豪猪源肠侵袭型大肠杆菌的分离鉴定及致病性研究

Isolation, Identification and Pathogenicity of Enteroinvasive Escherichia coli from Porcupine

【目的】查明引起福建省龙岩市某豪猪养殖场致豪猪腹泻死亡的病原菌及其特征,为该病的科学防控及合理用药提供参考依据。【方法】分离腹泻死亡豪猪的病原,并结合形态特征、生理生化试验、16S rRNA基因扩增、种系发育分群鉴定分离菌株;通过毒力基因检测、动物致病性试验及药敏试验对分离菌株的致病性和耐药性进行研究。【结果】从发病死亡豪猪肝脏组织中分离到1株大肠杆菌,命名为Fj/Porcupine2018,该分离菌株与22株不同来源的参考菌株16S rRNA序列之间的相似度为97.4%~99.8%,其中与禽源分离株(登录号:MN022583)和人源分离株(登录号:MW881377)的序列相似度高达99.8%;种系发育分群证实该分离菌株属于B1群。毒力基因检测结果显示,该分离菌株除检出肠侵袭型大肠杆菌毒力基因EinV外,还检出papC、iroC、Afa、luxs、stx2f及ompA 6种毒力相关基因。动物致病性试验显示,该分离菌株对小鼠具有较强的致病性,小鼠在攻毒后31 h内全部死亡,且肝脏、脾脏、肺脏及肠道等器官组织均有明显的病理损伤;在对常见抗菌药物的药敏试验中,该分离菌株对大环内酯类、四环素类、喹诺酮类、磺胺类、头孢类、氨基糖苷类、青霉素类7类11种药物表现出不同程度的耐药,耐药率为25%~100%,仅对头孢曲松和头孢西叮2种药物表现敏感。【结论】本研究分离获得了1株腹泻死亡豪猪的病原菌大肠杆菌,通过一系列生物学特性研究证实该分离菌为1株具有较强致病力且呈现多重耐药性的B1群肠侵袭型大肠杆菌,为豪猪等野生动物大肠杆菌病的防控提供了重要的参考依据。

【Objective】 This study was aimed to identify the pathogenic bacteria and its characteristics that caused diarrhea and death of porcupine in a porcupine farm in Longyan city, Fujian province, and provide a reference for scientific prevention and control of the disease and rational drug use.【Method】 The pathogens of diarrhea-dead porcupines were isolated, and the isolates were identified by combining morphological characteristics, physiological and biochemical tests, 16 S rRNA gene amplification and phylogenetic grouping.The pathogenicity and drug resistance of the isolates were studied by virulence gene detection, animal pathogenicity test and drug sensitivity test.【Result】 A strain of Escherichia coli was isolated from liver of the morbid and dead porcupine, and named as Fj/Porcupine2018.The sequence homology between the isolate and 22 reference strains from different sources of 16 S rRNA was 97.4% to 99.8%.Among them, the sequence homology with the avian isolate(accession No.:MN022583) and the human isolate(accession No.:MW881377) were as high as 99.8%.The phylogenetic grouping confirmed that the isolate belonged to B1 group.The virulence gene test results showed that that in addition to the enteroinvasive Escherichia coli virulence gene EinV,6 virulence-related genes of papC,iroC,Afa,luxs,stx2f and ompA were also detected.The results of animal pathogenicity tests showed that the isolate had strong virulence to the challenged mice, and all the mice died within 31 h after the challenge, and obvious pathological damage was caused to liver, spleen, lung, intestine and other organs and tissues of the dead mice.In the drug susceptibility test, the isolate showed different degrees of resistance to 7 classes of 11 drugs, including macrolides, tetracyclines, quinolones, sulfonamides, cephalosporins, aminoglycosides and penicillins, the drug resistance rate was 25% to 100%,and it was only sensitive to ceftriaxone and ceftriaxone.【Conclusion】 In this study, a pathogenic Escherichia coli strain of diarrhea-dead porcupines was isolated and obtained.Through a series of biological characteristics studies, it was confirmed that the isolate was a group B1 enteroinvasive Escherichia coli with strong pathogenicity and multi-drug resistance, which provided a certain reference for the prevention and control of colibacillosis in wild animals such as porcupine.