一株新的多重耐药福氏志贺菌噬菌体生物学特性及基因组分析

doi:10.13560/j.cnki.biotech.bull.1985.2021-1549

摘要/Abstract

摘要：

抗生素的大量使用和滥用造成细菌耐药性问题愈发严峻,噬菌体疗法作为一种精准治疗多重耐药病原菌的有效方法开始被人们日益重视。采用双层琼脂平板法从活性污泥中分离出多重耐药福氏志贺菌（Shigella flexneri）噬菌体P003,鉴定为肌尾噬菌体科（Myoviridae）。通过生物学特性测定表明P003的最佳感染复数为10,潜伏期约10 min;在4-70℃、pH 3.0-10.0的条件下保持活性。全基因组测序表明,噬菌体基因组长约68 721 bp,GC含量46.14%,预测编码99个开放阅读框（open reading frame,ORF）,未发现已知耐药基因和毒力基因。全基因组多序列线性与进化树分析结果表明,P003与大肠杆菌噬菌体有较近的亲缘关系,但不能侵染大肠杆菌。从活性污泥原位分离到一株新的多重耐药福氏志贺菌噬菌体P003,为利用噬菌体疗法防治多重耐药病原菌S. flexneri感染提供了新的菌种资源。

关键词: 噬菌体, 福氏志贺菌, 抗生素耐药性, 生物学特性, 全基因组分析

Abstract:

Antibiotic resistance of bacteria caused by over use and abuse of antibiotics has been more and more seriously,and phage therapy as an precision treatment to treat multi-drug-resistant pathogens has being received more attentions. Double-layer agar plate method was applied to isolate a phage P300,a multi-drug resistant Shigella flexneri,from activated sludge and identified as Myoviridae. The measurement of biological characteristics demonstrated that the optimal multiplicity of infection（MOI）of phage P003 was 10,and the latent period was 10 min. Phage P003 remained active under the conditions of 4-70℃ and pH 3.0-10.0. The whole genome sequencing of phage P003 revealed that the length was 68 721 bp,contained 99 ORFs and 46.14% GC content,and no known drug resistance genes and virulence genes were discovered. Whole genome multi-sequence linear comparison and phylogenetic tree analysis showed that P003 was highly similar to Escherichia coli phage,but it cannot infect E. coli,indicating that P003 was a novel phage. In sum,a novel multidrug resistant phage S. flexneri P003 is isolated from active sludge,which may provide a new strain resource for the treatment of multidrug-resistant pathogen S. flexneri infection by phage therapy in the future.

Key words: phage, Shigella flexneri, antibiotic resistance, biological characteristics, whole genome sequencing

文畅, 刘晨, 卢诗韵, 许忠兵, 艾超凡, 廖汉鹏, 周顺桂. 一株新的多重耐药福氏志贺菌噬菌体生物学特性及基因组分析[J]. 生物技术通报, 2022, 38(9): 127-135.

WEN Chang, LIU Chen, LU Shi-yun, XU Zhong-bing, AI Chao-fan, LIAO Han-peng, ZHOU Shun-gui. Biological Characteristics and Genome Analysis of a Novel Multidrug-resistant Shigella flexneri Phage[J]. Biotechnology Bulletin, 2022, 38(9): 127-135.

图/表 10

表1 宿主菌耐药性

Table 1 Antibiotic sensitivity of host bacteria

抗生素名称 Antibiotic name	抗生素浓度Antibiotic concentration /（μg·mL^-1）	药敏性Sensitivity
四环素 Tetracycline	2	R
氯霉素 Chloramphenicol	16	R
氨苄青霉素 Ampicillin	100	R
卡那霉素 Kanamycin	100	S
阿莫西林 Amoxicillin	64	R
硫酸链霉素 Streptomycin	30	R
庆大霉素 Gentamicin	10	R
红霉素 Erythromycin	15	R
利福平 Rifampin	100	R

图1 噬菌体P003噬菌斑（A）及透射电镜形态（B）

Fig.1 Plaque（A）and morphology by TEM（B）of phage P003

图2 噬菌体P003最佳感染复数（A）及一步生长曲线（B）

Fig.2 Optimal multiplicity of infection（A）and one-step growth curve of phage P003（B）

图3 噬菌体P003温度（A）、pH（B）及紫外（C）稳定性

Fig. 3 Temperature (A), pH (B) and UV (C) stability of phage P003

表2 噬菌体P003裂解谱

Table 2 Lytic range of phage P003

菌株 Bacterial strain	敏感性 Sensitivity
Shigella flexneri B003	+
Shigella flexneri M001	—
Shigella flexneri M002	—
Shigella flexneri M003	—
Escherichia coli HB101	—
Escherichia coli DH5α	—
Escherichia coli K12	—
Escherichia coli KN	—
Escherichia coli OP50	—
Escherichia LFHY_s J001	—
Escherichia hermannii	—
Enterobacter cloacae subsp	—
Serratia nematodiphila	—
Serratia marcescens	—
Klebsiella africana	—
Klebsiella pneumoniae subsp	—

图4 噬菌体P003全基因组图谱

Fig.4 Genome profile of phage P003

图5 噬菌体P003基因功能注释

Fig. 5 Gene function annotation of phage P003

表3 噬菌体P003与数据库中病毒基因组比较分析

Table 3 Comparative analysis of phage P003 with virus database based on genome

Accession No.	Escherichia phage	Coverage/%	Identity/%
NC_048194.1	vB_EcoM_WFH	96	99.18
MH051335.1	vB_EcoM-Ro157lw	94	97.22
JX128258.1	ECML-117	92	97.04
MK903282.1	Mansfield	92	96.36
NC_048073.1	FEC19	94	95.32

图6 噬菌体P003与3株大肠杆菌噬菌体全基因序列比对

Fig. 6 Whole-gene sequence alignment of phage P003 with 3 Escherichia phages

图7 基于噬菌体P003主要衣壳蛋白系统发育树

Fig.7 Phylogenetic tree based on the major capsid protein of phage P003

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