兔源大肠埃希菌噬菌体分离鉴定与生物学特性研究

doi:10.3969/j.issn.1004-1524.2022.08.04

浙江农业学报 ›› 2022, Vol. 34 ›› Issue (8): 1599-1608.DOI: 10.3969/j.issn.1004-1524.2022.08.04

兔源大肠埃希菌噬菌体分离鉴定与生物学特性研究

王志鹏¹(), 赵剑², 黄盼², 崔雪梅², 南黎², 宋厚辉¹, 鲍国连², 刘燕²^,^*

1.浙江农林大学动物科技学院·动物医学院,浙江省畜禽绿色生态健康养殖应用技术研究重点实验室,动物健康互联网检测技术浙江省工程实验室,浙江杭州 311300
2.浙江省农业科学院畜牧兽医研究所,浙江杭州 310021

收稿日期:2021-04-03 出版日期:2022-08-25 发布日期:2022-08-26
通讯作者: 刘燕
作者简介:*刘燕,E-mail:ly-liuyan@qq.com
王志鹏(1990—),男,黑龙江海林人,硕士研究生,主要从事兽医微生物研究。E-mail: 130640516@qq.com
基金资助:
浙江省重点研发计划(2019C02052);国家兔产业技术体系岗位科学家项目(CARS-43-C-2)

Isolation, identification and biological characteristics of rabbit-derived Escherichia coli bacteriophage

WANG Zhipeng¹(), ZHAO Jian², HUANG Pan², CUI Xuemei², NAN Li², SONG Houhui¹, BAO Guolian², LIU Yan²^,^*

1. Key Laboratory of Application Technology of Ecological Green and Healthy Livestock and Poultry Breeding in Zhejiang Province, Zhejiang Provincial Engineering Laboratory for Animal Health Inspection and Internet Technology, College of Animal Science and Technology·College of Veterinary Medicine, Zhejiang A&F University, Hangzhou 311300, China
2. Institute of Animal Husbandry and Veterinary Medicine, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China

Received:2021-04-03 Online:2022-08-25 Published:2022-08-26
Contact: LIU Yan

摘要/Abstract

摘要：

对浙江省某兔场腹泻病兔进行病原分离鉴定,经革兰氏染色和PCR鉴定,分离出致病性大肠埃希菌1株,命名为ZJE1株。ZJE1株对链霉素、环丙沙星敏感。以ZJE1株为宿主菌,分离纯化出噬菌体4株,分别命名为ZRP2、ZRP3、ZRP4和ZRP5,并对其生物学特性、形态与结构特征进行研究。结果显示,4株噬菌体分离株均由六角形的头部、可收缩的尾鞘和尾管组成,均属于肌尾噬菌体科。采用双层平板法测定噬菌体分离株的宿主谱、最佳感染复数、pH稳定性、热稳定性和一步生长曲线。结果显示,ZRP2、ZRP3、ZRP4和ZRP5具有宿主特异性,在37~55 ℃的条件下可保持良好活性。ZRP2、ZRP3在pH值7的条件下可保持良好活性,ZRP4、ZRP5在pH值5~7的条件下可保持良好活性。ZRP2、ZRP3、ZRP5的潜伏期为11~19 min,均能在体外裂解ZJE1株。研究成果可为应用噬菌体治疗兔大肠埃希菌病提供研究基础与数据支持。

关键词: 肠致病性大肠埃希菌, 噬菌体, 分离鉴定, 生物学特性

Abstract:

In this study, one enteropathogenic Escherichia coli strain was isolated and identified via Gram staining and PCR from a rabbit farm in Zhejiang Province, China, which was named as ZJE1. ZJE1 was sensitive to streptomycin and ciprofloxacin. Four bacteriophages were isolated with the ZJE1 strain, which were named as ZRP2, ZRP3, ZRP4 and ZRP5, respectively. The biological and morphological characteristics of these bacteriophages were analyzed. It was shown that the bacteriophages were composed of hexagonal head, contractible tail sheath and tail tube. All the four bacteriophages belonged to Myoviridae family. The host spectrum, optimal multiplicity of infection (MOI), pH stability, thermal stability and one-step growth curve of these four bacteriophages were determined by double-layer plate method. The results showed that ZRP2, ZRP3, ZRP4 and ZRP5 all showed good activity under 37-55 ℃ with high host specificity. ZRP2, ZRP3 could maintain good activity under pH value of 7, while ZRP4 and ZRP5 could maintain good activity under pH value of 5-7. ZRP2, ZRP3, ZRP5 showed good lytic activity in vitro, with incubation period of 11-19 min. These findings provided research basis for the treatment of rabbit enteropathogenic E. coli with bacteriophage.

