京津冀部分地区致犊牛腹泻大肠杆菌毒力基因、耐药基因检测及药物敏感性分析

doi:10.16431/j.cnki.1671-7236.2022.12.030

摘要/Abstract

摘要： 【目的】探明京津冀地区犊牛腹泻大肠杆菌毒力基因与耐药基因流行情况，筛选敏感药物。【方法】于2020年12月至2021年7月从京津冀地区部分牛场采集146份犊牛腹泻样本，通过细菌分离纯化、革兰氏染色镜检及16S rRNA测序进行大肠杆菌分离鉴定；采用PCR方法对分离菌进行毒力基因（F17、K99、F41、STa、stx1、irp2和fyuA基因）和耐药基因（aac（6'）-Ⅰb、bla_CTX-M、bla_TEM、OqxB、tetA和sul1基因）检测；采用K-B纸片法进行药物敏感性试验。【结果】分离菌在鉴别培养基上的生长形态及革兰氏染色镜检结果均符合大肠杆菌生理生化特性，分离菌16S rRNA测序结果呈单一峰值，对拼接序列在NCBI中进行BLAST比对后发现，与大肠杆菌相似性均>96%，确定分离菌为大肠杆菌。试验共分离鉴定大肠杆菌142株，其中有88株携带毒力基因，占61.97%（88/142），毒力基因F17、K99、F41、STa、stx1、irp2、fyuA阳性率分别为24.65%、0.70%、0、2.11%、1.41%、45.07%和21.83%，其中F17、irp2、fyuA为优势毒力因子，同时携带多重毒力因子的大肠杆菌检出率较低。aac（6'）-Ⅰb、bla_CTX-M、bla_TEM、OqxB、tetA和sul1 6种耐药基因皆被检出，bla_TEM基因检出率最高，为45.77%，aac（6'）-Ⅰb和OqxB基因检出率最低，均为9.15%，分离菌株主要携带1~3种耐药基因。药物敏感性试验结果显示，142株分离菌对诺氟沙星敏感率最高，其次为环丙沙星，对青霉素敏感率为0，耐药现象严重，耐2种以上抗菌药物的菌株达86.62%。【结论】京津冀地区犊牛腹泻大肠杆菌毒力基因与耐药基因流行广泛，耐药普遍，多重耐药现象严重。本研究可为京津冀地区犊牛腹泻的防治提供理论依据。

关键词: 犊牛腹泻; 大肠杆菌; 毒力基因; 耐药基因; 耐药表型

Abstract: 【Objective】 The purpose of this study was to explore the prevalence of virulence genes and drug resistance genes of Escherichia coli (E.coli) isolated from diarrheic calves in Beijing-Tianjin-Hebei regions, providing basis for the screening of sensitive drugs.【Method】 In this study, 146 fecal samples of diarrheic calves were collected from different cattle farms in Beijing-Tianjin-Hebei regions from December 2020 to July 2021. E.coli was isolated, purified and identified by Gram staining microscopy and 16S rRNA sequencing.The virulence genes (F17, K99, F41, STa, stx1, irp2 and fyuA genes) and drug resistance genes (aac(6')-Ⅰb, bla_CTX-M, bla_TEM, OqxB, tetA and sul1 genes) were identified by PCR.The drug sensitivity test was performed using K-B paper method.【Result】 The growth morphology of the isolates on the medium and the gram staining results was consistent with the physiological and biochemical characteristics of E.coli.The 16S rRNA sequencing results of the isolates showed a single peak, and more than 96% of the sequences were identical to E.coli genome using NCBI BLAST, indicating the clinical isolates were E.coli.A total of 142 strains of E.coli were isolated, of which 88 strains carried virulence genes, accounting for 61.97% (88/142).The positive rates of virulence genes F17, K99, F41, STa, stx1, irp2 and fyuA were 24.65%, 0.70%, 0, 2.11%, 1.41%, 45.07% and 21.83%, respectively.Therefore, F17, irp2 and fyuA were the dominant virulence factors, while E.coli carrying multiple virulence factors was less frequently detected.Six resistance genes aac(6')-Ⅰb, bla_CTX-M, bla_TEM, OqxB, tetA and sul1 were detected.bla_TEM gene had the highest detection rate of 45.77%.In addition, aac(6')-Ⅰb and OqxB genes had the lowest detection rate of 9.15%, and the isolated strains mainly carried 1 to 3 resistance genes.The results of drug susceptibility test on 142 isolates showed the highest susceptibility rate of norfloxacin, followed by ciprofloxacin.The susceptibility rate to penicillin was 0, and the drug resistance phenomenon was severe, with 86.62% of isolates resistant to more than 2 antimicrobial agents.【Conclusion】 The virulence genes and drug resistance genes of E.coli isolated from diarrheic calves in Beijing-Tianjin-Hebei regions were widespread distributed and highly drug resistance.The phenomenon of multiple drug resistance was severe.This study provided a theoretical basis for the prevention and treatment of E.coli induced calf diarrhea in Beijing-Tianjin-Hebei regions.

Key words: calf diarrhea; Escherichia coli; virulence genes; drug resistance genes; drug resistance phenotype

中图分类号:

S852.61^＋2

贾垌, 张迪, 张兆天, 陈攀亮, 张杰, 李莹慧, 朱悦, 秦建华, 李妍. 京津冀部分地区致犊牛腹泻大肠杆菌毒力基因、耐药基因检测及药物敏感性分析[J]. 中国畜牧兽医, 2022, 49(12): 4820-4831.

JIA Tong, ZHANG Di, ZHANG Zhaotian, CHEN Panliang, ZHANG Jie, LI Yinghui, ZHU Yue, QIN Jianhua, LI Yan. Detection of Virulence and Resistance Genes and Drug Sensitivity Analysis of Escherichia coli Derived from Diarrheic Calves in Beijing-Tianjin-Hebei Partial Regions[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(12): 4820-4831.

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