犊牛源肠外致病性大肠杆菌分离鉴定、致病性及耐药性分析

doi:10.16431/j.cnki.1671-7236.2023.12.024

摘要/Abstract

摘要： 【目的】确定新疆喀什地区某规模化牛场致犊牛死亡的细菌性病原体及其生物学特性,为该地区规模化牛场细菌病的防治和合理用药提供参考。【方法】对死亡犊牛进行病理解剖及病原分离,挑选肝脏(KS-1)与肛拭子(KS-2)来源的单菌落纯化培养,通过形态观察、生化试验、16S rRNA基因扩增及系统进化分群鉴定分离株,并通过毒力基因检测、致病性试验及药敏试验对分离株的致病性和耐药性进行研究。【结果】死亡犊牛肺脏、肝脏、心血、关节液和肠系膜淋巴结以及健康犊牛肛拭子样品接种后菌落生长形态一致,结合选择性培养基上菌落生长形态、革兰染色镜检形态特点、生化特性及大肠杆菌16S rRNA的验证,鉴定分离菌株为大肠杆菌。系统进化分群试验中,KS-1为A群,KS-2为B1群。毒力基因检测结果显示,KS-1携带crl、papC、hlyE、eaeA、ompA、ompC、iucD和iutA共8种毒力相关基因,KS-2携带crl、hlyE和ompF共3种毒力相关基因。致病性试验结果显示,腹腔注射1×10⁸ CFU分离株感染小鼠,KS-1组小鼠在16 h内全部死亡,KS-2组小鼠无死亡现象出现。药敏试验结果显示,KS-1对哌拉西林/他唑巴坦、头孢唑林、头孢呋辛等17种药物敏感,对氨苄青霉素/舒巴坦和哌拉西林中度敏感,对氨苄西林和复方新诺明耐药;KS-2对哌拉西林/他唑巴坦、头孢替坦、亚胺培南、美罗培南、阿米卡星和呋喃妥英敏感,对妥布霉素中度敏感,对其余14种药物耐药。【结论】本研究分离出2株犊牛源大肠杆菌,其中KS-1毒力较强,且携带丰富的毒力基因,对大部分常用抗生素敏感,对氨苄西林和复方新诺明具有耐药性;KS-2携带毒力基因较少,呈现多重耐药。

关键词: 犊牛; 肠外致病性大肠杆菌; 分离鉴定; 毒力基因; 耐药性

Abstract: 【Objective】 The purpose of this study was to identify the bacterial pathogens and its biological characteristics causing the death of calves in a large-scale cattle farm in Kashi,Xinjiang,so as to provide reference for the treatment of bacterial-associated diseases and rational drug for the area.【Method】 The pathological dissection and pathogen isolation on dead calves were conducted,single colony from liver (KS-1) and anal swab (KS-2) were selected for purification and culture.The isolates were identified by morphological observation,biochemical test,16S rRNA gene amplification together with phylogenetic group.The pathogenicity and drug resistance of the isolates were evaluated by virulence gene detection,pathogenicity test and drug sensitivity test.【Result】 After inoculation,the lung,liver,heart blood,joint fluid and mesenteric lymph nodes of dead calves showed a consistent colony growth pattern,as did anal swab samples from healthy calves.The isolated strains were identified as Escherichia coli(E.coli) by the growth morphology of the colony on selective medium,the morphological features of Gram staining microscopy,the biochemical properties and the verification of E.coli 16S rRNA.Phylogenetic analysis showed that KS-1 belonged to group A and KS-2 to group B1.KS-1 contained 8 virulence-related genes (crl,papC,hlyE,eaeA,ompA,ompC,iucD and iutA),and KS-2 contained 3 virulence-related genes (crl,hlyE and ompF).The pathogenicity test results showed that following intraperitoneal injection of 1×10⁸ CFU of the isolate,all mice in KS-1 group died within 16 h,however no mice in KS-2 group died.The results of drug sensitivity tests showed that KS-1 was sensitive to 17 drugs,including piperacillin/tazobactam,cefazolin,cefuroxime,cefuroxime,etc.,it was moderately sensitive to ampicillin/sulbactam and piperacillin,and resistant to ampicillin and cotrimoxazole.KS-2 was sensitive to piperacillin/tazobactam,cefotetan,imipenem,meropenem,amikacin and nitrofurantoin,moderately sensitive to tobramycin,and resistant to the other 14 drugs.【Conclusion】 In this study,two calf-derived E.coli strains were isolated,of which KS-1 was more virulent and carried abundant virulence genes,it was sensitive to most commonly used antibiotics,and was resistant to ampicillin and sulfamethoxazole,while KS-2 carried few virulence genes and showed multi-drug resistance.

Key words: calves; extraintestinal pathogenic Escherichia coli; isolation and identification; virulence genes; drug resistance

中图分类号:

S852.61

史唯地, 康立超, 刘彩霞, 寇丽君, 吕双飞, 任慧杰, 钱瑞宣, 马勋, 王静, 蒋建军, 高盛杰. 犊牛源肠外致病性大肠杆菌分离鉴定、致病性及耐药性分析[J]. 中国畜牧兽医, 2023, 50(12): 5022-5031.

SHI Weidi, KANG Lichao, LIU Caixia, KOU Lijun, LYU Shuangfei, REN Huijie, QIAN Ruixuan, MA Xun, WANG Jing, JIANG Jianjun, GAO Shengjie. Isolation,Identification,Pathogenicity and Drug Resistance Analysis of Extraintestinal Pathogenic Escherichia coli from Calves[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(12): 5022-5031.

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