Biological Characterization Of A Bacteriophage Of Klebsiella Pneumonia And Screening Of Resistant Strains To The Phage

Klebsiella pneumonia is an important species producing1,3-propanediol from glycerol. However, phage infection could seriously influence cell growth and metabolism, which affected the production and the quality of products and caused great economic losses to the fermentation industry. The previous reports about phage infection in fermentation mainly focused on dairy and a few bio-based chemicals. To date there are not any reports about phage infection in the fermentation of1,3-propanediol. This paper will study the phage that caused abnormal fermentation by Klebsiella pneumonia. The preventive measures from phage infection were also discussed. The resistant strains to the phage would be screened by plasma.Firstly, lysogenic phages were identified by mitomycin C induction. The sensitive indicator bacterium was selected. A K. pneumoniae phage was isolated by using Adams double plate method from the infected fermentation broth of1,3-propanediol. Phage titer could get1011PFU/mL after fission. Its insensitivity to chloroform showed that it has no coat and there is no lipid in capsid. Electron microscope observation showed that the phage has an isometric polyhedral head (about60-70nm in diameter) and a long noncontractile tail (about160nm long). The genome of the phage was extracted and cut off by dsDNA restriction enzyme EcoR1and HindⅢ. The number of enzyme digested fragments is three and seven respcectively. Its complete size is about42kb.Secondly, the physiological characteristics of the phage were tested. The optimal multiplicity of infection was1, the one-step growth curves showed the latent phase and rise phase were both50min, and the burst size was343. The optimal growth temperature was37℃and the phages were died under70℃for60min. It could survive in wide range of pH but be sensitive to UV. There were basically no surviving phages after12min UV radiation.Thirdly, the phage infected fermentation was carried out and compared with normal fermentation. It showed that cell growth was delayed about8h and the highest bacteria concentration decreased by33%. The metabolic flow tended to the lactic acid and other organic acids. The effects of Ca2+, three kinds of surfactants, and different concentrations of ampicillin were also investigated to restrain and control the phage infection. The results showed that the three methods had no effective role on the phage restraining and controlling. Finally, a resistant strain was selected by spontaneous mutation. Although no bacterial growth stagnation occurred in fermentation, the metabolism was similar to the infected group. Based on the complex treatment of plasma and LiCl. a mutant strain of K pneumoniae was isolated. The concentration of1.3-PD was increased by13.1%. It was proved to be a simple and effective method for obtaining phage-resistant mutants.