Isolation And Identification Of Virulent And Temperate Bacteriophage Of Streptococcus Suis Serotype 2

Streptococcus suis(SS) is an important pathogen of human and animal. Thirty-five capsular serotypes or S.suis have been described(types 1/2 and 1 through 34).Streptococcus suis type 2(SS2) is considered to be the most-virulent serotype and prevalenced in wordwide.In China,SS2 was first isolated in 1991,later,human Streptococcus suis infection in Jiangsu province and Sichuan Province happened in 1998 and 2005,respectively.Study on the phages of SS2 would play a crucial role in the research on the ecology and evolution of such Gram-positive bacteria.However, very little is known of the phages of SS2 for that.only one paper about the temperate phage of SS2 has been published by far.A SS2 bacteriophage,named SMP,was isolated from nasal swabs of healthy Bama minipigs and was characterized at the microbiological and molecular level. Phage SMP had an isometric head of 50 nm,a noncontractile tail of approximately 135 nm.The host range of phage SMP was limited to 2 of 24 SS2 strains tested.The activity of SMP declined dramatically when heated above 40℃and stable at pH6-10. SMP nucleic acid belonged to double-stranded DNA and could be digested with BglⅡ, EcoRⅠ,NcoⅠ,BamHⅠand PstⅠbut not with NotⅠ.One-step growth curve for SMP showed a latent period of about 20min,a rise period of 120min,and an average burst size of about 77 PFU/cell.SS2 virulent phage SMP particles were purified with glycerol gradient centrifugation,and its genomic DNA was extracted in order to construct the shot-gun library.The randomly picked clones under went genome sequencing.The sequence was reconstructed,and then analyzed for the genome ends with restrictive endonuclease BamHⅠspectrum under denaturing Pannealing conditions.ORFs were predicted and phylogeny of phage-associated gene sequences was analyzed to determine the class of bacteriophage SMP.The genome of phage SMP is a 36216bp of linear double-stranded DNA.The base constituents of the genome were A= 31.72%[11487];G=23.24%[8418];T=26.67%[9659];C=18.37%[6652]; Ambiguous=[0];A+T=58.39%[21146];C+G=41.61%[15070]respectively.A total of 169 open reading frames(ORFs),of larger than 150 bp and 48 coding sequences, were detected in the phage SMP genome.A search using BlastP identified some sequences of high homology,including head protein,terminase large subunit,putative replication protein,single strand binding protein and major tail protein.The genome size of Siphophages range mostly from 22 to 121 kb and the study of molecular characteristics of phage SMP suggested that it should to be a member of the Siphoviridae family.To study the distributional characteristics of phage integrase gene in SS2,a PCR assay was developed and a 323bp distinct DNA target can be amplified in 25 strains of virulent SS2,while can not be amplified in avirulent strain T15,5 strains of other serotypes(SS1,SS7,SS9) and strains of group C streptococcus strains from pigs. The sequencing and restriction endonuclease analysis of the PCR products were also done.Complete cell lysis was observed with SS2 virulent strains but not with avirulent strain T15.Electron microscopy analysis of the lysate from SS2 virulent strains HA9801 and ZY05719 revealed the presence of phage particles.The induced phage has a small isometric nucleocapsid approximately 50 nm in diameter and has no tail and is therefore a member of the Tectiviridae family.The temperate phage and phage integrase gene can only detected from SS2 virulent strains but not from avirulent strain,and the detection of phage integrase gene was related to the virulence-associate factors of SS2,such as the muramidase-released protein gene (mrp),which suggested that the temperate phage of SS2 may be related to the pathogenicity of SS2.