Isolation,Identification And Biological Characteristics Of Klebsiella Pneumoniae From Giant Panda

Klebsiella pneumoniae（KP）is a common conditional pathogen.It belongs to Enterobacteriaceae.It is a gram-negative bacterium.It is widely distributed in human and animal intestines and natural environment.It is a pathogen of zoonosis.According to relevant documents,the infection of Klebsiella pneumoniae is second only to Escherichia coli and Pseudomonas aeruginosa.Klebsiella pneumoniae can infect the respiratory system,digestive system,urinary system and blood circulation system of humans,domestic livestock and poultry（pigs,cattle,chickens,etc.）and wild animals（giant pandas,minks,pangolins,red pandas,black and white colobus monkeys,etc.）,It is a common zoonotic pathogen.So far,there are few studies on the biological characteristics of Klebsiella pneumoniae from giant pandas at home and abroad.In this study,some healthy giant panda feces collected by Chengdu Giant Panda Breeding Research Base were isolated and identified,and the isolated Klebsiella pneumoniae was identified by morphology,16S r RNA and specific primer khe gene biology.Further study the selected biological characteristics of Klebsiella pneumoniae from giant panda,such as high viscosity phenotype,virulence gene,drug resistance phenotype and pathogenicity,in order to provide experimental data and reference basis for clinical prevention and control of the transmission of such bacteria in giant panda.The results are as follows:1.Isolation and identification of Klebsiella pneumoniae from giant pandaIn this study,the fecal samples of some healthy giant pandas collected by Chengdu Research Base of giant panda breeding were isolated by plate scribing method.Then 46strains of Klebsiella pneumoniae were identified according to the colony morphology,Gram staining microscopic examination results,16S r RNA and hemolysin（khe）gene PCR amplification of Klebsiella pneumoniae.It provides materials for further research on Klebsiella pneumoniae from giant panda.2.Study on some biological characteristics of Klebsiella pneumoniae from giant panda46 strains of Klebsiella pneumoniae isolated and identified were stained for capsule,and the high viscosity phenotype,virulence gene,biofilm forming ability and drug resistance phenotype were studied.The results of capsule staining showed that 46 strains of Klebsiella pneumoniae could form capsule.The results of high viscosity phenotype showed that 12 of the 46 strains of Klebsiella pneumoniae were high viscosity Klebsiella pneumoniae,and the other 34 strains were non high viscosity Klebsiella pneumoniae.The 46 strains were multidrug resistant.Among them,ampicillin,vancomycin and oxacillin were 100%resistant,and some cephalosporins,doxycycline,compound xinnomine and trimethoprim were resistant to varying degrees.The detection rates of rmp A、mag A、fim H、mak D、wab G、ug E、ybt A、ure A、iuc A、alls、iut A、ent B、aero、iuc B、kfu of Klebsiella pneumoniae were 2.17%（1/46）,2.17%（1/46）,67.39%（31/46）,84.78%（39/46）,82.61%（38/46）,80.44%（37/46）,17.39%（8/46）,91.30%（42/46）,2.17%（1/46）,36.96%（17/46）,0%（0/46）,63.04%（29/46）,0%（0/46）,respectively 2.17%（1/46）、36.96%（17/46）;Among them,the highest detection rate was ure A gene,which was 91.30%（42/46）,and the detection rate of rmp A,mag A,iuc A and iuc B was 2.17%（1/46）.It existed in one isolated strain,and iut A and aero were not detected.One isolate carried 13 virulence genes,31isolates carried 5～7 virulence genes,and 3 isolates did not detect any virulence genes.The biofilm forming ability results showed that among the 46 strains of Klebsiella pneumoniae,13 strains of Klebsiella pneumoniae were isolated with medium strength biofilm,17 strains of Klebsiella pneumoniae had weak biofilm forming ability,but 16 strains of Klebsiella pneumoniae did not form biofilm.This study identified the main biological characteristics of Klebsiella pneumoniae from giant panda.3.Study on pathogenicity of Klebsiella pneumoniae from giant pandaTaking the main biological characteristics related to pathogenicity of Klebsiella pneumoniae,whether it has mucus,biofilm forming ability,whether it has capsule,drug resistance and the carrying of virulence genes as the preliminary judgment criteria of pathogenic strains,select a strain pneumoniae-x-5 with mucus,capsule,multiple drug resistance,strong biofilm forming ability and carrying up to 13 virulence genes at the same time,To study its pathogenicity to Kunming mice.By measuring the LD₅₀of the strain on Kunming mice and analyzing the pathological changes of tissues and organs of infected dead mice.Based on the main pathogenicity related biological characteristics of Klebsiella pneumoniae,whether it has viscosity,biofilm forming ability,whether it has capsule,drug resistance and the carrying of virulence genes,a strain with viscosity,capsule,multiple drug resistance,strong biofilm forming ability and carrying up to 13 virulence genes was selected to study its pathogenicity to Kunming mice.The LD₅₀ of the strain to Kunming mice was determined,and the pathological changes of tissues and organs of infected dead mice were analyzed.Kunming mice were infected with test strain pneumoniae-X-5 and Klebsiella pneumoniae reference strain ATCC700603 by intraperitoneal injection in the same way and in different dose groups with the same number of bacteria.Results within 3～6 hours after the challenge,the mice in the test strain group and the reference strain ATCC700603 group began to suffer from mental depression,lying prone in the cage,unwilling to move,disordered and lusterless hair,abdominal breathing,half open eyes and no light in the eyes.Some mice had symptoms such as exudate and abdominal diarrhea in the eyes,and their intake of food and water decreased significantly.Mice in high dose group(7×10⁸ CFU～7×10¹⁰ CFU)began to die in succession after 5 hours of challenge.The dead mice were found to have swelling of lungs,liver and spleen with bleeding after autopsy.The bleeding of spleen was obvious,the stomach was inflated,the content of intestinal tract was thin,the wall of small intestine became thin,and the intestinal tract was bleeding.The mice in the control group of Klebsiella pneumoniae reference strain ATCC700603 died after 12 hours of challenge,but compared with the mice challenged by the test strain,the clinical symptoms were lighter and the course of disease was slower.According to the modified Coriolis method,the median lethal dose of the test strain to mice was 1.7×10⁴ CFU/m L.The results showed that the tested strain pneumoniae-X-5 had strong pathogenicity to mice.The liver,spleen and lung sections of the dead mice were observed after he staining.A small amount of inflammatory cells infiltrated in the lungs of the test strain group and the alveolar septum thickened;The hepatocytes in the test strain group and the standard positive control group were all degenerated,necrotic and disintegrated;Splenic hyperemia;The duodenal mucosa epithelium and lamina propria of the test strain group were separated,and some cells of lamina propria of the intestinal mucosa were necrotic;In the standard positive control group,the central chylous duct was slightly dilated.This study confirmed the strong pathogenicity of Klebsiella pneumoniae from giant pandas with obvious biological characteristics and carrying a variety of virulence genes,suggesting that attention should be paid to the prevention and control of Klebsiella pneumoniae in rare animals such as giant pandas.