Isolation And Biological Properties Identification Of The Bacteriophages For E.Coli K88and K99

The pathogenic E.coli has resulted in serious losses to the aquaculture industry in the world since the E.coli was first discovered. The E. coli K88and K99are the most widely serotypes in a large number of E. coli, and there is no effective drug to prevent and control them until now. Phage, a bacteria viruses, can specific lysis of the host bacteria, and this feature allows researchers to find new ideas and methods for the treatment of bacterial. Recent research shows that therapeutic phage preparations have the antibiotics incomparable advantages, such as a small dosage, efficacy, toxic side, fast effect and so on. In2007, the U.S. FDA approved the first phage preparations for E. coli0157:H7, which marks that the phage preparations entered a new stage of clinical research.In this test, double-layer agar plate was used to separate and purificate phage, and we has isolated a strong-lysis E.coli K88phage named as Bp8A0811and two strong-lysis E.coli K99phage named as Bp9B1226and Bp9C1213. The titers of Bp9B1226and Bp9C1213are above1010pfu/ml, and of Bp8A0811is109pfu/ml which a little lower than Bp9B1226and Bp9C1213. The plaque is relatively small at the beginning, but after further purification, the plaque significantly increased and enhanced the ability of lysis to the host bacteria. After determination of the spectrum and pyrolysis curve about three phage E.coli K88, E.coliK99and chicken E.coli, We found that Bp8A0811can not lysis E.coli K99and chicken E.Coli, Bp9B1226and Bp9C1213can lysis E.coli K99and K88. All the three phage can not lysis of chicken E. Coli, and the OD600value of three phage and bacteria mixture has shown a rising trend.In the biological characteristics of Bp9B1226, we found that its thermal is stable at60℃water bath for30min. However, when the water bath time is extended to60min its titer decreased rapidly.Bp9B1226titer in the range of pH6-9is relatively stable, and the titer will decreased obviously when the pH less than5and pH greater than10. And the alkaline solution easier to make Bp9B1226titer decreased. We also studied the different preservation methods on the phage and found that the phage with30%Glycerine stored at4℃Cor-80℃was better than at-20℃and room temperature, their titer in the six months decreased by2-3orders of magnitude. So we can store them with30%Glycerine at-20℃for3months, and at4℃Cor-80℃for6months.