多价噬菌体分离筛选及靶向灭活土壤病原菌的应用研究
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摘要
农田土壤中残留和滋生多种病原菌会对人体健康和生态环境带来显著的安全隐患,开展针对性的生物修复研究十分迫切。噬菌体疗法靶向灭活土壤中病原菌的技术为修复此类污染土壤提供了全新途径。本研究以南京城郊某奶牛场牛粪堆积池周边,粪肠杆菌和假单胞菌复合污染农田土壤为例,首先筛选和纯化获得两株专一型噬菌体(YSZ1和YSZ5),再人为加速其宿主谱的表达过程,获得对应的多价噬菌体(YSZ1R和YSZ5K),并进行生物学特性(形态、核酸、最佳感染复数、一步生长曲线等)鉴定,结果表明:在水相和污染土壤中不同噬菌体对于同步灭活病原菌的能力依次为YSZ5K>YSZ1R>YSZ5>YSZ1,并且施用多价噬菌体疗法有助于维护和改善修复后土壤微生物生态功能多样性与稳定性。本研究结果可为噬菌体疗法靶向灭活土壤中多种病原菌提供切实可行的修复技术。
【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(YSZ1 R and YSZ5 k) 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 YSZ5 K>YSZ1 R>YSZ5>YSZ1. In addition, compared with the control, host-specific phage inoculation(YSZ5 and YSZ1) slightly decreased the AWCD value, while polyvalent phage inoculation(YSZ5 K and YSZ1 R) significantly increased the AWCD value after 120 h of incubation. Shannon index showed the same trend of YSZ5 K/YSZ1 R> 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(YSZ5 K and YSZ1 R) 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.
【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(YSZ1 R and YSZ5 k) 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 YSZ5 K>YSZ1 R>YSZ5>YSZ1. In addition, compared with the control, host-specific phage inoculation(YSZ5 and YSZ1) slightly decreased the AWCD value, while polyvalent phage inoculation(YSZ5 K and YSZ1 R) significantly increased the AWCD value after 120 h of incubation. Shannon index showed the same trend of YSZ5 K/YSZ1 R> 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(YSZ5 K and YSZ1 R) 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.
引文
[1]Zhu J C,Zhang Z Q, Fan Z M,et al. Biogas potential, cropland load and total amount control of animal manure in China. Journal of Agro-Environment Science,2014,33(3):435-445
[2]Sharon I, Morowitz M J, Thomas B C, et al. Time series community genomics analysis reveals rapid shifts in bacterial species, strains, and phage during infant gut colonization. Genome Research, 2013,23(1):111-120
[3]Alves D R. Development and characterisation of a responsive polyvalent bacterio-phage therapeutic.Bath, UK:University of Bath, 2015
[4]Bhattacharjee A S,Choi J,Motlagh A M.Bacteriophage therapy for membrane biofouling in membrane bioreactors and antibioticresistant bacterial biofilms. Biotechnology andBioengineering, 2015, 112(8):1644-1654
[5]Elshibiny A, Elsahhar S, Adel M. Phage applications for improving food safety and infection control in Egypt. Journal of Applied Microbiology, 2017,123(2):556-567
[6]Jones J B, Vallad G E, Wernsing M H, et al. 1SFp05 Considerations for phage therapy for bacterial plant pathogens associated with foliar plant diseases.Japan:The Society for Biotechnology, 2013
[7]Hyman P, Abedon S T. Bacteriophage host range and bacterial resistance. Advances in Applied Microbiology, 2010, 70:217-248
[8]Alexa R,Samantha W,Paul H. More is better:Selecting for broad host range bacterio-phages bacteriophages. Frontiers in Microbiology, 2016,DOI:10. 3389/fmicb. 2016. 01352
[9]尹春艳,骆永明,滕应,等.典型设施菜地土壤抗生素污染特征与积累规律研究.环境科学,2012,33(8):2810-2816Yin C Y, Luo Y M, Teng Y, et al. Pollution characteristics and accumulation of antibiotics in typical protected vegetable soils. Environmental Science, 2012, 33(8):2810-2816
[10]Xu J,Chen M,He L,et al. Isolation and characterization of a T4-like phage with a relatively wide host range within Escherichia coli. Journal of Basic Microbiology, 2016, 56(4):405-421
[11]Jensen E C,Schrader H S,Rieland B,et al.Prevalence of broad-host-range lytic bact-eriophages of Sphaerotilus natans,Escherichia coli,and Pseudomonas aeruginosa. Applied and Environmental Microbiology, 1998, 64(2):575-580
[12]Yu P, Mathieu J, Li M, et al. Isolation of polyvalent bacteriophages using sequential multiple host approaches. Applied and Environmental Microbiology, 2015, 82(3):808-815
[13]尹雅菲,张乐,庆宏,等.新广谱大肠杆菌噬菌体IME11的分离及鉴定.生物医学工程与临床,2013,17(5):483-486Yin Y F,Zhang L, Qing H, et al. Isolation andclassification of new broad spectrum Escherichia coli bacteriophage IME11. Biomedical Engineering and Clinical Medicine, 2013, 17(5):483-486
[14]Hyman P,Abedon S T. Practical Methods for determining phage growth parameters. Methods in Molecular Biology, 2009, 501:175-202
[15]Wu M, Qin H, Chen Z, et al. Effect of long-term fertilization on bacterial composition in rice paddy soil. Biology and Fertility of Soils, 2011, 47(4):397-405
[16]Thingstad T F, Vage S,Storesund J E, et al. A theoretical analysis of how strain-specific viruses can control microbial species diversity. Proceedings of the National Academy of Sciences of the United States of America, 2014, 111(21):7813-7818
[17]Bukreyev A A, Chandran K, Dolnik O, et al.Discussions and decisions of the 2012-20 14International Committee on Taxonomy of Viruses(ICTV)Filoviridae Study Group, January 2012-June 2013. Archives of Virology, 2014, 159(4):821-830
