Studies On Phages Against E.coli O157:H7 And Salmonella Spp.and Their Endolysin And Holin

Escherichia coli O157:H7 and Salmonella spp.are major foodborne pathogenic bacteria,which cause severe threat on public health and safety.The two bacteria can generate cross-adaptation induced by stresses in food processing and storage,so they can survive in subsequent sterilization or antibacterial treatments.Moreover,an increasing number of bacteria appear resistance due to the long-term and extensive use of antibiotics.With the increasing demand for food production without use of chemical preservatives,phage therapy as a natural biological control method provides a new option for disinfection and sterilization in food industry.However,phage therapy has some shortcomings in application.The proliferation of phage depends on the growth rate of host bacteria and does not occur in non-growing host cells.So,the key point for application of phage therapy is determined by optimal time and dose of phage use.In addition,pathogenic bacteria easily develop resistance to phage and appear phage insensitive mutants(PIMs).The emergence of PIMs and high specificity of phage also hinder the application of phage,which can be solved by preparation of phage cocktail.However,as the functional proteins of phage lysis system,phage endolysin and holin have the advantages of rapid action,wider lysis spectrum than parent phage,no cytotoxicity,low possibility of bacteria to generate resistance,synergy with existing antibiotics and so on.Therefore,endolysin and holin can solve problems of phage therapy in practical application,and have potential to become new antibacterial agent.In this article,we used E.coli O157:H7 and Salmonella spp.as host bacteria to isolate phages and evaluate antibacterial potential of phage.The biological information and antibacterial potential of endolysin and holin of EC-p9 and SM-p2 were also investigated.The main results are listed as follows:(1)Thirteen phages were isolated in Jialing river,Chongqing.Nine phages against E.coli O157:H7 were named as EC-p1?EC-p2?EC-p3?EC-p4?EC-p5?EC-p6?EC-p7?EC-p8 and EC-p9.Four phages against Salmonella spp.were named as SM-p1?SM-p2?SM-p3 and SM-p4.All phages had different latent periods and burst sizes.EC-p9(10 min,333.33 PFU/cell)and SM-p2(10 min,119.56 PFU/cell)had the shortest latent period and the largest burst size among phages.The two phages also had high specificity.In addition,EC-p4,EC-p6,EC-p9,SM-p2 and SM-p4 had high stability of temperature and p H,and inhibited growth of planktonic cells completely at least for 10 h.Therefore,the five phages were selected to control the biofilm of host bacteria.The results showed that these phages inhibited biofilm formation and disintegrated formed-biofilm of host bacteria on stainless steels.EC-p9 and SM-p2 had the greatest ability to control biofilm of host bacteria.(2)EC-p9 and SM-p2 were identified by TEM and random primer amplification,encoding genes of endolysin and holin of the two phages were obtained by PCR,physicochemical properties of endolysin and holin were predicted by molecular biology techniques,and four recombinant proteins were expressed by E.coli expression system.The results showed that the two phages belong to ds DNA Myoviridae.Endolysin and holin of EC-p9 or SM-p2 were named as Lys 9 and Hol 9or Lys 2 and Hol 2,respectively.Lys 9 and Lys 2 with spherical structures were N-acetylmuramic acid enzymes.Lys 9 and Lys 2 had a N-terminal enzyme activity domain which belonged to the Muraidase family,and also had a binding domain which belonged to the PG?binding?1 family.Hol 9 and Hol 2 with a transmembrane structure were typical type III holin and belonged to Bacteriophage T holin family.Lys 9,Lys 2,Hol 9 and Hol 2 were water-soluble proteins.After expressing these recombinant proteins under optimum expression condition and purifying them by nickel affinity column,concentrations of Lys 9?Lys 2?Hol 9 and Hol 2 were 5.331 mg/m L,5.419mg/m L,3.145 mg/m L and 2.191 mg/m L,respectively.In addtion,the expression of Hol 9 and Hol 2 had toxic effect on BL21(DE3).(3)The function of endolysin and holin were researched.Lys 9,Hol 9,Lys 2 and Hol 2 had good antibacterial activity.The four recombinant proteins had broad-spectrum antibacterial activity against variety of gram-positive and gram-negative bacteria,which were far wider than the lytic spectrum of parent phage.The lytic activity of Lys 9,Hol 9,Lys 2 and Hol 2 maintained stable after 30,20,40 and 30 min,respectively.The highest lytic activity of four recombinant protein were61.348%,81.120%,74.251% and 83.356%,respectively.Moreover,these proteins have high lytic activity after treatment at 4,25 and 37 ? for 15 min and even can lysis bacteria after treatment at 50 ? for 15 min.All recombinant proteins showed the highest lytic activity under neutral conditions and had high stability at p H 4-10.Furthermore,EDTA can significantly increase lytic activity of Lys 9 and Lys 2(P <0.05).E.coli O157:H7 and Salmonella spp.which treated by the four recombinant proteins did not appear resistance.The above results showed that Lys 9,Hol 9,Lys 2and Hol 2 had broad application prospects.