Detection Test Of Serotype And Virulence Genes In Klebsiella Pneumoniae From Mink And Its Pathogenesis In Mice And Mink

Klebsiella pneumoniae(K.Pneumoniae),both a conditional pathogens that can cause a variety of animals and a pathogen of diseases in humans and animals.In this study,23 strains of K.Pneumoniae were stored in the laboratory for detection of serotypes and virulence genes by the reported polymerase chain reaction(PCR).The positive virulence gene amplification products of 15 strains were selected as the reference strains after sequencing in GenBank,and constructing the homology and system developmental trees of positive serotypes and virulence genes of K.Pneumoniae.The results showed,it was the first to identify serotype K2 ofK.pneumoniawas prevalent in mink in China,resulting in diseases.Animals experiments demonstrated that showed high virulence for mice and mink.Therefore,the etiology and epidemiological surveillance of K.pneumoniae pathogens in mink should be strengthened for people’s public health.As follows1.Detection of serotypesThe PCRs were performed to amplify serotype K1,K2,K5,K20,K54 and K57,which are those most associated with virulence in humans.The result showed that 22 of the 23 K.pneumoniae isolates,belonged to capsular serotype K2.However,K.pneumoniae-SD-15 was neither any of the above serotypes.The positive virulence gene amplification products of K.pneumoniae-SD-1 to K.pneumoniae-SD-13,K.pneumoniae-SD-15 and K.pneumoniae-SD-21 of 15 strains were selected and sequenced as the reference strains after sequencing in GenBank,and constructing the homology and system developmental trees of positive serotypes and virulence genes of K.Pneumoniae.The results showed that the serotype K2 genes showed 99.7-100% identity among the 14 isolates except K.pneumoniae-SD-15,strains 23,HK787,ATCC43816 and VGH525.Therefore,that K2 serotype,which was able to infect minks and was popular on mink in Shandong.2.Detection of virulence genesThe PCRs were performed to amplify 12 virulence genes,which was rmpA,aerobactin,wcaG,ybtA,iucB,iroNB,ureA,uge,kfuBC,fim,wcaG and allS.The results showed that thehighest rate of detection of ureA gene 100%(23/23);the rmpA,aerobactin,wabG and uge genes 96%(22/23);the IucB gene 87%(20/23)and the lowest rate of the detection of ybtA gene 13%(3/23).But the five genes,fim,iroNB,wcaG,alls and kfuBC,gave a negative PCR reaction in the 23 isolates.The positive virulence gene amplification products of 15 strains were selected and sequenced as the reference strains after sequencing in GenBank,and constructing the homology and system developmental trees of positive serotypes and virulence genes of K.Pneumoniae.14 strains of the positive rmpA gene in the 15 strains were closely related to the reference strain TMV-5(isolated from human organs).Fifteen strains of the positive gene were identified with the reference strain CG43(isolated from human 15)of the positive wabG gene representing the strain with the reference strain CG43(isolated from human blood),NTUH-K2044(isolated from human liver and blood),ED23(separated from human blood)12 strains of the positive IucB gene in the representative strain were closely related to the reference strain RJF999(isolated from human blood),T69(isolated from human sputum);15 strains representing 2 The positive ybtA gene is closely related to the reference strain NTUH-K2044(isolated from human liver and blood).The results showed that the wabG genes 98.4-100% among the 15 isolates,strains NTUH-K2044,KCTC2242,ED23,CAV1217 and CAV1042,the ureA genes 98.2-100% among the 15 isolates,CG43,RJF293,CAV1042 and U41,the rmpA gene 99.1-100% among the 14 isolates except K.pneumoniae-SD-15,NTUH-K2044,KCTC2242,ED23 and RJF293,the aerobactin gene 98.2-100% among the 14 isolates except K.pneumoniae-SD-15,ATCC43816,CG43 and ED23,shared the highest nucleotide sequence identity the uge gene 97.8-100% among with the 14 isolates except K.pneumoniae-SD-3,KCTC2242,RJF293,CAV1453 and KP52.145.the IucB gene 94.6-99.7%among the 12 isolates except K.pneumoniae-SD-12 to K.pneumoniae-SD-13,and K.pneumoniae-SD-15,RJF293 and sb227,and the ybtA gene 99.3-99.7% among K.pneumoniae-SD-12,K.pneumoniae-SD-13 NTUH-K2044,KP36,11 and ED23.Therefore that K.pneumoniae of mink was similar to that of humans’.3.Pathogenicity test ofK.pneumoniae from mink studyTo clarify the pathogenicity of the K.pneumoniae isolates in mouse and in mink,three strains,K.pneumoniae-SD-12,K.pneumoniae-SD-15 and K.pneumoniae-SD-21,were selected,according to the presence or absence of serotype K2 and the differences of thevirulence genes.Some of the mice and mink in the other inoculated groups gradually showed clinical designs on days2-8 p.i,including partial loss of appetite,coarse fur,sneezing and coughing,and the mouse death reached a peak on days 4-7 p.i.The dead mice showed Lung hemorrhage,liver hemorrhage and swelling and slight bleeding point in brain,but no liver abscess.The results showed that K.pneumoniae-SD-12,K.pneumoniae-SD-21 showed high virulence for mice,the LD50 were that 5.0×102CFU and 3.2×100CFU;and for mink,the LD50 were that 1.6×103CFU and 3.2×101CFU;whereas K.pneumoniae-SD-15 was avirulent for mice and mink,the LD50 of which was more than 10 8.0 CFU.So the strain mainly contained capsular serotype K2,hypermucoviscosity,aerobactin and rmpA virulence genes,which have high pathogenicity.