一株烈性沙门氏菌噬菌体的生物学特性及基因组分析

doi:10.13560/j.cnki.biotech.bull.1985.2020-1385

摘要/Abstract

摘要：

随着抗生素耐药问题的日益加重,使得致病菌的生物防治愈发困难,新型抗菌剂的研发迫在眉睫。使用噬菌体控制致病菌重新获得了极大关注。为获取具有防治沙门氏菌潜力的噬菌体,采用双层琼脂平板法从河水中分离出一株烈性噬菌体PSM6,在肠炎沙门氏菌的菌苔可形成直径约1.5 mm-2 mm的透亮均匀的噬菌斑;具有宽裂解谱,可裂解多种血清型沙门氏菌、部分大肠杆菌和宋内志贺氏菌;最佳感染复数为0.01;繁殖活性强,潜伏期约20 min,爆发量约56 PFU/cell;在40-60℃、pH=5-10时,效价较为稳定。基因组核酸为双链DNA,基因组大小为90 730 bp,G+C含量为39.57%,有133个开放阅读框,含有holin-lysin裂解系统,未发现毒力因子相关基因和抗生素抗性基因。透射电镜观察和基因组测序均显示PSM6属于肌尾噬菌体科。

关键词: 沙门氏菌, 噬菌体, 分离, 基因组, 生物学特性

Abstract:

With the situation of antibiotic resistance becoming more and more serious,it is increasingly difficult to prevent and cure pathogenic bacteria,thus research and development of new antibacterial agents is urgent. The use of bacteriophages to control pathogenic bacteria has received huge attentions. In order to obtain bacteriophages with potential applications in the biological control of Salmonella,a virulent phage PSM6 was isolated from river water by double-layer agar plate method,and transparent and uniform plaques with diameters of about 1.5-2 mm were formed on the lawn of Salmonella enteritidis. Phage PSM6 showed lytic activities on Salmonella of multiple serotypes,some Escherichia. coli strains and Shigella sonnei,indicating a broad-spectrum lytic activity. Phage PSM6 showed the maximal lytic activity to Salmonella with a MOI of 0.01. It demonstrated strong reproductive activity,of which the incubation period was about 20 min and the outbreak amount was about 56 PFU/cell. Phage PSM6 presented a good stability in 40-60℃ and kept a stable titer at pH5-10. The genome nucleic acid was double-stranded DNA. The genome was of 90 730 bp with a G+C content of 39.6%,133 ORFs and the holin-lysin lysis system,did not contain known virulence-associated and antibiotic resistance genes. Phage PSM6 should belong to Myoviridae according to the results of transmission electron microscopy and genome sequencing.

Key words: Salmonella, phage, isolation, genome, biological characteristics

黄景晓, 尚俊康, 陈慧敏, 沈嘉旻, 黎圆圆, 喻玉立, 倪进东, 林伯坤. 一株烈性沙门氏菌噬菌体的生物学特性及基因组分析[J]. 生物技术通报, 2021, 37(6): 136-146.

HUANG Jing-xiao, SHANG Jun-kang, CHEN Hui-min, SHEN Jia-min, LI Yuan-yuan, YU Yu-li, NI Jin-dong, LIN Bo-kun. Biological Characterization and Genome Analysis of a Lytic Phage Infecting Salmonella[J]. Biotechnology Bulletin, 2021, 37(6): 136-146.

图/表 13

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宿主菌 Host bacterium	宿主菌编号 Host No.	PSM6裂解活性 Infectivity of PSM6
Salmonella Typhimurium	J25、J29、J45、J47、J49	++
Salmonella Derby	J07	++
Salmonella Derby	J23	+
Salmonella Rissen	J13、J14、J20、J26	-
Salmonella Corvallis	J15、J30	++
Salmonella Acina	J18	-
Salmonella London	J19、J24	++
Salmonella Agona	J22、J58	-
Escherichia coli	ATCC25922、DH5α、BL21	++
Shigella Sonnei	CMCC(B)51592	+
Shigella Flexneri	CMCC(B)51572	-
Shigella Dysenteriae	CMCC(B)51105	-
Shigella	ATCC14029	-

宿主菌 Host bacterium	宿主菌编号 Host No.	PSM6裂解活性 Infectivity of PSM6
Salmonella Typhimurium	J25、J29、J45、J47、J49	++
Salmonella Derby	J07	++
Salmonella Derby	J23	+
Salmonella Rissen	J13、J14、J20、J26	-
Salmonella Corvallis	J15、J30	++
Salmonella Acina	J18	-
Salmonella London	J19、J24	++
Salmonella Agona	J22、J58	-
Escherichia coli	ATCC25922、DH5α、BL21	++
Shigella Sonnei	CMCC(B)51592	+
Shigella Flexneri	CMCC(B)51572	-
Shigella Dysenteriae	CMCC(B)51105	-
Shigella	ATCC14029	-

感染复数 MOI	噬菌体效价 Titer of phage (PFU·mL^-1)	宿主菌浓度 Concentration of host bacteria (CFU·mL^-1)	培养3 h后的噬菌体效价 The titer of phage after 3 h culture (PFU·mL^-1)
100	1.0×10⁵	1.0×10³	(9.4±0.2)×10⁸
10	1.0×10⁵	1.0×10⁴	(2.2±0.5)×10⁸
1	1.0×10⁵	1.0×10⁵	(6.2±0.6)×10⁷
0.1	1.0×10⁵	1.0×10⁶	(8.1±0.5)×10⁸
0.01	1.0×10⁵	1.0×1⁰7	(1.2±0.6)×10¹⁰
0.001	1.0×10⁵	1.0×10⁸	(4.7±0.3)×10⁹

感染复数 MOI	噬菌体效价 Titer of phage (PFU·mL^-1)	宿主菌浓度 Concentration of host bacteria (CFU·mL^-1)	培养3 h后的噬菌体效价 The titer of phage after 3 h culture (PFU·mL^-1)
100	1.0×10⁵	1.0×10³	(9.4±0.2)×10⁸
10	1.0×10⁵	1.0×10⁴	(2.2±0.5)×10⁸
1	1.0×10⁵	1.0×10⁵	(6.2±0.6)×10⁷
0.1	1.0×10⁵	1.0×10⁶	(8.1±0.5)×10⁸
0.01	1.0×10⁵	1.0×1⁰7	(1.2±0.6)×10¹⁰
0.001	1.0×10⁵	1.0×10⁸	(4.7±0.3)×10⁹

噬菌体名称 Phage name	大小Genome length/bp	GC含量 GC content/%	编码区 Coding region	编码区平均大小 Coding region length/bp	编码区占百分比 Encoding area percent/%	转运RNA Transfer RNA	GenBank登录号 Accession No.
PSM6	90730	39.57%	133	601.8	88.2	25	This study
Salmonella phage vB_SPuM_SP116	87510	38.29%	126	604.1	87.0	22	KP010413.1
Escherichia phage vB_EcoM_AYO145A	87372	39.00%	131	592.6	88.9	20	KR014248.1
Escherichia phage VpaE1_ev108	87749	38.92%	129	596.1	87.6	26	LR597658.1
Escherichia phage VpaE1_ev035	88002	38.96%	132	591.4	88.0	26	LR699048.1
Escherichia phage NBEco005	88272	38.84%	130	593.6	87.4	19	MN994499.1
Shigella phage Z31	89355	38.88%	132	594.7	87.9	26	MN655999.1
Salmonella phage D1-2	86878	38.73%	128	577.6	85.1	23	MN481367.1