Key words: enteropathogenic Escherichia coli, bacteriophage, isolation and identification, biological characteristics

中图分类号:

S858.291

王志鹏, 赵剑, 黄盼, 崔雪梅, 南黎, 宋厚辉, 鲍国连, 刘燕. 兔源大肠埃希菌噬菌体分离鉴定与生物学特性研究[J]. 浙江农业学报, 2022, 34(8): 1599-1608.

WANG Zhipeng, ZHAO Jian, HUANG Pan, CUI Xuemei, NAN Li, SONG Houhui, BAO Guolian, LIU Yan. Isolation, identification and biological characteristics of rabbit-derived Escherichia coli bacteriophage[J]. Acta Agriculturae Zhejiangensis, 2022, 34(8): 1599-1608.

图/表 12

参考文献 22

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基因 Gene	片段长度 Length/bp	上游引物序列 Forward primer sequences(5'→3')	下游引物序列 Reverse primer sequences(5'→3')
16S rRNA	584	GGGATGAAAGTTAATACCTTTGCTC	TTCCCGAAGGCACATTCT
eaeA	384	GACCCGGCACAAGCATAAGC	CCACCTGCAGCAACAAGAGG
bfpA	224	AATGGTGCTTGCGCTTGCTGC	GCCGCTTTATCCAACCTGGTA
stx	150	CTGGATTTAATGTCGCATAGTG	AGAACGCCCACTGAGATCATC
ipaH	600	GTTCCTTGACCGCCTTTCCGATACCGTC	GCCGGTCAGCCACCCTCTGAGAGTAC
st	186	TCTGTATTGTCTTTTTCACC	TTAATAGCACCCGGTACAAGC
lt	322	TCTCTATGTGCATACGGAGC	CCATACTGATTGCCGCAAT
aatA	630	CTGGCGAAAGACTGTATCAT	CAATGTATAGAAATCCGCTGTT

基因 Gene	片段长度 Length/bp	上游引物序列 Forward primer sequences(5'→3')	下游引物序列 Reverse primer sequences(5'→3')
16S rRNA	584	GGGATGAAAGTTAATACCTTTGCTC	TTCCCGAAGGCACATTCT
eaeA	384	GACCCGGCACAAGCATAAGC	CCACCTGCAGCAACAAGAGG
bfpA	224	AATGGTGCTTGCGCTTGCTGC	GCCGCTTTATCCAACCTGGTA
stx	150	CTGGATTTAATGTCGCATAGTG	AGAACGCCCACTGAGATCATC
ipaH	600	GTTCCTTGACCGCCTTTCCGATACCGTC	GCCGGTCAGCCACCCTCTGAGAGTAC
st	186	TCTGTATTGTCTTTTTCACC	TTAATAGCACCCGGTACAAGC
lt	322	TCTCTATGTGCATACGGAGC	CCATACTGATTGCCGCAAT
aatA	630	CTGGCGAAAGACTGTATCAT	CAATGTATAGAAATCCGCTGTT

生化试验 Biochemical test	结果 Result
葡萄糖 Glucose	+
乳糖 Lactose	+
麦芽糖 Maltose	+
甘露醇 Mannitol	+
山梨醇 Sorbic alcohol	+
鸟氨酸脱羧酶Ornithinede carboxylase	+
枸橼酸 Citric acid	-
H₂S	-
尿素 Urea	-
甲基红 Methyl red	+
VP试验 VP test	-
吲哚试验Indole test	-

