Biological Characteristics and Whole Genome Analysis of Klebsiella pneumoniae Phage kp6
[Objective]The purpose of this study was to explore the biological characteristics and whole genome characteristics of Klebsiella pneumoniae phage isolated from sewage samples,and provide a basis for the prevention and control of clinical Klebsiella pneumoniae infection and the development of new phage related alternative antibody products.[Method]A Klebsiella pneumoniae TJAU-K11 was used as the host bacteria,the phage of Klebsiella pneumoniae was isolated and purified from sewage samples by double plate method.The morphology of the isolated phage was observed by transmission electron microscopy,and the host spectrum,optimal multiplicity of infection(MOI)and one-step growth curve were determined,and the stability of phage was evaluated.The effect of phage on the biofilm formation and cleavage of Klebsiella pneumoniae TJAU-K11 was investigated,and the whole genome was sequenced to understand the biological characteristics and whole genome characteristics of phage.[Result]3 Klebsiella pneumoniae phage were successfully isolated from sewage samples and named kp4,kp5 and kp6,respectively.Transmission electron microscopy showed that phage kp6 was a short-tailed phage with regular icosahedral morphology.The one-step growth curve showed that the incubation period of phage kp6 was 20 min and the outbreak period was 30 min.The phage titer remained stable at-20-50 ℃,pH 4.0-10.0 and UV irradiation for 12 min.The results of single bacteriophage inhibition test in vitro showed that the D600 mm value of phage kp6 groups with different MOI was always less than 0.4 within 7 h.The results of in vitro bacteriostatic test of phage cocktail showed that the D600 nm value of phage kp6 within 12 h was less than 0.1 when MOI was 1 and 100.The amount of host bacteria biofilm formation in phage kp6 groups with different MOI was extremely significantly lower than that in positive control group(P＜0.01),and both single phage kp6 and bacteriophage cocktail could lysate host bacteria biofilm.The whole genome sequencing showed that the phage kp6 genome was double-linked looped DNA,with a total length of 40 971 bp and GC content of 53.13％.Among 49 coding sequences,23 encoded proteins with known functions,including gp12,a tail fibrillar protein for short tail iophage adsorption and injection,and lytic functional protein endolysin.[Conclusion]In this study,Klebsiella pneumoniae lytic phage kp6 was isolated,and there was an endolysin in its genome.The phage cocktail prepared by phage kp6 could completely inhibit the growth of host bacteria within 12 h.The results laid a foundation for the subsequent development of lytic enzymes and phage preparations.
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