulence genes,including act,aerA,aha,ahh,ahp,ahpA,alt,ast,hlyA,lip,and ompA.Simultaneously,an artificial infection study was carried out on healthy P.sinensis by injecting different concentrations of a bacterial suspension of the AH0421 strain identified as A.hydrophila.P.sinensis(body mass,~200 g)were randomly assigned to four treatment groups(6.8×10^(7),6.8×10^(6),6.8×10^(5)and 6.8×10^(4)CFU/mL)and a control group,with 10 P.sinensis in each group.The symptoms of the artificially infected turtles were identical to those of naturally infected turtles,and the concentration of strain AH0421 that was lethal for half of the test turtles(LC_(50))was 1.7×10^(6)CFU/mL.Drug susceptibility of the AH0421 strain was tested by the paper extension and agar punch methods using antibiotics and Chinese herbal medicines.The results show that the A.hydrophila strain is highly sensitive to the antibiotics norfloxacin,doxycycline,enrofloxacin,neomycin,cephalosporin,cefixime and ceftriaxone,and to the extract of Schisandra chinensis.In conclusion,A.hydrophila(AH0421)was the pathogen responsible for the mortality observed in the P.sinensis recirculating aquaculture system and cephalosporin antibiotics can be considered as therapeutic agents for the treatment of P.sinensis infected with A.hydrophila.