树鼩源大肠杆菌分离鉴定及耶尔森强毒力岛相关基因的检测及序列分析

doi:10.16431/j.cnki.1671-7236.2022.11.038

摘要/Abstract

摘要： 【目的】研究树鼩源大肠杆菌耶尔森强毒力岛（HPI）相关基因的携带情况及其耐药性，为树鼩的饲养管理及大肠杆菌病的防治提供一定思路。【方法】无菌采集树鼩肛拭子样品129份，采用麦康凯培养基、LB琼脂培养基、革兰氏染色和生化试验进行细菌分离鉴定，用PCR法对分离菌株进行HPI相关基因检测，经PCR鉴定后筛选HPI阳性菌株并对该菌株的主要结构基因irp2和fyuA进行相似性分析和系统发育树构建，采用纸片扩散法对分离菌株进行药敏试验。【结果】 129份树鼩肛拭子样品共分离得到123株大肠杆菌，分离率为95.35%（123/129）；革兰氏染色结果显示，分离菌株镜检为红色粗短杆菌；生化鉴定结果显示，分离菌株对乳糖、葡萄糖、麦芽糖、蛋白胨和甘露醇生化反应呈阳性，硫化氢和尿素酶生化反应呈阴性，均符合大肠杆菌特性；PCR产物电泳结果显示，HPI相关基因检出率为88.62%（109/123），irp2基因携带率为34.15%（42/123），fyuA基因携带率为47.97%（59/123）；主要结构基因序列分析结果显示，irp2和fyuA基因与GenBank中公开发表的irp2和fyuA基因序列相似性分别达到98.6%和98.9%以上；系统发育树结果显示，分离菌株与耶尔森菌属亲缘关系较近；耐药性分析结果显示，分离菌株对阿米卡星和氟苯尼考敏感，对阿莫西林和苯唑西林耐药，耐药率分别为87.80%和81.30%。【结论】 HPI相关基因在树鼩大肠杆菌中广泛分布，进一步证实HPI可发生水平转移，且主要结构基因irp2和fyuA的遗传具有较高保守性。

关键词: 树鼩; 大肠杆菌; 耶尔森强毒力岛

Abstract: 【Objective】 The purpose of this experiment was to study the carriage and drug resistance of Escherichia coli (E.coli) high pathogenicity island (HPI) related genes in artificially bred Tupaia belangeri and to provide some ideas for the breeding and management of Tupaia belangeri and the prevention and treatment of colibacillosis.【Method】 129 samples of Tupaia belangeri anal wipe were collected aseptically.The bacteria were isolated and identified by McConkey medium, LB agar medium, Gram staining and biochemical test.HPI-related genes were detected by PCR.HPI-positive strains were screened after PCR identification.The similarity analysis and phylogenetic tree construction of the main structural genes irp2 and fyuA of the isolates were performed.Disk diffusion method was used for drug sensitivity test of the isolates.【Result】 A total of 123 strains of E.coli were isolated from 129 Tupaia belangeri anal wipe samples, and 95.35% (123/129) were isolated.The results of Gram staining showed that the isolates were microscopically examined as stubby rubrum.The biochemical identification results showed that the isolates were positive for lactose, glucose, maltose, peptone, and mannitol biochemical reactions, and negative for hydrogen sulfide and urease biochemical reactions, all of which conformed to the characteristics of E.coli.The electrophoresis results of PCR products showed that the detection rate of HPI-related genes was 88.62% (109/123), the carrying rates of irp2 and fyuA genes were 34.15% (42/123) and 47.97% (59/123), respectively.The results of sequence analysis of the main structural genes showed that the irp2 and fyuA genes were more than 98.6% and 98.9% similar to the sequences published irp2 and fyuA gene nucleotide sequences in GenBank, respectively, and the phylogenetic tree results showed that the isolates were closely related to Yersinia spp.The results of drug resistance analysis showed that the isolated strains were sensitive to amikacin and florfenicol, and resistant to amoxicillin and oxacillin, the drug resistance rates were 87.80% and 81.30% respectively.【Conclusion】 HPI related genes were widely distributed in E.coli of Tupaia belangeri, which further confirmed that HPI could be horizontally transferred, and the inheritance of the main structural genes irp2 and fyuA were highly conserved.

Key words: Tupaia belangeri; Escherichia coli; high pathogenicity island

中图分类号:

S852.61^＋2

陈柄汛, 韩冬梅, 赵维薇, 张博, 邓静, 万全, 王喜, 肖鹏, 吕龙宝, 高洪. 树鼩源大肠杆菌分离鉴定及耶尔森强毒力岛相关基因的检测及序列分析[J]. 中国畜牧兽医, 2022, 49(11): 4466-4475.

CHEN Bingxun, HAN Dongmei, ZHAO Weiwei, ZHANG Bo, DENG Jing, WAN Quan, WANG Xi, XIAO Peng, LYU Longbao, GAO Hong. Isolation and Identification of Escherichia coli from Tupaia belangeri and Detection and Sequence Analysis of Genes Related to High Pathogenicity Island[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(11): 4466-4475.

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