鸽源性大肠杆菌的分离鉴定及茶多酚对其体外抑菌效果的研究

doi:10.16431/j.cnki.1671-7236.2022.10.038

摘要/Abstract

摘要： 【目的】确定广州市某规模化鸽场腹泻病例的病原菌及探讨茶多酚对该病原菌的体外抑菌效果。【方法】采集广州市某规模化鸽场腹泻病鸽的粪便,通过分离纯化、染色镜检、16S rRNA基因测序和构建系统进化树等方法对病原菌进行鉴定;采用药敏纸片法分析病原菌的耐药性;采用PCR技术检测病原菌携带的毒力基因;同时通过体外抑菌试验测定茶多酚对该病原菌的最小抑菌浓度(MIC)和最小杀菌浓度(MBC)以及不同浓度(0(空白对照)、125、250、500和1 000 μg/mL)茶多酚对该病原菌的生长、运动和毒力基因表达的影响,综合评定茶多酚对该病原菌的体外抑菌效果。【结果】从病料中分离出1株致病菌株,其生物学特性与大肠杆菌基本一致,命名为GG20210604;经16S rRNA基因测序和系统进化树进一步证实,该菌株为大肠杆菌;经检测,GG20210604对环丙沙星和氨苄西林耐药,并携带4种毒力基因:fliC、fimH、iroN和hlyF;体外抑菌试验结果显示,茶多酚对GG20210604的MIC和MBC均为4 000 μg/mL;与空白对照组相比,当茶多酚浓度为250、500和1 000 μg/mL时,GG20210604的生长和运动均被显著抑制(P＜0.05);250和500 μg/mL茶多酚均显著降低GG20210604的fliC、iroN和hlyF基因的表达(P＜0.05),250 mg/mL茶多酚显著降低fimH基因的表达(P＜0.05)。【结论】本研究分离得到1株鸽源性大肠杆菌,该菌株对环丙沙星和氨苄西林耐药并携带fliC、fimH、iroN和hlyF 4种毒力基因。茶多酚对该菌株具有良好的抑菌效果,可以显著抑制其生长、运动和毒力基因的表达。本研究为预防和缓解鸽因大肠杆菌引起的腹泻提供新的思路,并且为茶多酚作为植物性抑菌剂在养鸽生产中的应用提供理论依据。

关键词: 鸽; 大肠杆菌; 分离鉴定; 茶多酚; 抑菌效果

Abstract: 【Objective】 The purpose of this study was to identify the pathogen of diarrhea in a large-scale pigeon farm in Guangzhou and to explore the in vitro bacteriostatic effect of tea polyphenols on the pathogens.【Method】 The feces of sick pigeons in a large-scale pigeon farm in Guangzhou were collected,and the pathogen was identified by isolation and purification,staining and microscopic examination,16S rRNA gene sequencing and phylogenetic tree construction.The drug sensitivity of pathogen was detected by drug sensitivity test,and the virulence genes carried by pathogen were detected by PCR technology.At the same time,the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration(MBC) of tea polyphenols against pathogen and the effects of different concentrations (0 (the control group),125,500,and 1 000 μg/mL) of tea polyphenols on the growth,movement and virulence gene expression of the pathogen were determined by in vitro bacteriostatic test,so as to comprehensively evaluate the in vitro bacteriostatic effect of tea polyphenols on the pathogen.【Result】 From the pathologic materials,a pathogenic strain was isolated and named GG20210604,which biological characteristics were basically consistent with the Escherichia coli.16S rRNA gene sequencing and phylogenetic tree results further confirmed that the strain was Escherichia coli.After detection,GG20210604 was resistant to ciprofloxacin and ampicillin,while carrying a total of four virulence genes,including fliC,fimH,iroN and hlyF.The results of in vitro bacteriostatic test showed,the MIC and MBC values of tea polyphenols against GG20210604 were both 4 000 μg/mL.Compared with the control group,the growth and movement of GG20210604 were significantly inhibited in 250,500 and 1 000 μg/mL of tea polyphenols (P＜0.05);the GG20210604 genes expression of fliC,iroN and hlyF were significantly decreased in 250 and 500 μg/mL of tea polyphenols (P＜0.05),and the genes expression of fimH was also significantly decreased in 250 μg/mL of tea polyphenols (P＜0.05).【Conclusion】 A strain of Escherichia coli was isolated from pigeon.The strain was resistant to ciprofloxacin and ampicillin,while carrying a total of four virulence genes,including fliC,fimH,iroN and hlyF.Tea polyphenols had positive bacteriostatic effect on this strain,which could significantly inhibit its growth,movement and virulence gene expression.This study provided a new idea for preventing and alleviating pigeon diarrhea caused by Escherichia coli,and provided a theoretical basis for the application of tea polyphenols as a plant bacteriostatic agent in pigeon production.

Key words: pigeon; Escherichia coli; isolation and identification; tea polyphenols; bacteriostatic effect

中图分类号:

S858.39

钟雅静, 朱少平, 陈艾玲, 马婷婷, 邓珊, 黄燕华, 付晶. 鸽源性大肠杆菌的分离鉴定及茶多酚对其体外抑菌效果的研究[J]. 中国畜牧兽医, 2022, 49(10): 4052-4062.

ZHONG Yajing, ZHU Shaoping, CHEN Ailing, MA Tingting, DENG Shan, HUANG Yanhua, FU Jing. Isolation and Identification of Escherichia coli from Pigeon and Study on the Bacteriostatic Effect in vitro of Tea Polyphenols[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(10): 4052-4062.

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