中华草龟恶臭假单胞菌的分离鉴定及药物敏感性分析

doi:10.16431/j.cnki.1671-7236.2019.10.034

摘要/Abstract

摘要： 为探明海口某龟鳖养殖场送检的发病中华草龟的病原，本试验从患病中华草龟体内分离得到一株优势生长菌（命名为J1）。经菌落培养、革兰氏染色观察、生理生化鉴定及16S rRNA序列比对分析，并进行攻毒及耐药性试验验证其致病性及药物敏感性。结果显示，该分离菌株在普通营养琼脂培养基上形成圆形、光滑、湿润黏稠、灰黄色、不透明的菌落；革兰氏染色镜检结果显示，该分离菌为革兰氏阴性短杆状；生理生化试验结果显示，分离菌具有运动性，果糖、柠檬酸盐、硝酸盐、氧化酶、接触酶和精氨酸双水解酶呈阳性，而葡萄糖产气、葡萄糖产酸、鸟氨酸脱羧酶、乳糖、麦芽糖、V-P试验等呈阴性；16S rRNA基因序列分析显示，该分离菌株与GenBank中登录的假单胞菌同源性最高；系统进化树显示，该分离菌与恶臭假单胞菌聚为一簇；人工攻毒试验证实分离菌可使中华草龟发病并死亡，临床症状和病理变化与自然发病龟相似；耐药性分析发现，分离菌株对妥布霉素、头孢拉定、庆大霉素等药物敏感，对克林霉素、四环素、氨苄西林、头孢克洛等多种药物耐药。本试验结果可为龟鳖疾病的诊断、预防和治疗提供有力的技术支撑和实践指导。

关键词: 中华草龟; 恶臭假单胞菌; 分离鉴定; 16S rRNA; 致病性; 药敏特性

Abstract: In order to find out the pathogen of Chinemys reevesii which was sent for examination from a turtle farm in Haikou city,a dominant growth bacteria (named as J1) was isolated from diseased Chinemys reevesii.Colony culture,Gram staining observation,physiological and biochemical identification and 16S rRNA sequence alignment analysis were used to identify the isolotes,and the pathogenicity and drug sensitivity were verified by virus attack and drug resistance test.The results showed that the strain formed round,smooth,wet,sticky,grey-yellow and opaque colonies on the agar medium.The results of Gram staining microscopy showed that the isolate was short rod-shaped Gram-negative.Physiological and biochemical test results showed that the isolates were sporadic,fructose,citrate,nitrate,oxidase,contact enzyme and arginine hydrolase were positive,while glucose gas production,glucose acid production,ornithine decarboxylase,lactose,maltose,V-P test were negative.16S rRNA gene sequence analysis showed that the isolate had the highest homology with Pseudomonas spp.registered on GenBank.The phylogenetic tree showed that the isolate was clustered with Pseudomonas putida.Artificial challenge test confirmed that the isolated bacteria could cause the disease and death of Chinemys reevesii,and the clinical symptoms and pathological changes were similar to those of naturally occurring turtles.Drug resistance analysis showed that the isolates were sensitive to tobramycin,cefradine and gentamicin,and resistant to clindamycin,tetracycline,ampicillin and cefaclor.The results could provide strong technical support and practical guidance for the diagnosis,prevention and treatment of turtle diseases.

Key words: Chinemys reevesii; Pseudomonas putida; isolation and identification; 16S rRNA; pathogenicity; drug sensitivity

中图分类号:

S947

方振华, 代小梅, 丁利, 洪美玲. 中华草龟恶臭假单胞菌的分离鉴定及药物敏感性分析[J]. 中国畜牧兽医, 2019, 46(10): 3115-3123.

FANG Zhenhua, DAI Xiaomei, DING Li, HONG Meiling. Isolation,Identification and Drug Susceptibility Analysis of Pseudomonas putida from Chinemys reevesii[J]. China Animal Husbandry and Veterinary Medicine, 2019, 46(10): 3115-3123.

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