Isolation And Identification Of Klebsiella Pneumoniae And Study On Pathogenicity To Mice

Klebsiella pneumoniae(Klebsiella pneumonia, K. pneumonia, KP) is a conditioned pathogen, and can be found in normal surroundings. It can cause acute zoonotic diseases, which often occurs in pigs, cattle, sheep, chickens, ducks, geese, mink, panda, and other livestock, poultry and wildlife. It is also one of the major pathogens causing specific pathogen-free animals (SPF) contamination. Infection with KP may usually intefere the experiment on rat and mice, or even cause animal death, especially when they are in bad immune situation or in stress (poor surroundings, being tested).The national standard (GB14926-2001) provides that SPF Laboratory rat and mice must be detected to exclude KP infection. Conventional tests need several steps (eg, sample collection, culture, staining, biochemical reactions) and several days to get a results. In order to meet the need of rapid and high-throughput detection, we design this study.This study combine microbiology, pathology, and polymerase chain reaction (PCR) to detect mice KP infection. By calculating the sensibility and specificity, and testing the clinical samples, we first establish a fast and accurate way to make gene diagnosis of KP, providing a new effective way for SPF laboratory mice microbiological quality control, and KP infection clinical diagnosis.According to the KP phoE gene sequence, we design a pair of specific primers. Using PCR techniques to amplify phoE gene fragment, we extract the DNA of KP, Streptococcus, Staphylococcus aureus, salmonella, Escherichia coli, Pasteurella and other non-KP strains for PCR amplification. The PCR product of KP possesses specific DNA fragment, but others not. This PCR system can detect107pgKP-DNA, and takes only4hours from DNA extration to electrophoresis. The detectable rate is2.44%(3/123). The concordance rate with bacteriological assay is99.19%.In this study,PCR test,biochemical assays were used to identify the isolated strain from mice which were suspicious of infected by Klebsiella pneumoniae, the identification result of isolated strain is Klebsiella pneumoniae.Then inoculate KPm0912and standard strains KP1300into ICR mice respectively. Then we study the animal model from clinical features, tissue changes, bacteriology and histopathology to investigate the pathogenicity. The median lethal dose (LD50) of KPm0912and KP1300for ICR mice are1.1×105CFU/ml and1.3×106CFU/ml respectively. The infected mice are found with subcutaneous abscess, intrathoracic jelly-like exudation, pulmonary congestion, edema, and swelling and congestion in liver, spleen and other organs with purulent exudate on the serosa side. In histopathological sections, a large number of inflammatory cells infiltration are visible in lung, liver, spleen.The above test results show that PCR method has a high specificity and sensitivity, it is also easy to operate, timesaving. Thus, it is recommended to quick disease diagnosis and epidemiological survey for SPF laboratory rat and mice monitoring. The image data which get from the histopathology,providing a theoretical basis for the research of the pathogenicity to mice.