Preliminary Research Of Identification And Biological Characteristics Of Klebsiella Pneumoniae Phages

Background and Object:Bacteriophages constitute a group of viruses with the ability to invade various bacterial species as their specific hosts. Moreover, bacteriophages are universally observed in open and coastal waters, marine sediments, terrestrial ecosystems such as soil, and the bodies of humans, animals and insects. Once a bacteriophage has infected the host cell, the resultant infection may be either lytic, reprogramming and soon destroying the infected cell, or lysogenic, where the bacteriophage genome is integrated into the bacterial genome and passed on to further generations of bacteria.Klebsilla pneumoniae, a gram-negative bacteriod, is a member of family of Enterbactericea. It has been shown that half a century of global antibiotics abuse has equipped the surviving bacteria with"supergenes"that enable them to quickly resist new classes of antibiotics, even those to which they have never been exposed. As a consequence, in recent years, by the time newer antibiotics have gone through clinical trails and have reached the marked, 20% or more of clinical isolates, including Klebsilla pneumoniae, in the hospitals are already resistant to them at the time of regulatory approval, and within a few more years the majority of strains are resistant. In this study, we have isolated two strains of phage direct against Klebsilla pneumoniae from swage using plaque forming test and the biological properties of two strains of phage were analyzed. These basically experimental achievements support the view that bacteriophages could be useful in the treatment of human infections particularly those caused by antibiotic-resistant strains of bacteria.Methods:1. Isolation and Identification of phages direct against Klebsilla pneumoniae:CaCl2 were added to 1000 ml of swage to a final concentration at 1 mmol/L, after centrifugation, the supernatant liquid were mixed with 16 strains of Klebsilla pneumoniae at 37℃condition for 16-18 hours. Semi-solid medium were selected for observation of plaque formation.2. Detection of Crossing test for screen host range:Phages were mixed with 16 trains of Klebsilla pneumoniae, respectively, then plaques were checked and host range was identified.3. Extract of phage genome DNA and analysis of electrophoresis:After destroying host cells, bacterial DNA must be digested by DNase first. The structural proteins of phage were lysed by protease, and extraction of phage DNA were carried out. Extracted DNA were digested with restrict enzyme, the digested DNA were analyzed on electrophoresis.4. Electro microscope of phages:Isolated phages were under observation using electro microscope to find out the their shape, size and so on.5. Detection of multiplicity of infection (MOI):Solution containing phages were diluted by serious steps, and diluted phages were mixed with host bacteria. MOI were identified according to the amount of plaques.6. Growth curve of phages:On the basis of MOI, after interaction among phages and bacteria, growth curve of phages were drawn at different times.7. Lytic activity of isolated phages:After phages infected host cells, the number of host bacteria were counted at different time to estimated lytic activity of phages.8. Sensitivity of phages to physical agents:(1) sensitivity to pH value(2) sensitivity to temperature(3) sensitivity to ultravioletResults and DiscussionIn this study, we have isolated two strains of Klebsilla pneumoniae phages, K1BP108 and K1BP109, from swage. Some biological properties of the isolated phages were analyzed and described. Through plaque test, the titer of these phages is 4.9×1011PFU/ml and 4.9×1011PFU/ml. It suggested that a relatively purified phages was obtained. Phages tend to have a relatively narrow host range, posing certain disadvantages. Therefore, we selected crossing test to screening phage, which exhibits a wide host range direct to different serotypes of Klebsilla pneumoniae. The results showed that phage K1BP108 may lyse 4 strains of host bacteria among 16 strains of Klebsilla pneumoniae. Under the observation of electro microscope, these phages have a head with a cubic symmetry and a tail with a filamentous form. Electrophoresis illustrated that the size of DNA from phage K1BP108 is about 25~30kb, and genome of phage K1BP 108 may be digested with BamHI, EcoRI, HindIII and XbaI. Multiplicity of infection (MIO) reflects efficacy in which phage infect and lyse host cells. In this experiment, the MIO of phage K1BP108 is 0.1. When MIO is 10, growth curve of phage K1BP108 was drawn, from which the latent phase is about 23 minutes, the burst phase is about 47 minutes, and the lytic amount is 90. The data involved in lytic activity of K1BP108 is potent, because the number of host cells decreased dramatically in 240 minutes. The phage K1BP108 is stable between pH 6~pH 10, and activity of K1BP108 may lost at 80°C. The phage K1Bp108 can be inactivated by ultraviolet within 12~18 minutes. These data support a possibility that we should make further research in genetic and molecular scope of phages.