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Isolation and Characterization and in Vitro Bactericidal of Bacteriophage for Klebsiella Pneumonia

Author: ZhangHongMing
Tutor: FuWeiLing
School: Third Military Medical University
Course: Clinical Laboratory Science
Keywords: Klebsiella pneumoniae phage isolation bactericidal biofilm
CLC: R446.5
Type: Master's thesis
Year: 2008
Downloads: 154
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Abstract

Klebsiella pneumoniae is a gram-negative rod-shaped bacteria, and clinically the most important member of the Klebsiella genus of Enterobacteriaceae. It is among the most common gram-negative bacteria encountered by physicians worldwide. It is a common hospital-acquired pathogen, causing urinary tract infections, nosocomial pneumonia, and intraabdominal infections. Klebsiella pneumoniae is also a potential community-acquired pathogen. It can cause pneumonia although it is more commonly implicated in hospital-acquired urinary tract and wound infections, particularly in people with weakened immune systems. It is an increasing problem on hospitals because of the problem of antibiotic resistant strains.Bacteriophage is bacterial virus, which created evolutionally intimate relationship with its host bacterium. Firstly, the lysogenic bacteriophages, can alter the biologic features of host bacterium by its inheritance material. Meanwhile, they can change host’s inheritance material constituent in the process of the DNA recombination, integration, transposition, and incision in host genome. As a result, bringing the microbe’s gene diversity. This kind of gene horizontal. Secondary, both lytic and lysogenic phages (the later could be induced into lytic growth cycle) having ability to lyses host bacteria, bacteriophage is in the hope to be developed as anti-bacteria pharmaceutics.Isolation of the new phage and described its basic biological characteristics is the primary task.In this study, bacteriophage of klebsiella pneumoniae was isolated from sewage by double-layer agar plate method, identified lytic or lysogenic capacity by method of ultraviolet induce and method of mitomycin C induce, performed one-step growth experiments, decided the optimal multiplicity of infection, and its structure was observed with electron microscope, at last, assessed its capability in vitro bactericidal. The major research and experimental results include the following:1. To isolate the bacteriophage of Klebsiella pneumoniae, a clinical isolates of K. pneumoniae was used. the bacteriophage was isolated from the sewage and was named KPNP1. Its plaque was transparent and 4～7 mm in diameter, its titer is 6.1×1011PFU/ml. KPNP1’s electron microscope results showed that it has a polyhedron-dimensional symmetric head, and the head is about 180 nm in diameter, no envelope, with a long tail about 210 nm at length and 30nm in width, indicating that the isolated bacteriophage belongs to Stylovinidae.2. The multiplicity of infection (MOI) is the ratio of the number of the phage to the number of the host bacteria, also called multiple infections. The optimal multiplicity of infection is the best MOI ,which can produce best results. In this study, the optimal multiplicity of infection of KPNP1 was 4.3. One step growth-experiments showed that: when incubated at 37℃, after 15 min, the adsorption rate was 72%. The latent period of infection was 35 min and burst period was 40min, the average burst size was about 94 PFU/cell.4. Mitomycin-C and UV induced bacteria cannot format the plaque, suggesting that KPNP1 is not lysogenic phage, but lytic phage.5. KPNP1 has relatively narrow host spectrum, only sensitive to a few (1/30) Klebsiella pneumoniae, also sensitive to some of Escherichia coli.6. Bactericidal experiments in vitro showed that KPNP1 has a obvious effect on KPN suspension, when the phage concentration is 1.8×1010PFU/ml, react with KPN suspension 100 minutes later, the killing efficiency was 81.1%; when the phage concentration is 1.8×109PFU/ml, react with KPN suspension 100 minutes, the killing efficiency was 82.3%. Low concentration of phage solution can obtain the same bactericidal effect when extended the reaction time.7. Surgery commonly used silicone rubber urinary catheter was used is this experiments, we successfully constructed the biofilm on the surface of the catheter, and handled the biofilm with KPNP1 2 hours, the results showed that: KPNP1 plays a obvious role on KPN biofilm, the efficient bactericidal rate is 71.8%. The electron microscopy results also showed that before treating with KPNP1, the bacterium grows flourishing, and agglomerate on the surface of the catheter, but when treated with KPNP1 for two hours, the bacterium on the surface of catheter decreased obviously, and agglomerate growth was missing.

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