海南省奶源和养殖环境主要肠杆菌科细菌的分布及基因分型

doi:10.11843/j.issn.0366-6964.2023.12.028

畜牧兽医学报 ›› 2023, Vol. 54 ›› Issue (12): 5184-5197.doi: 10.11843/j.issn.0366-6964.2023.12.028

海南省奶源和养殖环境主要肠杆菌科细菌的分布及基因分型

郝若晨^1,2, 唐敏嘉², 刘光亮^1,3, 张艳¹, Muhammad Shoaib², 尚若锋², 曹宗喜^1*, 蒲万霞^2*

1. 海南省农业科学院畜牧兽医研究所海南省热带动物繁育与疫病研究重点实验室, 海口 571100;
2. 中国农业科学院兰州畜牧与兽药研究所/农业农村部兽用药物创制重点实验室/甘肃省新兽药工程重点实验室, 兰州 730050;
3. 中国农业科学院兰州兽医研究所, 兰州 730046

收稿日期:2023-02-23 出版日期:2023-12-23 发布日期:2023-12-26
通讯作者: 曹宗喜,主要从事家畜传染病学研究,E-mail:caozongxi@163.com;蒲万霞,主要从事细菌耐药性研究,E-mail:puwanxia@caas.cn.
作者简介:郝若晨(1998-),女,河南省睢县人,硕士,主要从事病原微生物研究,E-mail:hrc_zh@163.com
基金资助:
海南省热带动物繁育与疫病研究重点实验室开放课题计划任务书(HKL20200302);海南省重点研发项目(ZDYF2023XDNY038)

Distribution and Genotyping of Major Enterobacteriaceae Bacteria in Milk Sources and Dairy Farm Environment of Hainan Province

HAO Ruochen^1,2, TANG Minjia², LIU Guangliang^1,3, ZHANG Yan¹, MUHAMMAD Shoaib², SHANG Ruofeng², CAO Zongxi^1*, PU Wanxia^2*

1. Hainan Provincial Key Laboratory of Tropical Animal Breeding and Disease Research, Institute of Animal Science and Veterinary Medicine, Hainan Academy of Agricultural Sciences, Haikou 571100, China;
2. Gansu Province Key Laboratory of New Animal Drug Project, Ministry of Agriculture and Rural Affairs Key Laboratory of Veterinary Drug Discovery/Lanzhou Institute of Husbandry and Pharmaceutical Sciences of CAAS, Lanzhou 730050, China;
3. Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China

Received:2023-02-23 Online:2023-12-23 Published:2023-12-26

摘要/Abstract

摘要： 本研究从2021年海南省两个奶牛场采集的52份样本中分离肠杆菌科细菌,并对分离菌株进行了毒力基因、质粒型和生物被膜形成能力检测及肠杆菌科细菌基因间重复序列分子分型(ERIC-PCR)研究,以期明确牛奶及奶牛场环境中肠杆菌科细菌分布流行状况及其生物学特性。首先,采用16S rDNA PCR和ERIC-PCR对分离的肠杆菌科细菌进行鉴定和去重,结果显示52份样本中分离到肠杆菌科细菌49株,其中大肠埃希菌(Escherichia coli)32株,肺炎克雷伯菌(Klebsiella pneumoniae)13株,阴沟肠杆菌(Enterobacter cloacae)3株,产气肠杆菌(Enterobacter aerogenes)1株;其次,采用PCR方法进行了分离菌株的毒力基因检测,结果显示,ompA的检出率最高;之后,采用CARATTOLI创建的基于PCR的质粒复制子分型,结果显示K质粒的检出率最高;采用刚果红琼脂法对分离菌株进行了生物被膜形成能力定性检测,发现49株肠杆菌科细菌中有37株指示有生物被膜形成能力;最后,通过ERIC-PCR分型进行了肠杆菌科细菌的亲缘关系和遗传多样性分析,发现49株肠杆菌科菌株可划分为17型(Ⅰ~ⅩⅦ),Ⅵ型为优势型(均为Escherichia coli),共20株。整体研究表明肠杆菌科细菌在奶源环境中广泛存在、生物特性多样,推测具有一定的致病力和耐药性,引起人类和动物疾病风险较高,提示养殖、运输中的卫生和规范操作至关重要。

关键词: 肠杆菌科细菌, 分子分型, 质粒型, 生物被膜

Abstract: In this study, major bacteria in Enterobacteriaceae were isolated from 52 samples collected from two dairy farms in Hainan Province in 2021. The virulence genes detection, plasmid type, biofilm-forming ability and enterobacterial repetitive intergenic consensus (ERIC) molecular typing of isolates were studied in order to clarify the distribution, prevalence and biological characteristics of the isolates in Enterobacteriaceae in milk and dairy farm environment. First, the isolates in Enterobacteriaceae were identified by 16S rDNA PCR. The results showed that 49 strains of Enterobacteriaceae were isolated from 52 samples, including 32 strains of Escherichia coli, 13 strains of Klebsiella pneumoniae, 3 strains of Enterobacter cloacae and 1 strain of Enterobacter aerogenes. The results of virulence gene testing showed that ompA had the highest detection rate. Plasid typing is performed by PCR-based replicon typing created by CARATTOLI. The results showed that the detection rate of K plasmid was the highest among 49 isolated bacteria. The biofilm-formating ability of isolates was qualitatively detected using the Congo Red Agar method. Thirty-seven isolates were positive. The genetic relationship and diversity of Enterobacteriaceae were determined by ERIC-PCR. The results showed that 49 strains of Enterobacteriaceae were divided into 17 types (I-XVI), and the dominant type was type VI (all Escherichia coli), with a total of 20 strains. The overall study showed that Enterobacteriaceae widely existed in the milk and environment, causing a high risk of human and animal diseases. It is suggested that hygiene and standardized operation in breeding and transportation are crucial.

Key words: Enterobacteriaceae, molecular typing, plasmid type, biofilm

中图分类号:

S852.6

郝若晨, 唐敏嘉, 刘光亮, 张艳, Muhammad Shoaib, 尚若锋, 曹宗喜, 蒲万霞. 海南省奶源和养殖环境主要肠杆菌科细菌的分布及基因分型[J]. 畜牧兽医学报, 2023, 54(12): 5184-5197.

HAO Ruochen, TANG Minjia, LIU Guangliang, ZHANG Yan, MUHAMMAD Shoaib, SHANG Ruofeng, CAO Zongxi, PU Wanxia. Distribution and Genotyping of Major Enterobacteriaceae Bacteria in Milk Sources and Dairy Farm Environment of Hainan Province[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(12): 5184-5197.

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海南省奶源和养殖环境主要肠杆菌科细菌的分布及基因分型

Distribution and Genotyping of Major Enterobacteriaceae Bacteria in Milk Sources and Dairy Farm Environment of Hainan Province

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