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

doi:10.3969/j.issn.1004-1524.2022.08.04

Acta Agriculturae Zhejiangensis ›› 2022, Vol. 34 ›› Issue (8): 1599-1608.DOI: 10.3969/j.issn.1004-1524.2022.08.04

• Animal Science • Previous Articles Next Articles

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

Abstract

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

CLC Number:

S858.291

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.

Figures/Tables 12

References 22

[1]	GORDILLO ALTAMIRANO F L, BARR J J. Phage therapy in the postantibiotic era[J]. Clinical Microbiology Reviews, 2019, 32(2): e00066.
[2]	LOC-CARRILLO C, ABEDON S T. Pros and cons of phage therapy[J]. Bacteriophage, 2011, 1(2): 111-114. DOI URL
[3]	GOLKAR Z, BAGASRA O, PACE D G. Bacteriophage therapy: a potential solution for the antibiotic resistance crisis[J]. Journal of Infection in Developing Countries, 2014, 8(2): 129-136. DOI URL
[4]	CHATTERJEE A, DUERKOP B A. Beyond bacteria: bacteriophage-eukaryotic host interactions reveal emerging paradigms of health and disease[J]. Frontiers in Microbiology, 2018, 9: 1394. DOI URL
[5]	YAP M L, ROSSMANN M G. Structure and function of bacteriophage T4[J]. Future Microbiology, 2014, 9(12): 1319-1327. DOI URL
[6]	NILSSON A S. Phage therapy: constraints and possibilities[J]. Upsala Journal of Medical Sciences, 2014, 119(2): 192-198. DOI URL
[7]	LUZ MARÍA CHACÓN J, LIZETH TAYLOR C, CARMEN VALIENTE A, et al. A DNA pooling based system to detect Escherichia coli virulence factors in fecal and wastewater samples[J]. Brazilian Journal of Microbiology, 2012, 43(4): 1319-1326. DOI URL
[8]	AL-MAMUN A, MILY A, SARKER P, et al. Treatment with phenylbutyrate in a pre-clinical trial reduces diarrhea due to enteropathogenic Escherichia coli: link to cathelicidin induction[J]. Microbes and Infection, 2013, 15(13): 939-950. DOI URL
[9]	FARFÁN-GARCÍA A E, ARIZA-ROJAS S C, VARGAS-CÁRDENAS F A, et al. Virulence mechanisms of enteropathogenic Escherichia coli[J]. Revista Chilena De Infectologia, 2016, 33(4): 438-450.
[10]	SLATER S L, SÅGFORS A M, POLLARD D J, et al. The type Ⅲ secretion system of pathogenic Escherichia coli[J]. Current Topics in Microbiology and Immunology, 2018, 416: 51-72.
[11]	SLATER S L, SAGFORS A M, POLLARD D J, et al. The type Ⅲ secretion system of pathogenic Escherichia coli[M]// FRANKEL G, RON E Z. Escherichia coli, a versatile pathogen. Cham, Switzerland: Springer Cham, 2018.
[12]	SOLANS L, ARNAL J L, SANZ C, et al. Rabbit enteropathies on commercial farms in the Iberian peninsula: etiological agents identified in 2018-2019[J]. Animals, 2019, 9(12): 1142. DOI URL
[13]	HYMAN P, ABEDON S T. Bacteriophage host range and bacterial resistance[J]. Advances in Applied Microbiology, 2010, 70: 217-248.
[14]	SKURNIK M, PAJUNEN M, KILJUNEN S. Biotechnological challenges of phage therapy[J]. Biotechnology Letters, 2007, 29(7): 995-1003. DOI URL
[15]	BRUTTIN A, BRÜSSOW H. Human volunteers receiving Escherichia coli phage T4 orally: a safety test of phage therapy[J]. Antimicrobial Agents and Chemotherapy, 2005, 49(7): 2874-2878. DOI URL
[16]	KORTRIGHT K E, CHAN B K, KOFF J L, et al. Phage therapy: a renewed approach to combat antibiotic-resistant bacteria[J]. Cell Host & Microbe, 2019, 25(2): 219-232.
[17]	SULAKVELIDZE A, ALAVIDZE Z, MORRIS J G JR. Bacteriophage therapy[J]. Antimicrobial Agents and Chemotherapy, 2001, 45(3): 649-659. DOI URL
[18]	ZHANG J C, LI Z, CAO Z H, et al. Bacteriophages as antimicrobial agents against major pathogens in swine: a review[J]. Journal of Animal Science and Biotechnology, 2015, 6(1): 39. DOI URL
[19]	BRÜSSOW H. Phage therapy: the Escherichia coli experience[J]. Microbiology (Reading, England), 2005, 151(Pt 7): 2133-2140. DOI URL
[20]	CHIBANI-CHENNOUFI S, SIDOTI J, BRUTTIN A, et al. In vitro and in vivo bacteriolytic activities of Escherichia coli phages: implications for phage therapy[J]. Antimicrobial Agents and Chemotherapy, 2004, 48(7): 2558-2569. DOI URL
[21]	TANJI Y, SHIMADA T, FUKUDOMI H, et al. Therapeutic use of phage cocktail for controlling Escherichia coli O157: H7 in gastrointestinal tract of mice[J]. Journal of Bioscience and Bioengineering, 2005, 100(3): 280-287. DOI URL
[22]	LIU N, LEWIS C, ZHENG W M, et al. Phage cocktail therapy: multiple ways to suppress pathogenicity[J]. Trends in Plant Science, 2020, 25(4): 315-317. DOI URL

