大肠埃希菌裂解性噬菌体vB_EcoP-R1的特性及其抗细菌生物膜活性分析

Characterization of Escherichia coli lytic phage vB_EcoP-R1 and its anti-biofilm activity

目的从医院污水中分离大肠埃希菌特异性噬菌体,分析其生物学特性,并检测其对细菌生物膜的裂解作用。方法通过双层琼脂平板法分离纯化噬菌体,透射电镜观察其形态,并测定其一步生长曲线、体外裂解曲线、温度及酸碱稳定性。全基因测序分析噬菌体基因组组成及特性,结晶紫染色法评估噬菌体对宿主菌生物膜的清除效果。结果从医院污水中新分离1株大肠埃希菌特异性噬菌体,命名为vB_EcoP-R1;透射电镜下显示形态与尾病毒目足病毒科噬菌体成员相似,在-20~50℃的温度范围内以及在pH值5.0~11.0酸碱条件下能稳定存活。vB_EcoP-R1基因组全长为40875 bp,GC含量为49.94%,不含毒力基因和耐药基因。vB_EcoP-R1能够裂解临床分离的大肠埃希菌,且显示高度种属特异性。噬菌体作用于宿主菌预成形生物膜4 h后,对生物膜的清除率达到80.16%。结论vB_EcoP-R1是1株新的大肠埃希菌特异性噬菌体,裂解性强且种属特异性高,可裂解宿主菌已形成的生物膜,有望用于大肠埃希菌感染的替代治疗。

Objective To isolate Escherichia coli-specific bacteriophages from hospital waste water,analyze their biological characteristics and detect their cleavage effects on bacterial biofilms.Methods The phages were isolated and purified by double-layer agar plate method and their morphology was observed by transmission electron microscopy.Their one-step growth curve,in vitro lysis curve,temperature and acid-base stability were determined.Whole genome sequencing was used to analyze the genome composition and characteristics of bacteriophages.The crystal violet staining was used to evaluate the scavenging effects of phage on host biofilms.Results A novel Escherichia coli-specific bacteriophage named vB_EcoP-R1 was isolated from hospital sewage.Transmission electron microscopy revealed that the morphology of vB_EcoP-R1 was similar to that of Podoviridae,a member of Caudovirales,and it could survive stably in the temperature range of-20℃to 50℃and pH 5.0 to 11.0.The total length of the vB_EcoP-R1 genome was 40875 bp and the GC content was 49.94%.There were no virulence genes and drug resistance genes in the phage genome.vB_EcoP-R1 was capable of lysing clinically isolated Escherichia coli and showed high species and genus specificity.After the phage acted on the host bacteria preformed biofilm for 4 h,the clearance rate of the biofilm reached 80.16%.Conclusion vB_EcoP-R1 should be a novel Escherichia coli-specific bacteriophage with strong lysis and high specificity for species and genus.The bacteriophage could lyse the biofilm formed by the host bacteria,and is expected to be used as an alternative treatment for Escherichia coli infection.