瑶山亚种树鼩源嗜水性气单胞菌的分离鉴定及生物学特性研究

doi:10.16431/j.cnki.1671-7236.2020.05.031

摘要/Abstract

摘要： 为确诊广西中医药大学瑶山亚种树鼩人工驯化养殖中心的瑶山亚种树鼩发病死亡原因，并调查分离菌株的致病性与耐药情况，试验通过平板划线分离获得病原菌，进一步对分离菌进行形态观察、培养特性、生化试验及16S rRNA序列分析，纯化获得一株致病性嗜水性气单胞菌；利用昆明小鼠的致病性试验测定了该分离菌株的半数致死量（LD₅₀），利用药敏纸片法分析该分离株对常用药物的敏感性，并应用PCR方法进行菌株耐药基因的检测。结果显示，从病死瑶山亚种树鼩分离到一株嗜水性气单胞菌，镜检显示为革兰氏阴性菌，形态呈短杆状。该菌16S rRNA序列与日本淡水湖源嗜水性气单胞菌SWCY-3.27株同源性高达100%，遗传进化关系最近。该分离菌对成年昆明小鼠的LD₅₀为1×10⁷ CFU，毒力高于模式菌株ATCC 7966，近似于中国流行株NJ-35和J-1等，致病性试验鉴定该菌为强毒力菌株。生化鉴定七叶苷、阿拉伯糖、氧化酶呈阳性，各种生化鉴定符合嗜水气单胞菌生化特性。药敏检测显示，该菌株对米诺环素、链霉素、妥布霉素等9种药物敏感，对四环素、红霉素、青霉素等10种药物耐药。以PCR扩增基因方法对分离菌进行6类12种耐药基因的检测，发现分离菌存在氨基糖苷类（Aph-（3）-lia）和β-内酰胺类（mecA）两类抗生素的耐药基因，与药敏试验结果基本吻合。本研究结果表明，引起此次树鼩死亡的病原菌为嗜水性气单胞菌，可用高敏药物进行针对性治疗，为防治嗜水性气单胞菌感染导致的树鼩疾病提供参考依据。

关键词: 树鼩; 嗜水性气单胞菌; 16S rRNA; 致病性; 耐药基因

Abstract: In order to confirm the cause of death of Tupaia belangeri yaoshanensis in the artificial domestication and breeding experiment center,the pathogenicity and drug resistance of the isolated strain were investigated.Pathogenic bacteria was isolated and obtained by plate drawing,and further morphological observation,culture characteristics analysis,biochemical test and 16S rRNA sequence analysis were carried out on the isolated bacteria.A pathogenic Aeromonas hydrophila was purified and obtained.The lethal dose 50%(LD₅₀) of the isolated strain was determined by pathogenicity test on Kunming mice.At the same time,the sensitivity of Aeromonas hydrophila isolates to common drugs was analyzed using the paper sensitive method,and the drug resistance genes of the isolates were detected by PCR.The results showed that a strain of Aeromonas hydrophila was isolated from the diseased tree shrews and the microscopic examination showed Gram-negative bacteria in a short rod shape.The 16S rRNA sequence of this strain showed that a strain of Aeromonas hydrophila was isolated from the diseased tree shrew,which was 100% homologous to the Aeromonas aeruginosa collected by GenBank SWCY-3.27 isolated from freshwater lake,and had the closest genetic evolution.The LD₅₀ of the isolates to adult Kunming mice was 1×10⁷ CFU,which was more virulent than the model strain ATCC 7966 and similar to the prevalent strains NJ-35 and J-1,etc.in China.The pathogenicity test identified the isolates as highly virulent.The biochemical identification of esculin,arabinose and oxidase was positive,and various biochemical identifications were consistent with the biochemical characteristics of Aeromonas hydrophila.The drug sensitivity test showed that the strain had high sensitivity to 9 drugs such as minocycline,streptomycin and tobramycin,completely resistant to 10 drugs such as tetracycline,erythromycin and penicillin.PCR amplification was used to detect 12 drug resistance genes of 6 major categories in the isolated bacteria,and it was found that there were two antibiotic resistance genes of aminoglycoside (Aph-(3)-lia) and β-lactams (mecA) in the isolated bacteria,which were basically consistent with the drug sensitivity test results.This study indicated that the pathogen causing the death of tree shrew was Aeromonas hydrophila,which could be treated with high-sensitivity drugs,and provided reference for the prevention and treatment of tree shrew diseases caused by Aeromonas hydrophila infection.

Key words: Tupaia belangeri; Aeromonas hydrophila; 16S rRNA; pathogenicity; drug resistance gene

中图分类号:

S852.61⁺7

唐海波, 运晨霞, 梁亮, 袁小芳, 黄正团, 曹颖颖, 冷静. 瑶山亚种树鼩源嗜水性气单胞菌的分离鉴定及生物学特性研究[J]. 中国畜牧兽医, 2020, 47(5): 1560-1570.

TANG Haibo, YUN Chenxia, LIANG Liang, YUAN Xiaofang, HUANG Zhengtuan, CAO Yinyin, LENG Jing. Isolation and Identification and Biological Characterization of Aeromonas hydrophila from Tupaia belangeri yaoshanensis[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(5): 1560-1570.

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