一株可裂解耐黏菌素大肠杆菌噬菌体的分离鉴定及全基因组分析

Isolation,identification and complete genomic analysis of a bacteriophage capable of lysing colistin-resistant Escherichia coli

为研究耐黏菌素大肠杆菌的噬菌体治疗方法,本研究以耐黏菌素大肠杆菌为宿主菌,采用双层琼脂平板法从山东烟台某养鸡场污水样中分离纯化得到裂解性噬菌体SDYTW1-F1-2-2。通过透射电镜观察,最佳感染复数,一步生长曲线,温度稳定性,酸碱度稳定性等生物学特性测定,并对其进行全基因组测序分析。结果表明:1)噬菌体SDYTW1-F1-2-2噬菌斑呈现透亮,外周无晕圈,形态学观察显示其具有可伸缩性尾鞘。2)生物学特性分析表明,噬菌体最佳感染复数为0.1,潜伏期为50 min,爆发期为40 min,爆发量为(331±9)PFU/cell。在温度4~40℃,pH为4~12的环境下,噬菌体活性较为稳定。3)通过噬菌体全基因组测序及核酸类型鉴定显示,该噬菌体基因组全长为74752 bp,双链DNA,GC含量42.1%。在噬菌体基因组中共鉴定出121个编码蛋白(CDS),包括结构组成、DNA代谢与复制和包装以及细菌裂解相关的功能基因,发现一个tRNA基因,且未检测到如stx-1等致病性基因和mcr-1等耐药基因相关的基因。综上,噬菌体SDYTW1-F1-2-2裂解谱较宽,具有良好的酸碱性、热稳定性以及安全性。本研究为后续开展针对耐黏菌素大肠杆菌的噬菌体治疗研究提供依据。

To study bacteriophage(phage)therapy for colistin-resistant Escherichia coli(E.coli),phage SDYTW1-F1-2-2 was isolated and purified by double AGAR plate method using colistin-resistant E.coli as host bacteria from sewage samples collected from a chicken farm in Yantai,Shandong Province.The biological characteristics of the bacteriophage were analyzed by transmission electron microscopy,optimum infection complex number,one-step growth curve,temperature stability and pH stability,and the whole genome was sequenced.The results showed as follows:1)The plaque of bacteriophage SDYTW1-F1-2-2 was transparent,halo free and had a flexible tail sheath.2)The analysis of biological characteristics showed the optimal number of phage infection was 0.1,the incubation period was 50 min,the outbreak period was 40 min,and the amount of outbreak was(331±9)PFU/cell.The phage activity was stable when the temperature was 4-40℃and the pH was 4-12)The total length of the bacteriophage genome was 74752 bp,with double strands of DNA,GC content of 42.1%.A total 121 sequence coding for amino acids in protein(CDS)were identified in the genome of phage,including functional genes associated with structure composition,nucleotide metabolism and replication,packaging and bacterial lysis,a tRNA genome were discovered and no genes associated with pathogenicity genes such as stx-1 and drug resistant genes such as mcr-1 were identified.In summary,phage SDYTW1-F1-2-2 has a wide cleavage spectrum,strong acid and base tolerance,thermal stability and safety.This study provides a basis for further research on phage therapy of colistin resistant E.coli.