中国农业大学学报2024，Vol.29Issue(9)：102-111,10.DOI:10.11841/j.issn.1007-4333.2024.09.09
肺炎克雷伯菌噬菌体分离鉴定及抑菌效果研究
Isolation,identification and antibacterial effect study of Klebsiella pneumoniae phage
摘要
Abstract
The study was to explore the phage as a new type of biological antibacterial agent to control Klebsiella pneumoniae,which cause dairy cow mastitis and contaminate dairy products and their processing,and the ability of biofilm clearance.This study utilized ATCC 700603 as the host bacteria isolated a strain of Klebsiella pneumoniae phage vB_KpnM-YP11 from a water sample of a cattle farm and the morphological characteristics of the phage were observed by transmission electron.The host spectrum of phage was determined by double-layer plate method.The biological characteristics of phages were assessed through an optimal multiplicity of infection,one-step growth curve,as well as temperature and pH stability tests.The structual characteristics and evolutionary relutionary of the genome were analyzed by whole genome sequencing.The inhibitory and scavenging effects of phage on biofilms were determined using crystal violet staining and plate counting methods.The antibacterial effect of the phage on Klebsiella pneumoniae in milk was analyzed.The results showed that:1)The Klebsiella pneumoniae phage vB_KpnM-YP11 isolated from the water samples of cattle farms could form clear and translucent plaques.The typical structure of tailed phage was observed by TEM.2)Determination of host spectrum showed that vB_KpnM-YP11 could lyse 18/34 clinical isolates of Klebsiella pneumoniae from cattle.3)The optimal multiplicity of infection(MOI)of this phage was 0.1.The one-step growth curve showed that the latent period was 30 min,and the average lysis amount was 69 PFU/cell.The phage was stable at 4-60 ℃ or pH 3-11.4)The full-length genome of vB_KpnM-YP11 was 166 607 bp,and the GC content was 39.52％.The annotation results revealed that the phage contained 276 ORF and 15 tRNA,while lacking drug resistance and virulence genes.5)When the MOI of vB_KpnM-YP11 was 0.1,it had the ability to inhibit the biofilm formation of Klebsiella pneumoniae.The mature biofilm were cleared by 108 PFU/mL and 109 PFU/mL.At room temperature,the bacterial concentration in milk decreased by 98％within 12 h.In conclusion,Phage vB_KpnM-YP11 with short incubation period and high lysis efficiency not only has a good lytic effect on Klebsiella pneumoniae in milk,but also can effectively remove the biofilm formed by Klebsiella pneumoniae.It has the potential to be used as a biological antibacterial agent for Klebsiella pneumoniae.关键词
肺炎克雷伯菌/噬菌体/生物学特性分析/基因组分析/生物被膜Key words
Klebsiella pneumoniae/phage/biological characteristics/genomic analysis/biofilm分类
农业科技引用本文复制引用
孔祥宇,杨文静,庞申雨,苑庆欣,潘顺圆,高东阳,宋军,王金涛..肺炎克雷伯菌噬菌体分离鉴定及抑菌效果研究[J].中国农业大学学报,2024,29(9):102-111,10.基金项目
国家自然科学基金青年科学基金项目(31802226) （31802226）
黑龙江省自然科学基金联合引导项目(LH2022C072) （LH2022C072）
黑龙江省牛病防制重点实验室开放课题(PCBD201710) （PCBD201710）
