Preliminary Research On Isolation And Biological Characteristics Of A Lytic Bacteriophage Against Enterobacter Cloacae

The bacteriophages are viruses of bacteria, and this kind of virus widelydistributes in nature. The restrict specificity and high efficient cleavage to hostbacterium make them became new antimicrobial agents. Enterobacter cloacae areGram-negative bacilli, and Enterobacter cloacae are common pathogen of sepsis,respiratory tract infections, urinary tract infections, peritonitis and other diseases. Inrecent years, due to the their production of β-lactamase enzymes, Enterobactercloacae show high resistance rate to antibotics such as quinolones andaminoglycoside, which make the treatment of Enterobacter cloacae very difficult. Butthe study on phages of Enterobacter cloacae has not been carried out in-depth athome and abroad.In this study,4phages of Enterobacter cloacae were isolated from living sewage,and all of them were purified repeatedly by a plaque method. Among of them, a strongtoxic and lytic one named as ФEc53was identified of biological characteristics, and itwas used to treat Enterobacter cloacae infected animal.Biological characteristics of the bacteriophage ФEc53ФEc53induces a round, tansparent plaque with clear boundary in dourble agarafter3to5purifications, and the plques are approximately2~4mm in diameter.Under the electroscope, ФEc53has a spherical head and a long tail, and the head isabout60nm in diameter and the tail is about130nm in length. According to newclassification rules, ФEc53belongs to Candovirales, Siphoviridae. The genome ofФEc53is a double strander DNA, and the length of genome is about37.7kbcontaining many restriced endonuclease recognizing sites. The SDS-PAGE of ФEc53shows that the bacteriophage particles containing at least eight kinds of capsid protein, among of them, and a22kD capsid protein has a high proportion.The bacteria are infected by ФEc53at MOI0.1,0.01,0.001,0.0001,0.00001under37°C shaking culture, and the MOI0.01has the most efficient lysis to hostbacteria. The MOI0.01was used to perform single step growth of ФEc53. A triphisiccurve, including the latent phase, rise phase, and plateau phase, was obtained. Thelatent time is5minutes, and the burst size of phage ФEc53is6.06×103PFU/cell. Theplateau phase reaches in50minutes.The concentration of phage can be increased and the maximum of endotoxincan be removed by ultrafiltration. The ФEc53has a narrow host range, and just onestrain of Enterobacter cloacae susceptible to it. And the ФEc53can be stored at4℃and-70℃for a few months without biological characteristics changing.Bacteriophage treatment experiments in vitroThe rabbits were infected by Enterobacter cloacae53by subcutaneousinjection, and the ФEc53was injected into the same part of skin immediately afterbacteria infection. Then the development of abscess was observed, and bacteria andphage in liver and spleen were tested. Accompanying with the increasing phage titerand decreasing titer of bacteria, the area of abscess of phage therapy group decreasesgradually.In summary, we isolated and identified a bacteriophage ФEc53, and thebiological characteristics and its anti-infection treatment will provide some clues forfuture in-depth study of the phage.