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

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

RichHTML

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

Figures/Tables 12

References 22

Related Articles 15

Recommended Articles

Metrics

Comments

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

[1]	LI Xudong, LIU Yongtao, YANG Xianle, YANG Yibin, AI Xiaohui. Analysis on pathogens of frogs with crooked head, broken head or white eye [J]. Acta Agriculturae Zhejiangensis, 2022, 34(8): 1617-1625.
[2]	CHEN Shiyu, XU Meiyu, DENG Zhengyu, WANG Feng, ZHANG Qilin, DENG Xianyu, LIN Lianbing. Treatment of diseased broilers with Shigella bacteriophage ΦDS8 and the effect on their intestinal flora [J]. Acta Agriculturae Zhejiangensis, 2022, 34(7): 1386-1395.
[3]	YANG Ling, SHA Nanjing, PAN Pengju, WU Bozhi. Identification and main biological characteristics of pathogen of Clematis leaf blight in Yunnan [J]. Acta Agriculturae Zhejiangensis, 2022, 34(7): 1449-1456.
[4]	WANG Xiaoli, ZHAO Yingwei, KONG Xiaona, CAO Zilin. Isolation and identification of mycorrhizal fungi in rhizosphere and their effect on growth and photosynthetic characteristics of Eucalyptus globulus seedlings [J]. Acta Agriculturae Zhejiangensis, 2022, 34(5): 1015-1023.
[5]	CHEN Mengzhu, KANG Zhenya, GUO Xianghui, GENG Yi, BAI Minghuan, OUYANG Ping, CHEN Defang, HUANG Xiaoli, LAI Weimin. Isolation and biological characteristics of a pathogenic ST-251 Aeromonas hydrophila from Procypris Rabaudi (rock carp) [J]. Acta Agriculturae Zhejiangensis, 2021, 33(12): 2286-2294.
[6]	CHEN Runchen, WANG Yining, LIU Xiaowen, WANG Hongyan, DING Qiang, WANG Honglei. Identification, artificial cultivation and nutritional analysis of wild Pholiota adiposa [J]. Acta Agriculturae Zhejiangensis, 2021, 33(12): 2330-2338.
[7]	LI Xuqing, YAN Jianli, RUAN Songlin. Identification and biological characteristics of anthracnose pathogen on Tetrastigma hemsleyanum [J]. , 2020, 32(11): 2009-2019.
[8]	YUAN Xianyu, YANG Longbin, HE Zanzan, MAO Tianjiao, HE Changsheng, ZHAN Songhe, SUN Pei, WEI Jianzhong, LI Yu. Isolation and identification of pseudorabies virus and molecular characterization of its main virulence genes in Anhui [J]. , 2020, 32(1): 43-56.
[9]	YI Keke, YIN Wenqi, ZHOU Yuancheng, JIANG Jinzhen, ZHANG Baiyu, LI Zhongyin, YAN Qigui. Isolation and identification of 4 strains of porcine pseudorabies virus and analysis of main virulence genes [J]. , 2019, 31(9): 1429-1436.
[10]	YANG Yibin, AI Xiaohui, SONG Yi, DONG Jing, XU Ning, JIANG Lan. Preliminary study on hemolytic ascites disease of Pelteobagrus fulvidraco [J]. , 2019, 31(8): 1239-1248.
[11]	LI Xuqing, ZHANG Jingze, ZHANG Ya, WU Genliang. Identification of pathotype of Verticillium dahlida isolates on eggplant and their biological characteristics in Shaoxing City, Zhejiang Province [J]. , 2019, 31(5): 784-789.
[12]	CUI Yilong, SHI Yun, YANG Dahan, YIN Youqin, XUE Jiangdong, HUO Xiaowei, MA Dehui. Isolation,identification of horse Bacillus cereus and its virulence genes detection [J]. , 2019, 31(2): 216-221.
[13]	ZHOU Huiming, ZHANG Yanzhen, CHAI Hongmei, YANG Rongqing, TAN Ying, ZHANG Pingping, BAI Yuying, ZHAO Yilian, JIN Yujie. Identification and culture conditions of a wild-type Termitomyces aurantiacus strain from Lincang [J]. , 2019, 31(10): 1655-1662.
[14]	BA Shaobo, SHI Lin, LI Qunjing, LIU Zhengkui, CHEN Lin, WANG Xiaodu, SONG Houhui. Isolation, identification and molecullar epidemiological analysis of 17-ZJ-HZ strain of porcine reproductive and respiratory syndrome virus [J]. , 2018, 30(8): 1303-1311.
[15]	MA Xiaoping, YANG Qiuxia, YU Yan, LI Desheng, WANG Chengdong, LING Shanshan, GU Yu. Comparison of partial biological characteristics and drug sensitivity between Cladosporium cladosporioides wild strain (Z20) from giant panda and mutant strain (Zt) [J]. , 2018, 30(8): 1328-1335.