锦鲤源铜绿假单胞菌的分离鉴定及药敏试验

对从发病锦鲤分离出的病原菌主要形态与生理生化特性、16S rRNA基因序列及耐药特性等进行了研究，旨在为锦鲤疾病防治提供理论依据，对铜绿假单胞菌的进一步研究提供基本资料。发病锦鲤体长37~42 cm，体重1.5~2.0kg，眼球脓肿凸出，鳃丝被轻微腐蚀，其上可见附着物，肛门红肿并伴有黄色粘液流出。健康锦鲤体长7~10cm，体重(25±3)g。分离到的1株形态一致优势生长菌株（记为HL2）呈现圆形光滑、边缘整齐、中央隆起、略透明、乳白色,直径约1.4mm。分离菌经革兰氏染色镜检，均为革兰氏阴性、短杆状。肌肉注射感染后，供试健康锦鲤14d内全部死亡，并在试验后期注射部位轻微出血，眼球周边有白浊及化脓的现象。HL2有运动性，精氨酸脱羧酶、鸟氨酸脱羧酶、精氨酸双水解酶阳性，苯丙氨酸脱羧酶阴性，发酵麦芽糖、甘露醇、蔗糖、半乳糖等，不发酵侧金盏花醇与阿拉伯糖等。分离株HL2的序列长度为1461bp(GenBank登录号为KF413420)，在系统发育树上与铜绿假单胞菌（KC959478）聚为一支。头孢曲松、卡那霉素、四环素等35种药物有效抑制HL2，庆大霉素、强力霉素、头孢拉定等5种药物具有一定的抑菌作用，青霉素G、氨苄西林、头孢唑林等15种药物无效。使用抗生素时必须更加科学、合理、有效；从药物选择、使用剂量、用药疗程等方面着手，以减少耐药菌株的产生。

Isolation, Identification and Drug Sensitivity Test of Pathogenic Pseudomonas aeruginosa from Koi

To identify the pathogen that caused serious mortality of koi cultured in Jiangsu Province during August 2014, the pathogenic strain was isolated from diseased fish and labeled the HL2 strain. After purification, the isolated strain was cultured by incubation in LB medium. Healthy koi with body length 7-10 cm and body mass 25±3g were infected with strain HL2. Sixty healthy koi were randomly assigned into three control groups and three test groups, with 10 koi in each group. The koi in the test groups were injected with 0.2ml bacterium suspension at a concentration of 1×108 CFU/mL and the control groups were injected with an equal volume of LB medium. The mortality rate of each group was recorded daily and the pathogenic strain from dead koi was isolated to confirm the cause of death was infection by strain HL2. The isolated bacteria strain HL2 was highly pathogenic and healthy koi in the test groups were all dead within 14 days. Clinical symptoms in artificially infected koi were similar to those observed in koi naturally infected. Physiological and biochemical characteristics of the isolated HL2 strain were detected by Gram-stain and 16S rRNA gene analysis. Bacterial strain HL2 is gram-negative rod shape and motile and the length of the 16S rRNA gene sequence was 1461bp (Genbank login number: KF413420). Phylogenetic tree analysis shows that, based on the 16S rRNA sequence, HL2 clusters with and is a strain of Pseudomonas aeruginosa. Drug susceptibility of strain HL2 was tested by the disc agar diffusion (K-B) method using 55 test antibiotics: 35 antibiotics, including Ceftriaxone, Kanamycin and Tetracycline showed obvious inhibition; 5 antibiotics, including Gentamicin, Doxycycline and Cefradine showed moderate inhibition;15 antibiotics including Penicillin G, Ampicillin and Cefazolin showed no inhibition. Our study provides information for preventing and treating strain HL2 infection in koi and basic data for further research on Pseudomonas aeruginosa.