大口黑鲈源豚鼠气单胞菌的分离鉴定及全基因组分析

doi:10.16431/j.cnki.1671-7236.2024.04.038

摘要/Abstract

摘要： 【目的】探究患病大口黑鲈的致病菌及其主要生物学特性,为临床疾病防控提供技术支撑。【方法】首先对发病大口黑鲈进行病理组织学观察,进而利用平板划线法分离细菌,通过革兰染色、生化试验和16S rDNA序列测定对分离菌进行鉴定,通过致病性试验观察分离菌对小鼠主要脏器造成的病理损伤;而后分别利用PCR方法和Kirby-Bauer (K-B)纸片法对分离菌的常见毒力基因和耐药特征进行检测分析;最后利用二代测序技术绘制其全基因组草图,并通过生物信息学方法对其分子特征进行解析。【结果】自然感染大口黑鲈肝细胞肿大,空泡变性,肠黏膜下层出血,肠壁坏死;于患病黑鲈体内分离到1株优势菌,在血平板上呈乳白色,半透明,直径约1 mm的小菌落,呈β溶血,革兰染色为阴性短杆菌,两端钝圆,经生化试验和16S rDNA测序证实分离菌为豚鼠气单胞菌(Aeromonas caviae),将其命名为SCMS。试验小鼠接种后24 h内死亡率为100%,病理特征与自然感染大口黑鲈相似;SCMS株中共检测到9种毒力基因,分别为Aha、Ahp、Aer、Alt、gcaT、Act、Exu、Ela和eprCAI;药敏试验结果显示,分离菌对大多数四环素类、大环内酯类药物表现耐药或中度敏感;全基因组测序显示,分离菌全长4 329 602 bp,GC含量为61.5%,基因组信息在GenBank中的登录号为JARFUQ000000000,相似性分析显示分离菌与人源、食品源豚鼠气单胞菌具有高度相似性;病原毒力因子数据库(VFDB)比对显示,分离菌携带多种与黏附、侵入及分泌系统相关的毒力基因;耐药基因数据库(CGE)分析显示,菌株中携带7种耐药基因,部分基因与耐药表型存在不一致的现象。【结论】本试验通过全基因组测序技术解析了1株大口黑鲈源豚鼠气单胞菌的毒力基因和耐药基因,其结果可为大口黑鲈临床疾病防控提供技术支撑。

关键词: 大口黑鲈; 豚鼠气单胞菌; 全基因组测序; 毒力基因; 耐药基因

Abstract: 【Objective】 The purpose of the present study was to obtain the pathogenic bacteria and their main biological characteristics of diseased Micropterus salmoides,and provide technical support for clinical disease prevention and control.【Method】 Pathological changes of the naturally diseased Micropterus salmoides were observed firstly.After that,bacteria were isolated and identified through Gram staining,biochemical testing and 16S rDNA sequencing analysis.Pathogenicity of the isolate was performed on mice to explore the pathological lesions.Furthermore,PCR and Kirby-Bauer (K-B) disk methods were used to detect the common virulence genes and analyze the drug resistance characteristics of the isolated bacteria,respectively.The draft genome was obtained by second-generation sequencing technology,and its molecular characteristics were analyzed using bioinformatics methods.【Result】 Swollen hepatocytes,vacuolar degeneration and hemorrhagic necrotic lesions were observed in the naturally infected Micropterus salmoides.A dominant bacterial strain was isolated from the diseased Micropterus salmoides,which exhibited milky white,semitransparent,and with a diameter of approximately 1 mm colony and β hemolysis on the blood plate.Gram staining showed negative short bacilli,with blunt and round ends.Through biochemical tests and 16S rDNA sequencing,it was confirmed that the isolated strain was Aeromonas caviae,which was named SCMS.The mortality rate of experimental mice was 100% within 24 h after inoculation with SCMS.And the pathological characteristics were similar to those of naturally infected Micropterus salmoides.Nine virulence genes were detected in the SCMS strain,including Aha, Ahp, Aer, Alt, gcaT, Act, Exu, Ela and eprCAI genes.Drug sensitivity test results showed that the isolated bacteria showed resistance or moderate sensitivity to most tetracycline and macrolide drugs.The whole genome sequencing showed that the isolated strain had a total length of 4 329 602 bp and a GC content of 61.5%.The genome information was registered as JARFUQ000000000 in GenBank.Similarity analysis showed that the isolated strain had high similarity with human and food sources of Aeromonas caviae.The comparison of Virulence Factor Database (VFDB) showed that the isolate strain carried multiple virulence genes related to adhesion,invasion,and secretion systems etc.Also,7 drug resistance genes were harbored in the isolated strain when annoted by the drug resistance gene database(CGE).Inconsistency phenomenon was found between partial genes and its drug resistance phenotypes.【Conclusion】 In this experiment,virulence and drug resistance genes of an Aeromonas caviae strain isolated from diseased Micropterus salmoides were analyzed through whole genome sequencing technology,and all these findings could provide technical support for clinical disease prevention and control of Micropterus salmoides.

Key words: Micropterus salmoides; Aeromonas caviae; whole genome sequencing; virulence gene; drug resistance gene

中图分类号:

S852.61

王迎平, 赵静贤, 尚佳富, 郝成森, 左妤祺, 朱桓奕, 冯旭东, 徐婷婷, 倪兴维, 刘霞, 杨晓伟, 曹蓝云, 张立武, 谭昌藩, 曹礼静, 赵光伟. 大口黑鲈源豚鼠气单胞菌的分离鉴定及全基因组分析[J]. 中国畜牧兽医, 2024, 51(4): 1706-1716.

WANG Yingping, ZHAO Jingxian, SHANG Jiafu, HAO Chengsen, ZUO Yuqi, ZHU Huanyi, FENG Xudong, XU Tingting, NI Xingwei, LIU Xia, YANG Xiaowei, CAO Lanyun, ZHANG Liwu, TAN Changfan, CAO Lijing, ZHAO Guangwei. Isolation,Identification and Whole Genome Analysis of Aeromonas caviae from Micropterus salmoides[J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(4): 1706-1716.

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