| Pathogenic Escherichia coli causes colibacillosis,which results in significant economic losses to the livestock industry worldwide.Due to the drug residues and increased antibiotic resistance caused by antibiotic use,bacteriophages and other alternative therapeutic agents are expected to control pathogenic Escherichia coli infection.The use of phages as antibacterial agents is limited by the narrow host range and the difficulty of screening phages for specific pathogens rapidly.In this study,the tail fiber proteins of T4-like lysogenic phages and P2-like lysogenic phages were analyzed and modified to find the important region for the determination of receptor specificity.In this process,we established the platforms that enabled efficient genetic manipulation of lytic phage and lysogenic phage.Utilzing this platform,we successfully introduced the specific genetic mutations and demonstrated that these lytic phages and lysogenic phages showed much wider host ranges than the parental phages,meanwhile,we located the the important regions for the determination of receptor specificity of T4-like phage WG01 and P2-like phage P88.We investigated the therapeutic efficiency of a cocktail to APEC strain DE205B.This study lay some foundations to solve the problems in therapeutic phage agents application.1 Isolation,genome sequencing of coliphagesIn this study,isolation and genome sequencing of lytic phage and lysogenic phage was performed.The lytic phages were isolated from the feces from the farmers market in China.The lysogenic phages were obtained following mitomycin C induction of 54 lysogenic strains.More than 200 strains of phage were isolated.More than 40 strains of phage were observed by Electron Microscopy.8 strains of phage were sequenced.There were 6 strains of lysogenic phages in the sequenced 8 strains,which were T7-like phages P483 and P694,C3-like phage 172-1,T4-like phages QL01,WG01 and MX01.There were 2 strains of lysogenic phages in the sequenced 8 strains,which were P2-like phages P88 and pro 147.The date of this study lay some foundation for the subsequent research.2 Comparative genomics and functional analysis of two T7-like coliphages of avian pathogenic Escherichia coliPhages P483 and P694 were the first phages which host was APEC from the T7-like phage family to be reported.Comparative genomic analysis showed that most of the predicted proteins of these two phages showed strongest sequence similarity to the Enterobacteria phages BA14 and 285P,Erwinia phage FE44,and Kluyvera phage Kvpl;however,some proteins such as gp0.6a,gp1.7 and gp17 showed lower similarity(<85%)with the homologs of other phages in the T7 subgroup.We also found some unique characteristics of P483 and P694,such as the two types of the genes of P694.Our results serve to further our understanding of phage evolution of T7-like coliphages and provide the potential application of the phages as therapeutic agents for the treatment of diseases.3 Inducible prophage mutant of Escherichia coli can lyse new host and the key sites of receptor recognition identificationThe use of bacteriophages as therapeutic agents is hindered by their narrow and specific host range,and by a lack of the knowledge concerning the molecular mechanism of receptor recognition.Two P2-like coliphages,named P88 and pro 147,were induced from Escherichia coli strains K88 and DE147,respectively.A comparison of the genomes of these two and other P2-like coliphages obtained from GenBank showed that the tail fiber protein genes,which are the key genes for receptor recognition in other myoviridae phages,showed more diversity than the conserved lysin,replicase,and terminase genes.Firstly,replacing hypervariable region 2(HR2:amino acids 716-746)of the tail fiber protein of P88 with that of pro 147 changed the host range of P88.Then,replacing six amino acids in HR2 with the corresponding residues from pro 147 altered the host range only in these mutants with changes at position 730(leucine)and 744(glutamic acid).Thus we predicted that these amino acids are vital to establish the host range of P88.This study provided a vector of lysogenic bacteria that could be used to change or expand the phage host range of P88.These results illustrated that,in P2-like phage P88,the tail fiber protein determined the receptor recognition.Amino acids 716-746 and the amino acids at positions 730 and 744 were important for receptor recognition.4 Alterations in gp37 expand the host range of a T4-like phageThe use of phages as antibacterial therapeutic agents is limited by the narrow host range.The aim of this study was first to make a T4-like phage,WG01,to obtain the host range of another T4-like phage,QL01,by replacing its host determinant gene region with that of QL01.This process triggered a direct expansion of the WG01 host range.The offspring of WG01 obtained both the host ranges of QL01 and WG01,as well as the ability to infect eight additional host bacteria in comparison to the wildtype strains.WQD1 had the widest host range;therefore the corresponding fragments of QD could be used for constructing a homologous sequence library.Moreover,after a sequencing analysis of gene37,we identified two different mechanisms responsible for the expanded host range:1)the first generation of WG01 formed chimeras without mutations;and 2)the second generation of WG01 mutants formed from the chimeras.The expansion of the host range indicated that regions other than the C-terminus may indirectly change the receptor specificity by altering the supportive capacity of the binding site.Additionally,we also found that the subsequent generations acquired a novel means of expanding the host range through acquiring a wider temperature range for lysis by exchanging gene37.Our findings demonstrate a method for rapidly changing and expanding the host range of phages.The future clinical application for screening phages against a given clinical isolate could be achieved after an inducible phage library is established with more suitable homologous sequences.5 Phage therapy is effective to control avian colibacillosis and oral phages with translocation capacity could reduce the bacteria numbersIncreasing emergence of antibiotic-resistant bacteria isolates has provoked renewed interest in the possible use of bacteriophages to control bacterial infections.The present study is aimed to investigate the therapeutic efficiency of a cocktail to APEC strain DE205B.In this study,a cocktail of three different bacteriophages were selected for evaluating the protecting rate of artificial animal infection by APEC strain DE205B of ducks via three different ways:intramuscular injection,oral gavage and nasal drip.The results showed that the survival rate of the intramuscular injection treated group(100%)was more significant than the oral gavage treated group(28.7%)and the nasal drip treated group(0%).Meanwhile,only phage PHI could be recovered from the hearts,livers and lungs of ducks dead from oral gavage treated group at different time points after administration,from 34 to 60 hours post infection.The number of re-isolated phage PH1 in ducks varied between 3.07x107-1.07x109 PFU/g in the heart,between 2.16x105-4.33x107PFU/g in the liver and between 9.92x104-1.69x108PFU/g in the lung.These phages could reach high titers in vivo and could reduce the concentration of bacteria.In the three different methods of administration,intramuscular injection was a best way for administering treatment with survival rate of 100%.In the three phages used in this study,phage PHI had the translocation capacity that translocate from the gastrointestinal tract to the cardiovascular system.Intramuscular injection of this phage cocktail would be an optional way to administer treatment when face avian pathogenic Escherichia coli infection.When in the cases of prevention,phage treatment by oral administration could be an optional administration with further understanding of this mechanism of translocation. |
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