Isolation, Biological Characterization And Comparative Genome Analysis Of Staphylococcus Aureus Phages

Bovine mastitis is a global problem leading to huge financial losses to dairy industry.Staphylococcus aureus is one of the most important pathogens causing mastitis.Preventive strategies such as optimization of milking practices,culling,postmilking teat disinfection and antibiotic therapy are being applied;however,the prevalence of subclinical and chronic mastitis is still high.Bacteriophage are specific bacterial viruses that attach to their hosts and kill them by internal replication and bacterial lysis.Phages attack only their target bacterial hosts,and they cannot infect human or other eukaryotic cells.Discovery of bacteriophages suggested their application against bacterial infections of humans and animals or as an antiseptic material.Virulence factor genes encoded by temperate phage are not strictly associated to a specific phage and appear to be exchanged by horizontal gene transfer and recombination.So the temperate phage played an important role in the evolution of bacteria virulence.In this study,two strains of Staphylococcus bacteriophages were isolated and characterized from the milk of mastitis,named JS01 and WH01.We have investigated their morphological,physiological and molecular characteristics.The JS01 has a long non-contractile tail and an icosahedral head,which was assigned to the family Siphovirus in the order Caudovirales.While WH01 belongs to family Myoviridae with a short contractile tail and an icosahedral head.Phage JS01 could lyse a part of tested bovine S.aureus.The host range of WH01 included different S.aureus isolations from different hosts(bovine,monkey and duck)and other Staphylococcus(S.horomogenes and S.saprophyticus).It might have an application value in controlling S.aureus infection.Both of the phages were sensitive to high temperature and low pH.The MOI of both two phages was 1.JS01 had latent period of 10 mins,growing period of 30 mins and average burst size of 78 PFU/cell.WH01 had latent period of 60 mins,growing period of 20 mins and average burst size of 99 PFU/cell.To get thorough knowledge of phage JS01’s genetic background,we purified phage JS01 by glycerol gradient centrifugation and extracted its DNA.And then,we sequenced its genome using a 454 GS-FLX Titanium sequencing system.The JS01 genome was 43,458 bp long with a G+C content of 33.32%,66 open reading frames(ORFs)and no tRNA.34 ORFs are hypothetical or unknown protein due to insufficient database information about the functional genes of Staphylococcus aureus phage genomes.Annotation and functional analysis of the predicted ORFs revealed six functional modules as follow:structure and morphology,DNA replication and regulation,packaging,lysogeny,lysis and pathogenicity.Comparative analysis with the genomes of other Staphylophage and S.aureus revealed that JSO1 had high nucleotide identity with S.aureus MSSA476 and S.aureus prophage PVL.JSO1 encode two virulence factors which may impact the virulence of host bacteria by positive lysogenic conversion,since they provide novel functions and activities to the host.Autolysin encoded by ORF 7 is an enzyme that destroys the cell walls of bacteria.Autolysin could be an alternative to conventional antibiotic therapy against pathogens that are resistant to multiple antibiotics.We tried different expression vectors of prokaryotic expression system and failed to obtain lys in the supernatant.Renaturation of the lys inclusion did not lytic the bacteria.Staphylokinase encoded by ORF8 is a virulent factor which possessed significantly thrombolytic effects.purified sak supernatant had clear thrombolytic effects in vitro.