Identification Of Bacteriophages Of Serratia Marcescens And Efficacy Of Phage Therapy Against Serratia Marcescens Infection In Mice

Serratia marcescens, a member of Enterobacteriaceae family, is found in water, soil andhospital settings. S. marcescens is an important causative agent of nosocomial infections,especially in immunocompromised individuals, severe burn patients, transplant recipients andcancer patients, including urinary tract infection, respiratory tract infection, wound infection,bacteremia, endotoxin shock and endocarditis. In recent years, infections and outbreaks ofmultidrug-resistant S. marcescens in neonatal intensive care units (NICU) have been reported,an important warning for medical workers. On one hand infections of drug-resistant S.marcescens or other pathogenic bacteria make chemotherapy treatment of diseases caused bythese microorganisms difficult, on other hand it also increased the financial burden of patients.Bacteriophages are viruses specifically infecting bacteria and distribute widely in naturalenvironment. Recently, there have been renewed interests in the researches and applications ofbacteriophages as antibacterial agent, due to their specificity in targeting and lysing hostbacteria. On the basis of current data, there is less information involved in researches on S.marcescens phages. therefore, a comprehensive and thorough insight into both biologicalcharacteristics of S. marcescens phages and phage therapy against bacterial infections has animportantly realistic significance.In this paper, ten clinical strains of S. marcescens were used for phage isolation, and sevenbacteriophages of S. marcescens (designed ФSM41Y、ФSM254Y、ФSM23Y、ФSM13Y、ФSM01Y、ФSM9-3Y and ФSM22Y, respectively) were isolated from hospital sewage usingdouble-agar method. Transmission electron microscopy revealed that these bacteriophagescould be classified as a member of the Myoviridae family and the Caudovirale order. A furtherinvestigation on a virulent phage ФSM9-3Y was carried out, and its biological features isfollowing:(1) Phage ФSM9-3Y had an icosahedral head of70nm in diameter, a tail with50nm in length;(2) Restriction analysis indicated phage ФSM9-3Y was dsDNA with anapproximate genome size of54kb, and its genomic DNA could be cut by EcoRV、BamHI、SacII and NdeI;(3) Lytic test showed that phage ФSM9-3Y exhibited a relatively narrow host ranges (only3of10host cells lysed by ФSM9-3Y);(4) Multiplicity of infection of phageФSM9-3Y was1(MOI=1);(5) One-step experiment demonstrated that ФSM9-3Y had a latentperiod of30min, a burst period of nearly40min and a burst size of the629PFU/cell;(6)SDS-PAGE analysis exhibited13protein bands on gel, with molecular weights ranging from25to130kDa, and the most abundant one with molecular weight of48kDa was most likelythe major capsid protein of ФSM9-3Y.On the basis of investigation of biological characteristics of phage ФSM9-3Y, a murinemodel of bacteremia was induced by intraperitoneal administration of S. marcescens inBALB/c mice. Intraperitoneal administration of a single dose of ФSM9-3Y (1×109PFU/mL)at20min,40min and60min after S. marcescens infection was able to protect mice from death(survival rate:100%,60%and60%, respectively), providing a significant protection.Pharmacokinetics showed that the titer of ФSM9-3Y in blood was9.6×104PFU/mL at24hand6.5×104PFU/mL at48h, respectively, after phage ФSM9-3Y was alone administeredintraperitoneally. However, the titer of ФSM9-3Y in blood was sustained a level of6.9×106PFU/mL at24h and a level of7×105PFU/mL at48h, respectively, after both ФSM9-3Y(109PFU/mL) and S. marcescens (108CFU/mL) were administered simultaneously. Surveys ofbacteria counts for mice treated with both ФSM9-3Y and S. marcescens by the intraperitonealroute revealed that the bacteria were eliminated effectively from blood.The seven separated phages of S. marcescens are virulent phage which has the pyrolysischaracteristics of the host. Its morphological characteristics conform that these bacteriophagescould be classified as a member of the Myoviridae family and the Caudovirale order.Biological characteristics analysis showed that ФSM9-3Y had a latent period of30min, aburst period of nearly40min and a burst size of the629PFU/cell. Multiplicity of infection ofphage ФSM9-3Y was1(MOI=1). Phage ФSM9-3Y was dsDNA with an approximate genomesize of54kb. BALB/c mice by intraperitoneal infection MLD of S. marcescens, giving phage(Ф SM9-3Y) treatment, can obviously increase the animal survival rate. Pharmacokineticshows: Ф SM9-3Y with the host bacterium24h after the injection of animals at the same time,the phage drops is higher than that of single injection of phage drops degrees.Based on our researches, a conclusion may be drawn as followings:（1）The seven phages of S. marcescens belong to virulent bacteriophages capable oflysing host cells; their morphological characteristics coincide with these bacteriophages being classified as a member of the Myoviridae family, the Caudovirale order.（2）Analysis of biological characteristics showed that ФSM9-3Y had a latent period of30min, a burst period of nearly40min and a burst size of the629PFU/cell. Multiplicity ofinfection of phage ФSM9-3Y was1(MOI=1). Phage ФSM9-3Y was dsDNA with anapproximate genome size of54kb.（3）Phage treatment for infection caused by S. marcescens may significantly improvedsurvival of BALB/c mice.（4）Pharmacokinetics showed that the phage titers in the circulation was higher at24hafter the injection of bacteriophages mixed with host cells in comparison with that of singleinjection of phage.