[18]Azeredo J,Sillankorva S,Pires D P. Pseudomonas bacteriophage isolation and production//Pseudomonas Methods and protocols. New York:Springer, 2014:23-32
[19]Shao Y,Wang I N. Bacteriophage adsorption rate and optimal lysis time. Genetics, 2008,180(1):471-482
[20]Maslov S,Sneppen K. Population cycles and species diversity in dynamic Kill-the-Winner model of microbial ecosystems. Scientific Reports, 2017,DOI:10. 1038/srep 39642
[21]Abedon S T. Ecology of anti-biofilm agentsⅡ:Bacteriophage exploitation and biocontrol of biofilm bacteria. Pharmaceuticals, 2015,8(3):559-589
[22]Gomez P, Bennie J, Gaston K J, et al. The impact of resource availability on bacterial resistance to phages in soil. PLoS One, 2015, DOI:10. 1371/journal.pone. 0123752
[2]Sharon I, Morowitz M J, Thomas B C, et al. Time series community genomics analysis reveals rapid shifts in bacterial species, strains, and phage during infant gut colonization. Genome Research, 2013,23(1):111-120
[3]Alves D R. Development and characterisation of a responsive polyvalent bacterio-phage therapeutic.Bath, UK:University of Bath, 2015
[4]Bhattacharjee A S,Choi J,Motlagh A M.Bacteriophage therapy for membrane biofouling in membrane bioreactors and antibioticresistant bacterial biofilms. Biotechnology andBioengineering, 2015, 112(8):1644-1654
[5]Elshibiny A, Elsahhar S, Adel M. Phage applications for improving food safety and infection control in Egypt. Journal of Applied Microbiology, 2017,123(2):556-567
[6]Jones J B, Vallad G E, Wernsing M H, et al. 1SFp05 Considerations for phage therapy for bacterial plant pathogens associated with foliar plant diseases.Japan:The Society for Biotechnology, 2013
[7]Hyman P, Abedon S T. Bacteriophage host range and bacterial resistance. Advances in Applied Microbiology, 2010, 70:217-248
[8]Alexa R,Samantha W,Paul H. More is better:Selecting for broad host range bacterio-phages bacteriophages. Frontiers in Microbiology, 2016,DOI:10. 3389/fmicb. 2016. 01352
[9]尹春艳,骆永明,滕应,等.典型设施菜地土壤抗生素污染特征与积累规律研究.环境科学,2012,33(8):2810-2816Yin C Y, Luo Y M, Teng Y, et al. Pollution characteristics and accumulation of antibiotics in typical protected vegetable soils. Environmental Science, 2012, 33(8):2810-2816
[10]Xu J,Chen M,He L,et al. Isolation and characterization of a T4-like phage with a relatively wide host range within Escherichia coli. Journal of Basic Microbiology, 2016, 56(4):405-421
[11]Jensen E C,Schrader H S,Rieland B,et al.Prevalence of broad-host-range lytic bact-eriophages of Sphaerotilus natans,Escherichia coli,and Pseudomonas aeruginosa. Applied and Environmental Microbiology, 1998, 64(2):575-580
[12]Yu P, Mathieu J, Li M, et al. Isolation of polyvalent bacteriophages using sequential multiple host approaches. Applied and Environmental Microbiology, 2015, 82(3):808-815
[13]尹雅菲,张乐,庆宏,等.新广谱大肠杆菌噬菌体IME11的分离及鉴定.生物医学工程与临床,2013,17(5):483-486Yin Y F,Zhang L, Qing H, et al. Isolation andclassification of new broad spectrum Escherichia coli bacteriophage IME11. Biomedical Engineering and Clinical Medicine, 2013, 17(5):483-486
[14]Hyman P,Abedon S T. Practical Methods for determining phage growth parameters. Methods in Molecular Biology, 2009, 501:175-202
[15]Wu M, Qin H, Chen Z, et al. Effect of long-term fertilization on bacterial composition in rice paddy soil. Biology and Fertility of Soils, 2011, 47(4):397-405
[16]Thingstad T F, Vage S,Storesund J E, et al. A theoretical analysis of how strain-specific viruses can control microbial species diversity. Proceedings of the National Academy of Sciences of the United States of America, 2014, 111(21):7813-7818
[17]Bukreyev A A, Chandran K, Dolnik O, et al.Discussions and decisions of the 2012-20 14International Committee on Taxonomy of Viruses(ICTV)Filoviridae Study Group, January 2012-June 2013. Archives of Virology, 2014, 159(4):821-830
[18]Azeredo J,Sillankorva S,Pires D P. Pseudomonas bacteriophage isolation and production//Pseudomonas Methods and protocols. New York:Springer, 2014:23-32
[19]Shao Y,Wang I N. Bacteriophage adsorption rate and optimal lysis time. Genetics, 2008,180(1):471-482
[20]Maslov S,Sneppen K. Population cycles and species diversity in dynamic Kill-the-Winner model of microbial ecosystems. Scientific Reports, 2017,DOI:10. 1038/srep 39642
[21]Abedon S T. Ecology of anti-biofilm agentsⅡ:Bacteriophage exploitation and biocontrol of biofilm bacteria. Pharmaceuticals, 2015,8(3):559-589
[22]Gomez P, Bennie J, Gaston K J, et al. The impact of resource availability on bacterial resistance to phages in soil. PLoS One, 2015, DOI:10. 1371/journal.pone. 0123752
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