生化试验 Biochemical test	结果 Result
葡萄糖 Glucose	+
乳糖 Lactose	+
麦芽糖 Maltose	+
甘露醇 Mannitol	+
山梨醇 Sorbic alcohol	+
鸟氨酸脱羧酶Ornithinede carboxylase	+
枸橼酸 Citric acid	-
H₂S	-
尿素 Urea	-
甲基红 Methyl red	+
VP试验 VP test	-
吲哚试验Indole test	-

药剂 Antimicrobial agent	判定结果 Interpretation
氨苄西林Ampicillin	R
阿莫西林Amoxicillin	R
青霉素Penicillin	R
头孢噻吩Cefthiophene	R
头孢噻呋Ceftiofur	R
头孢噻肟Cefotaxime	I
链霉素Streptomycin	S
庆大霉素Gentamicin	R
阿米卡星Amikacin	R
卡那霉素Kanamycin	R
壮观霉素Spectinomycin	R
红霉素Erythromycin	R
替米考星Tilmicosin	R
强力霉素Doxycycline	R
四环素Tetracycline	R
氟苯尼考Florfenicol	I
林可霉素Lincomycin	R
复方新诺明Trimethoprim	R
恩诺沙星Enrofloxacin	I
环丙沙星Ciprofloxacin	S

兔源大肠埃希菌噬菌体分离鉴定与生物学特性研究

Isolation, identification and biological characteristics of rabbit-derived Escherichia coli bacteriophage

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参考文献 22

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菌株类型 Strain type	菌株名 Strain name	ZRP2	ZRP3	ZRP4	ZRP5
标准菌株	CVCC1495	-	-	-	-
Standard	CVCC232	+	+	-	-
strain	CVCC249	-	-	-	+
EHEC	PARX	-	-	-	-
	WZ01	-	-	-	-
EAEC	ZS185	-	-	-	-
	ZS85	-	-	-	-
EIEC	DC10	+	+	-	-
	DC16	-	-	-	-
	DC23	-	-	-	-
	FL15	+	+	-	+
	TCQX	+	+	-	-
EPEC	DCW	+	+	+	-
	ZB16	+	+	+	+
	ZHC1	+	+	+	-
	ZHC2	-	-	-	-
	ZK01	-	-	-	+
	ZK02	-	-	-	+
	ZK15	-	-	-	+
	ZK16	-	-	-	+
	ZK1901	-	-	-	+
	ZK1902	-	-	-	+
	ZK1903	-	-	-	+
	ZX01	+	+	-	-
NPEC	JX03	+	-	-	-
	JX04	-	-	-	-
	XCF1	-	-	-	-
	XCF2	+	+	-	-

噬菌体滴度 Bacteriophage titer/ (PFU·mL^-1)	细菌浓度 Bacteria concentration/ (CFU·mL^-1)	混合培养后噬菌体滴度 Bacteriophage titer after co-culture/(PFU·mL^-1)				噬菌体扩增比值 Bacteriophage amplification ratio
噬菌体滴度 Bacteriophage titer/ (PFU·mL^-1)	细菌浓度 Bacteria concentration/ (CFU·mL^-1)	ZRP2	ZRP3	ZRP4	ZRP5	ZRP2	ZRP3	ZRP4	ZRP5
1×10⁹	1×10⁸	9.31×10¹⁰	4.87×10¹⁰	4.15×10⁹	3.72×10¹⁰	93.1	48.7	4.15	37.2
1×10⁸	1×10⁸	1.24×10¹¹	5.53×10¹⁰	6.42×10⁸	5.48×10¹⁰	1 240	553	6.42	548
1×10⁷	1×10⁸	1.61×10¹¹	5.66×10¹⁰	1.23×10⁸	1.35×10¹¹	16 100	5 660	12.3	13 500
1×10⁶	1×10⁸	1.39×10¹¹	4.11×10¹⁰	6.49×10⁶	3.68×10¹¹	139 000	41 000	6.49	368 000
1×10⁵	1×10⁸	6.11×10¹⁰	1.76×10¹⁰	5.45×10⁵	1.55×10⁹	611 000	176 000	5.45	15 540
1×10⁴	1×10⁸	5.96×10⁸	2.89×10⁸	4.79×10⁴	7.24×10⁷	59 600	28 900	4.79	7 240

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