1株林麝源绿脓杆菌的分离鉴定及全基因组序列分析

doi:10.11843/j.issn.0366-6964.2021.09.020

摘要/Abstract

摘要： 旨在确定甘肃省天水市某林麝养殖场致死林麝的病原菌，并开展其致病性和耐药性研究及全基因组序列分析。采集病死林麝肺，通过细菌的分离纯化、生化鉴定和16S rRNA基因序列分析，对分离菌进行鉴定；随后对其致病性和药物敏感性进行分析，并在分离菌全基因组测序的基础上，对分离菌全基因组序列进行组装和注释，对毒力基因toxA和exoT进行遗传进化分析。结果表明，从病死林麝肺中分离到一株绿脓杆菌，命名为TS2019。致病性试验测得分离菌对小鼠的LD₅₀为2.82×10⁷CFU·mL^-1；药敏试验结果表明，TS2019具有多重耐药性，但对环丙沙星、洛美沙星等药物敏感。基因组测序表明，TS2019基因组全长为6 308 327 bp，编码5 929个基因，其中有1 035个编码产物参与新陈代谢途径；全基因组中有毒力因子编码基因875个，产物有黏附蛋白、调控因子、毒性蛋白等；有四环素类、氨基糖苷类等抗生素耐药相关基因5 288个。遗传进化分析表明，TS2019毒力基因toxA、exoT与GenBank中绿脓杆菌众多菌株相应基因序列相似性均高于99%，其中eoxT基因与中国杭州人源分离株P33的遗传关系最近，但处于独立分支。本研究从病死林麝肺分离鉴定到一株绿脓杆菌，并证实该菌有较强致病性和多重耐药性，其毒力基因toxA和exoT与GenBank中绿脓杆菌相应基因序列具有高度相似性。研究结果为林麝绿脓杆菌感染相关疾病的防治提供了理论支持，也为绿脓杆菌致病机制和耐药机制的深入研究奠定了基础。

关键词: 林麝, 绿脓杆菌, 分离鉴定, 致病性分析, 全基因组序列分析

Abstract: The purpose of this study was to identify the pathogenic bacteria that killed the forest musk deer in a forest musk deer farm in Tianshui, Gansu Province, and to analyze its pathogenicity, drug resistance and the whole genome sequence. The bacterial strains were isolated and purified from the lungs of dead forest musk deer. Then the isolate was identified through biochemical test and 16S rRNA gene sequence analysis. Subsequently, the pathogenicity and drug sensitivity of the isolate were analyzed. And based on the whole genome sequence, the whole genome sequence of the isolate was assembled and annotated. In addition, the phylogenetic analysis of the virulence genes toxA and exoT was completed. The results showed that a strain of Pseudomonas aeruginosa was isolated from the lung of the dead forest musk deer and named TS2019. The LD₅₀ of TS2019 in mice was 2.82×10⁷ CFU·mL^-1. The results of drug sensitivity test demonstrated that TS2019 had multiple drug resistance, but it was sensitive to ciprofloxacin, lomefloxacin, etc. The results of genome sequencing showed that the genome size of TS2019 was 6 308 327 bp, including 5 929 genes, of which 1 035 genes were involved in metabolic pathways. There were 875 genes encoding virulence factors which included adhesion proteins, regulatory factors, toxic proteins, etc. There are 5 288 genes related to antibiotic resistance such as tetracycline and aminoglycoside in the genome. The results of the phylogenetic analysis showed that virulent genes toxA and exoT of TS2019 shared over 99% homology with corresponding genes of many P. aeruginosa strains in GenBank. And the phylogenetic analysis of the eoxT gene showed the genetic relationship between TS2019 and P33 strain isolated from human in Hangzhou, was the closest, but it was an independent branch. In this study, a strain of P. aeruginosa was isolated and identified from the lungs of the dead forest musk deer, and the strong pathogenicity and multi-drug resistance of the isolate had been confirmed. The virulence genes toxA and exoT had high homology with the corresponding gene sequences of P. aeruginosa in GenBank. The results provided the theoretical support for the prevention and treatment of P. aeruginosa infection-related diseases of forest musk deer, and also laid a foundation for further study on the pathogenic mechanism and drug resistance mechanism of P. aeruginosa.

Key words: forest musk deer, Pseudomonas aeruginosa, isolation and identification, pathogenic analysis, genome sequence analysis

中图分类号:

S852.61

贺健, 邢小勇, 武小椿, 温峰琴, 张阳阳, 张生英, 刘佳, 包世俊. 1株林麝源绿脓杆菌的分离鉴定及全基因组序列分析[J]. 畜牧兽医学报, 2021, 52(9): 2569-2578.

HE Jian, XING Xiaoyong, WU Xiaochun, WEN Fengqin, ZHANG Yangyang, ZHANG Shengying, LIU Jia, BAO Shijun. Isolation and Identification of a Strain of Pseudomonas aeruginosa from Forest Musk and Analysis of the Whole Genome Sequence[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(9): 2569-2578.

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