肠侵袭性大肠埃希菌噬菌体DK-13的生物学特性及应用

doi:10.13560/j.cnki.biotech.bull.1985.2023-0902

摘要/Abstract

摘要：

【目的】 筛选并分离出能裂解肠侵袭性大肠埃希菌噬菌体，分析其生物学特性，并探究其对污染猪肉的杀菌作用。【方法】 采用双层平板法分离鉴定噬菌体，并测定其最佳感染复数，一步生长曲线等，对其基因组进行测序分析以及裂解功效的测定。【结果】 从医院污水中分离鉴定能特异裂解肠侵袭性大肠埃希菌（Enteroinvasive Escherichia coli coli，EIEC）的噬菌体并命名为DK-13，其呈典型的蝌蚪状外形，包含一个正二十面体的头部和一个可收缩的螺旋对称的尾部，属于肌尾噬菌体科（Myoviridae）噬菌体；生物学特性表明：最佳感染复数为0.01，潜伏期约为10 min，裂解期约为70 min，噬菌体DK-13能在50℃，pH 5.0-10.0条件下存活。全基因组测序表明，噬菌体基因组长约172 275 bp，GC含量为40.18%，预测其共有293个开放阅读框（open reading frames, ORF）,未发现已知耐药基因和毒力基因。应用试验表明：被污染的猪肉中表现的杀菌效果良好，宿主菌的数量明显减少。【结论】 分离并鉴定一株新的烈性噬菌体DK-13，DK-13具有潜伏期短、裂解效率高等优势，在食品安全方面具有很大应用潜力。

关键词: 肠侵袭性大肠埃希菌, 噬菌体, 噬菌体疗法, 食品安全

Abstract:

【Objective】 Selection and isolation of phage that can lyse Enteroinvasive Escherichia coli (EIEC), analyzing its biological characterization, and investigating its sterilizing effect on contaminated pork. 【Method】 The double-layer agar method was used to isolate phages, and then the optimal multiplicity of infection，one-step growth curve and other biological characteristics of the isolate were determined. The genetic genomes were sequenced and analyzed, and the lysis efficiency was evaluated.【Result】 A bacteriophage that specifically lysed EIEC was isolated from hospital sewage and named DK-13. It has a typical tadpole-like morphology, consisting of a head and a contractile helical symmetric tail. It belongs to the family Myoviridae. The biological characterization showed that the optimal multiplicity of infection was 0.01, the incubation period was about 10 min, the lysis period was about 70 min, and the phage DK-13 was able to survive at 50℃ and pH 5.0-10.0. The whole genome sequencing showed that the phage genome was about 172 275 bp in length, with a GC content of 40.18%, and was predicted to have a total of 293 open reading frames（ORFs）, and no resistance genes or virulence genes were found. the sterilization effect in the contaminated pork was good, and the number of host bacteria significantly reduced. 【Conclusion】 A new virile phage DK-13 was isolated and identified, which has the advantages of short incubation period and high cleavage efficiency, and has great potential for application in food safety.

Key words: Enteroinvasive Escherichia coli, phage, phage application, food safety

王梦雅, 刘家齐, 姜海霖, 李菁华, 赵春燕, 黄红兰. 肠侵袭性大肠埃希菌噬菌体DK-13的生物学特性及应用[J]. 生物技术通报, 2024, 40(3): 296-304.

WANG Meng-ya, LIU Jia-qi, JIANG Hai-lin, LI Jing-hua, ZHAO Chun-yan, HUANG Hong-lan. Biological Characteristics and Application of Enteroinvasive Escherichia coli Phage DK-13[J]. Biotechnology Bulletin, 2024, 40(3): 296-304.

图/表 7

图1 噬菌体DK-13的形态特征 A:噬菌体形态；B:透射电镜形态

Fig. 1 Morphological characteristics of phage DK-13 A: Morphology of phage DK-13. B: Transmission electron micrograph of phage DK-13

图2 噬菌体DK-13的生物学特性 A:噬菌体DK-13的MOI；B:噬菌体DK-13的一步生长曲线。不同小写字母表示处理组别间有显著差异（P<0.05)。下同

Fig. 2 Biological characters of phage DK-13 A: MOI of phage DK-13. B: Step growth curve of phage DK-13. Different lower letters indicate significant difference among each group (P<0.05). The same below

图3 噬菌体DK-13的生物学特性 A:噬菌体DK-13的热稳定性；B:噬菌体DK-13的酸碱稳定性

Fig. 3 Biological characteristics of phage DK-13 A: Temperature resistance of phage DK-13. B: pH resistance of phage DK-13

表1 噬菌体DK-13宿主范围

Table 1 Host range of DK-13 phage

菌株名称 Strain name	菌株类型 Strain type	噬菌体DK-13的裂解性 Phage DK-13 lytic ability
大肠埃希菌A1 Escherichia coli A1	O157:H7	-
大肠埃希菌A2 E. coli A2	EPEC	-
大肠埃希菌A3 E. coli A3	O26:H11	-
大肠埃希菌A4 E. coli A4	O26:H11	-
大肠埃希菌A5 E. coli A5	O26:H11	-
大肠埃希菌A6 E. coli A6	O26:H11	-
大肠埃希菌A7 E. coli A7	O26:H11	-
大肠埃希菌A8 E. coli A8	O46:H38	-
大肠埃希菌A9 E. coli A9	O11:NM	-
大肠埃希菌A10 E. coli A10	O157:H7	-
大肠埃希菌A11 E. coli A11	O157:H7	-
大肠埃希菌A12 E. coli A12	O157:H7	-
大肠埃希菌A13 E. coli A13	O157:H7	-
大肠埃希菌A14 E. coli A14	O157:H7	-
大肠埃希菌A15 E. coli A15	O157:H7	-
蜡样芽孢杆菌L1 Bacillus cereus L1	*	-
蜡样芽孢杆菌L2 B. cereus L2	*	-
金黄色葡萄球菌B1 Streptococcus aureus B1	*	-
金黄色葡萄球菌B2 S. aureus B2	*	-
金黄色葡萄球菌B3 S. aureus B3	*	-
大肠埃希菌（宿主菌）D1 E. coli D1	EIEC	+

图4 噬菌体DK-13基因组注释图谱箭头方向代表基因方向。红色：结构基因模块；棕色：包装基因模块；蓝色：核苷酸代谢基因模块；橙色：复制/重组基因模块；粉色：转录/翻译基因模块；绿色：其他功能模块；灰色：假定蛋白；黑色：GC含量

Fig. 4 Annotation diagram of the genome of DK-13 phage The direction of arrow indicates the direction of gene. Red: The structural gene module. Brown: The packaged genetic module. Blue: The nucleotide metabolism gene module. Orange: Duplication/ Recombination gene module. Pink: Transcription/ Translation gene module. Green: Other function module. Grey: Hypothetical protein. Black: GC content

图5 噬菌体DK-13的进化关系分析

Fig. 5 Phylogenetic analysis of phage DK-13

图6 不同温度下噬菌体DK-13对污染猪肉的杀菌效果 A: 4℃下噬菌体的杀菌效果；B：37℃下噬菌体的杀菌效果

Fig. 6 Sterilization effect of phage DK-13 at different temperatures on contaminated pork A: Sterilization effect of phage DK-13 at 4℃. B: Sterilization effect of phage DK-13 at 37℃

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