Isolation,Identification,Biological Characteristics And Genome Study Of Bacteriophage From Dogs In Heqing Wild Rats Plague Foci

ObjectiveConducting an investigation of the Yersinia pestis bacteriophages carried by the indicative animal ’dogs’ of Yunnan Heqing plague epidemic foci,and the purified proliferation of bacteriophages isolated from the dogs,and the research on their biological and genomic characteristics,was to provide the theoretical basis for studying their interactions with plague,preservation mechanism and micro-ecological effects,through which,the mechanism and function of plague bacteriophages in transmission of plague epidemic can be clarified.MethodsThe two-layer agar plate method was used to separate and purify the phages,and the phage state was observed by naked eyes.The titer of the obtained phages was determined by drop method,and the morphology was observed by ht770 electron microscope.The classical method was used to determine the optimal infection complex number and one-step growth curve.The single plate direct drop method was used to determine the fragmentation characteristics,which was rechecked by double-layer agar plate method.The genomic DNA of the phage was extracted according to the operation instructions of the kit.Several techniques such as NCBI,MEGA6 were utilized to analyze the whole genome biological characteristics of the obtained phages.Results1.Six strains of plague bacteriophages,namely HQG8、HQG12、HQG17、HQG27、HQG29 and HQG41,were separated and extracted out of 81 anal swap specimens collected from the indicative animal ‘Chinese Rural Dogs’ in Yunnan Heqin new Plague epidemic focus.2.The morphosis of Plague bacteriophage representative HQG8,HQG17 and HQG27 was observed by using transmission electron microscope.3 strains of phages were all in typical morphosis as the ’ tadpole shape ’,formed by hexahedralstructured head,ring-like middle collar and long shrinkable tail sheath.The diameter of head was from 50 nm to 70 nm,and the length of the tail part was from 140 nm to 160 nm.Thephage plaquesformedby HQG8,HQG27,HQG29 and HQG41 were in uniform size,round shape with neat edge and no aureole around,which defined as the typical characteristics of the lytic phage.3.HQG8,HQG27 and HQG29 barely lysed 20 strains of Yersinia pestis at 22℃,while lysed at 28℃ and 37℃.Both of HQG12 and HQG 17 lysed 20 strains of Yersinia pestis at 22℃,while being passive at 37℃.HQG41 lysed 20 strains of Yersinia pestis at all given temperature 22℃,28℃ and 37℃.HQG8,HQG27 and HQG41 illustrated the lysed characteristics against type-2a strain of Shigella flexneri at 22℃,28℃ and 37℃.HGQ8 and HQG27 showed lysed characteristics againsttype-X strain of Shigella flexneri at 22℃,28℃ and 37℃.HQG41 lysed type-1a strain of Shigella flexneri at all given temperature 22℃,28℃ and 37℃,also showing the lysed characteristics against type-X strain of Shigella flexneri at 37℃.HGQ12 and HQG17 presented the lysed characteristics against 5 clinical isolates strains of Serratia marcescens(6,24,26,36 and 38)at 22℃,28℃ and 37℃.HQG8 showed the lysed characteristics against Shigella boydii and Shigella sonnei respectively at 37℃,being passive against other bacterial in this research.HGQ27 lysed the strain of Shigella sonnei and the standard strain of Escherichia coli(ATCC35218)at 22℃ and37℃ respectively,being passive against otherbacterial in this research.HGQ41 lysedtype-1 strainof Shigella sonnei and standard strain of Escherichia coli(ATCC25922)at 37℃,being passive against otherbacterial in this research.In the condition of 28℃,HQG29 lysed the Type-2a strain and type-X strain of Shigella flexneri,being passive against otherbacterial in this research.HGQ17 lysed type-I,type-II,type-III strains of Yersinia pseudotuberculosis and Salmonella typhi strain at all given temperature 22℃,28℃ and 37℃,also lysed standard strain of Escherichia coli(ATCC25922)at 22℃ and 37℃,being passive against otherbacterial in this research.HGQ12 lysed standard strain of Yersinia enterocolitica(ATCC52301)at 22℃ and 28℃,also lysed standard strain of Escherichia coli(ATCC35218)and Salmonella typhi strain at 22℃,being passive against otherbacterial in this research.4.The optimal multiplicity of infection(MOI)for the plague bacteriophage representative HQG8 in this research was measured as 0.1,with an incubation of 40 minutes,a lysing period of 140 minutes.HQG8 was illustrated to be grown stably after 180 minutes.5.According to the biological analysis of 6 strains of obtained plague bacteriophage in this research,the genome size was measured from 32 kb to 283 kb,the total length of their annotated genes ranged from 28 kb to 283 kb,and the GC content ranged from 45.5% to 52.26%.HQG8,HGQ27 and HQG29 were demonstrated as 99.98% homology with the gene of Shigella flexneri C32(100% gene covered),and also high homology with the other species of Escherichia coli.HQG41 was demonstrated as 97.14% homology with the gene of avianinfluenza Salmonella(80% gene covered).Based on the whole genome comparison between HQG27 and HQG41,two stains of plague bacteriophage showed the 95.98% homology(74% gene covered).Based on the comparison of the gene sequence between the obtained plague bacteriophage in this research and 14 strains of bacteriophages published in NCBI database,HQG29 is in same cluster with phage L-413 C,showing the relatively close consanguinity,and HGQ8 has the close consanguinity with phage Ypsp-G,and HGQ27 is closely consanguine with phage Ypf,then HQG41 is in same cluster with Yep-Phi and close consanguinity.ConclusionThe existence of a certain amount of the plague bacteriophages in the indicative animal ’dogs’ of Yunnan Heqin new Plague epidemic foci was confirmed by this research,the discovery of which provided the theoretical basis for researchingplague preservation mechanism and micro-ecology.By the analysis of their biological characteristics,host range and whole genome analysis,the foundation can be drawn theoretically to realize the interaction between the plague bacteriophages and its host.