| Colibacillosis is caused by the avian pathogenic Escherichia coli(APEC),which results in substantial economic losses in the world.The rise of antibiotic resistance and drug residues rekindles interest in phage therapy as promising antibacterial alternatives.Nonetheless,the applications of phages were restricted by limited complete genome sequences,the restricted knowledge of undesired genes(DUG)as well as the narrow host ranges.In our study,23 phages were isolated and the host ranges of them were determined.QL01 was a unique virulent phage and chosen as the research object for the relatively broad lytic range which could lyse 41 of 78 APEC strains.The characterizations,genome information,comparative genomic analysis and clinical application against APEC infection were studied for further research on the mechanism of broad spectrum and the clinical application.1 The isolation and characterization of APEC phagesBacteriophages were isolated from duck feces,which were collected from different poultry markets in Nanjing,Jiangsu Province,China.In our study,23 phages were isolated and the host ranges of them were determined.QL01 was chosen as the research object for the relatively broad lytic range which could lyse 41 of 78 APEC strains,as well as the NMEC strain RS218.The morphological characterization revealed that phage QL01 possessed an icosahedral head(approximately 112x78 nm),a contractile tail(102x20 nm)and six tail fibers,which indicated that phage QL01 was assigned to the T4-like viruses genus,the Myoviridae family,and Caudovirales.The overlayer method was performed to obtain a high titer of phage suspension of 5.08×108PFU/mL.Assessment of thermal stability experiment revealed that QL01 was sensitive to heat.The optimal MOI for phage propagation was 1.2 Bacteriophage genome sequencing and analysisThe DNA sample was sequenced by Illumina MiSeq.The genome of QL01 consisted of a linear double-stranded DNA of 170527 bp with the average GC content of 39.6%.In total,275 ORFs were predicted.The genome of QL01 revealed high homology with other T4-like phages by comparative genome analysis.ORF142 and ORF143,encoding the IP9 and IP8,showed no homologies in many T4-like phages,especially the phages which showed high homology in the genome sequences.It was interesting that phage vB_EcoM_VR5,which was a Low-temperature T4-like coliphage,had similar 4 IP genes with QL01.This phenomenon indicated the occurrence of lateral gene transfer(LGT)between these phages.The genomic loci of the IP genes in phage QL01 and vB_EcoM_VR5 revealed a similarity in gene organization near IP8 and IP9,which were not very common in many T4-like phages,while rearrangement occurred near IP7 and IP3.3 Phage QL01 treatment of APEC infectionPhage therapy was conducted in artificially infected ducks to study the treatment effect against APEC infection.A preliminary experiment was conducted to determine the 50%lethal dose for challenge of ducks with APEC strain XM(2.12×104CFU).The challenge dose of the bacteria(3.6×104CFU)was determined according to the LD50 and multiple experiments.The 7-day-old ducks were infected and treated by different phage administration modes.The mortality in the intramuscular group was markedly lower than that of the positive group,indicating that this route is optimal for phage therapy.Ninety ducks were divided randomly into nine groups and treated intramuscularly after APEC infection.The survival condition of ducks was monitored for 7 days.The mean survival rate of the untreated group(63.33%)was lower than that of the treated group(90%)significantly(P<0.05).There was no mortality in the negative control group and no significant difference was observed between the treated groups and the negative controls(P>0.05).These observations indicated that phage administration mitigated the lethal effects of bacterial infection in ducks and decreased the mortality,providing new information on developing QL01 for phage therapy of APEC infections. |
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