多价噬菌体分离筛选及靶向灭活土壤病原菌的应用研究

【Objective】Given the drastic development of livestock industry in China, animal manure amount increased significantly in the last few decades. Without proper environmental management, the manure as well as relevant wastes have made the agricultural soil near the livestock site a hotspot of pathogens, which has posed potential threat against the public health and environmental safety. Therefore, it is urgent to carry out bioremediation technologies to effectively inactivate the targeted pathogens in the soil. Among various techniques, phage therapy provides a novel way for eliminating pathogenic bacteria in the soil. In this work, polyvalent phages were firstly isolated, and further screened for their capacity of inactivating various host pathogenic bacteria, in an attempt to apply the polyvalent phage therapy for controlling the pathogens contamination in the soil. 【Method】In this work, double-layer agar was employed to isolate phages from the soil applied with cow manure in a dairy farm in Nanjing Eastern China, which was contaminated by Enterobacteriaceae and Pseudomonas pathogenic bacteria. Two host-specific phages (YSZ1 and YSZ5) were isolated from the polluted soil. Then with the accelerated human-directed evolution in the laboratory, two polyvalent (broad host range) phages (YSZ1R and YSZ5k) were obtained each corresponding to YSZ1 and YSZ5. Meanwhile, the biological characteristics including morphology and molecular technology were carried out for the identification of the four phages obtained. Moreover, by studying the optimal multiplicity of infection and growth characteristics, the capacity of inactivating pathogenic bacteria among four phages were also determined. Meanwhile, Biolog ECO plates were used to investigate the impact of phage therapy on the diversity of the soil microbial community. 【Result】The morphological and molecular identification indicated that the four phages described above all belonged to Stylovinidaeacid. Meanwhile, in both aqueous system and contaminated soil, the four phages exhibited significacnt but varying capacity of inactivating fecal coliform and Pseudomonas, following the order of YSZ5K>YSZ1R>YSZ5>YSZ1. In addition, compared with the control, host-specific phage inoculation (YSZ5 and YSZ1) slightly decreased the AWCD value, while polyvalent phage inoculation (YSZ5K and YSZ1R) significantly increased the AWCD value after 120 h of incubation. Shannon index showed the same trend of YSZ5K/YSZ1R> CK> YSZ5/YSZ1. Therefore, polyvalent phages therapy applied in this work was able to maintain and increase the diversity and stability of the indigenous microbial community in the soil. 【Conclusion】The polyvalent phages isolated from the pathogen contaminated soil were determined as Stylovinidaeacid. Both polyvalent phages (YSZ5K and YSZ1R) could significantly stimulate the dissipation of the pathogenic bacteria in both aqueous and soil systems. Meanwhile, the diversity of the indigenous bacteria community in the soil could be clearly enhanced after polyvalent phage therapy, suggesting that the polyvalent phage therapy developed in this work was an applicable technique to inactivate broad range pathogenic bacteria in the soil.