特异性识别K1荚膜大肠杆菌的噬菌体PNJ1809-36生物学特性及全基因组分析

本研究以K1荚膜大肠杆菌DE058（avian pathogenic Escherichia coli）为宿主菌，从鸡粪中分离烈性噬菌体PNJ1809-36并对其进行生物学特性的研究和基因组测序及分析。将噬菌体PNJ1809-36进行分离纯化，通过形态学观察、最佳MOI测定、一步生长曲线测定、温度和酸碱耐受性测定、体外裂解能力测定以及全基因组测序及分析来研究该噬菌体的生物学特性和基因组特征。结果显示：噬菌体PNJ1809-36能够形成清晰透明的噬菌斑，其电镜照片显示为具有收缩尾部的肌尾科噬菌体；宿主谱分析结果显示，该噬菌体能够特异性裂解具有K1荚膜的大肠杆菌；最佳MOI为0.01；一步生长曲线结果显示，其潜伏期为10 min，爆发期为40 min；噬菌体PNJ1809-36在40和50℃下存活良好，60℃ 30 min可使噬菌体完全失活；噬菌体在pH3~11之间均可存活，最适pH为6；体外裂解试验表明，该噬菌体可在6 h内完全抑制宿主菌的生长。经基因组测序与分析，噬菌体PNJ1809-36基因组全长为152 343 bp，GC含量为39.11%，含有277个开放阅读框（ORF），11个tRNA基因。同源性分析显示其与早期发现的K1特异性的短尾噬菌体（如K1F、K1E）的同源性较低，而与噬菌体nepoznato、PVP-SE 1和phAPEC8等K1特异性噬菌体同源性较高，基因序列相似性达90%。噬菌体PNJ1809-36是一株特异性识别K1荚膜大肠杆菌的烈性噬菌体，属于肌尾科噬菌体新的系统发育分支。其针对K1荚膜的特性，提示该噬菌体在鉴定K1型大肠杆菌和治疗K1型大肠杆菌引起的疾病方面存在潜力。

Biological Characteristics and Whole Genome Analysis of Phage PNJ1809-36 Target Escherichia coli K1

Taking Escherichia coli (E. coli) K1 strain DE058 as a host, a virulent phage PNJ1809-36 was isolated from chicken feces. The biological characteristics and genome analysis of this phage were studied. Phage PNJ1809-36 was isolated and purified, whereafter, the biological characteristics of the phage were studied by analyzing the morphology, optimal MOI, one-step growth curve, temperature and acid-base tolerance, and lysis curve in vitro. Moreover, the whole genome of the phage was sequenced and analyzed. The results showed that phage PNJ1809-36 formed transparent plaque with a clear boundary. Under microscopy, phage PNJ1809-36 presented a contractile tail, indicating that it belongs to Myoviridae. The result of host range test showed that phage PNJ1809-36 specifically targeted E. coli with K1 capsule. The optimal MOI of phage PNJ1809-36 was 0.01. One-step growth curve showed that the latent and explosive period was 10 min and 40 min, respectively. Phage was stable at 40 and 50℃, while lost activity at 60℃ for 30 min. Phage PNJ1809-36 could maintain activity under conditions of pH 3-11, with the optimal pH 6; The lysis curve showed that the phage could continuously inhibit the growth of host bacteria for 6 h. Genomic analysis showed that it had low sequence identity with early reported E. coli K1-specific phage, such as K1F and K1E, but presented over 90% sequence homology with newly discovered E. coli K1-specific phage at nucleic acid level, such as nepoznato, PVP-SE1, and phAPEC8. PNJ1809-36, a lytic phage targeting E. coli K1, belongs to a new phylogenetic branch of Myoviridae. Due to the specificity to E.coli K1, phage PNJ1809-36 has the potential to identify and control the diseases caused by E. coli K1.