猪源ETEC/STEC杂合大肠埃希菌菌株的分离、毒力基因鉴定及耐药性分析

doi:10.16178/j.issn.0528-9017.20230291

摘要/Abstract

摘要：

仔猪断奶后腹泻(PWD)通常是由产肠毒素大肠埃希菌(ETEC)感染导致的疾病,该菌通常携带LT、STa及STb等肠毒素中的一种或多种。猪水肿病又被称为胃肠道水肿病,是由产志贺氏毒素大肠埃希菌(STEC)感染引发的一种急性致死性传染病,具有较高的死亡率,该菌通常携带Stx1及Stx2等毒素。文章诊断了一例仔猪腹泻与水肿病并发引起高死亡率的病例,分离鉴定了一株同时携带产肠毒素大肠埃希菌(ETEC)及产志贺氏毒素大肠埃希菌(STEC)毒力基因的杂合菌株。经鉴定,杂合菌株携带黏附素F18基因、肠毒素基因(STa、STb、LT)以及水肿病致病毒素基因(Stx2e)。药敏试验结果表明,该大肠埃希菌分离株对头孢噻肟、阿米卡星等5种药物敏感,对壮观霉素、环丙沙星等2种药物中度敏感,对恩诺沙星、链霉素等14种药物耐药。该分离菌株为产肠毒素型与产志贺氏毒素杂合型大肠埃希菌(ETEC/ESTEC),对多种抗菌药具有较强的耐药性。

关键词: 仔猪断奶后腹泻, 猪水肿病, 分离鉴定, 毒力基因

Abstract:

Post weaning diarrhea (PWD) in piglets is usually caused by infection with enterotoxigenic Escherichia coli (ETEC), which typically carries one or more of the enterotoxins LT, STa, and STb. Pigt edema disease (ED), also known as bowel edema or gut edema, is an acute and lethal infectious disease caused by infection with Shiga toxin-producing Escherichia coli (STEC), with a high mortality rate. This bacterium usually carries toxins such as Stx1 and Stx2. In this study, we diagnosed a case of PWD complicated by ED with high mortality in piglets, and isolated and identified a hybrid strain carrying both enterotoxigenic Escherichia coli (ETEC) and Shiga toxin-producing Escherichia coli (STEC) virulence genes. It shows that the hybrid strain carried the F18 adhesin gene, enterotoxin genes (STa, STb, LT), and edema disease causing toxin gene (Stx2e). The results of antimicrobial sensitivity test showed that the E. coli isolate was sensitive to five drugs, including cefotaxime and amikacin, moderately sensitive to two drugs, including spectinomycin and ciprofloxacin, and resistant to 14 drugs, including enrofloxacin and streptomycin. The isolate is a hybrid Escherichia coli (ETEC/STEC), and shows strong resistance to multiple antibiotics.

Key words: post weaning diarrhea, pig edema disease, isolation and identification, virulence gene

中图分类号:

S852.61

蒋利明, 陈怡洁, 袁秀芳, 李军星. 猪源ETEC/STEC杂合大肠埃希菌菌株的分离、毒力基因鉴定及耐药性分析[J]. 浙江农业科学, 2024, 65(8): 1939-1943.

JIANG Liming, CHEN Yijie, YUAN Xiufang, LI Junxing. Isolation, virulence gene identification, and antimicrobial resistance analysis of hybrid enterotoxigenic and Shiga toxin-producing Escherichia coli (ETEC/STEC) strain from pigs[J]. Journal of Zhejiang Agricultural Sciences, 2024, 65(8): 1939-1943.

图/表 6

表1 E.coli毒力因子引物序列

Table 1 Primers for virulence genes of E.coli

引物名称	引物序列(5'-3')	产物大小/bp
STa-F	GGGTTGGCAATTTTTATTTCTGTA	183
STa-R	ATTACAACAAAGTTCACAGCAGTA
STb-F	GCTACAAATGCCTATGCATCTACACA	125
STb-R	CATGCTCCAGCAGTACCATCTCTAAC
LT-F	GCACACGGAGCTCCTCAGTC	218
LT-R	TCCTTCATCCTTTCAATGGCTTT
Stx2e-F	GAATGAAGATGTTTATAGCGG	454
Stx2e-R	TTTTATGGAACGTAGGTATTACC
987P-F	GCCAGTCTATGCCAAGTGGATACTTC	391
987P-R	GTTTGTATCAGGATTCCCTGTGGTGG
F18-F	TGGCACTGTAGGAGATACCATTCAGC	334
F18-R	GGTTTGACCACCTTTCAGTTGAGCAG
K88-F	TGAATGACCTGACCAATGGTGGAACC	478
K88-R	GCGTTTACTCTTTGAATCTGTCCGAG
VT2-F	ATCCTATTCCCGGGAGTTTACG	584
VT2-R	GCGTATCGTATACACAGGAGC

图1 血琼脂平板β溶血大菌落

Fig.1 β hemolytic large colony on blood agar plate

图2 大肠埃希菌染色镜检(10×100)

Fig.2 Observation of Gram staining of E.coli

图3 致病菌STa、STb、LT、Stx2e毒力基因的PCR扩增 M-DNA ladder DL1000;1、2-致病菌分离株含STa毒力基因;5、6-致病菌分离株含STb毒力基因;9、10-致病菌分离株含LT毒力基因;13、14-致病菌分离株含Stx2e毒力基因;3、7、11、15-阳性对照;4、8、12、16-阴性对照。

Fig.3 PCR amplicon of virulence genes STa, STb, LT, Stx2e from the bacterial pathogen

图4 致病菌987P、F18、K88、VT2毒力基因的PCR扩增 M-DNA ladder DL1000;1、2-致病菌分离株未扩增出987P毒力基因;5、6-致病菌分离株含F18毒力基因;9、10-致病菌分离株未扩增出K88毒力基因;13、14-致病菌分离株含VT2毒力基因;3、7、11、15-阳性对照;4、8、12、16-阴性对照。

Fig.4 PCR amplicon of virulence genes 987P, F18, K88, VT2 from the bacterial pathogen

表2 药敏试验

Table 2 Antimicrobial sensitivity test

药敏片	抑菌圈直径/mm	耐药性
头孢噻肟	29	敏感
氟苯尼考	27	敏感
阿米卡星	22	敏感
恩诺沙星	21	耐药
环丙沙星	20	中介
安普霉素	18	敏感
庆大霉素	18	敏感
壮观霉素	14	中介
链霉素	12	耐药
红霉素	11	耐药
头孢噻呋	0	耐药
头孢噻吩	0	耐药
氨苄西林	0	耐药
阿莫西林	0	耐药
卡那霉素	0	耐药
多西环素	0	耐药
青霉素	0	耐药
四环素	0	耐药
替米考星	0	耐药
复方新诺明	0	耐药
林可霉素	0	耐药

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