The Study Of Virulent Characteristic Of Hypervirulent Klebsiella Pneumonia(hvKP)and The Effect Of Hvkp On The Expression Of Inflammatory Cytokines Of Retinal Pigment Epithelial Cells

Objective Endophthalmitis is a dangerous infectious blinding posterior eye disease,which often causes serious visual impairment Endogenous endophthalmitis results from the bacteria or fungi infection circulated through the blood into the eye,causing the inflammation of choroid,retina,vitreous and other parts of the eye,of which pathogenic spectrum continue to change in recent years.Hypervirulent Klebsiella pneumoniae(hvKP),a new pathogen of endogenous endophthalmitis has emerged,which was first found in Asia Pacific area,and now more and more recognized by Western countries.Although the symptoms of hvKP endophthalmitis were most common like eye swelling,redness or blurred vision of the sudden attack,its pathogenicity was significantly higher than that caused by the classic Klebsiella pneumonia(cKP),and it was more difficult to cure.Clinically,ophthalmologists master the distribution of bacteria and pathogenic characteristics of endogenous endophthalmitis is essential to the diagnosis,treatment and prognosis improvement of this disease.In this study,we compared the characteristics of hypervirulent strains with classical strains to investigate the virulent characteristic of hvKP and the effect of hvKP on the expression of inflammatory cytokines of retinal pigment epithelial(RPE)cells.Methods8 strains hvKP and 8 strains cKP screened through the "string test" were collected,then hvKP group and cKP group were divided.Firstly,virulence strength of two groups were compared through regulator of mucoid phenotype gene A(rmpA)testing,antiserum experiment and survival within macrophages experiment respectively.Secondly,the expression of Monocyte chemotactic protein-1(MCP-1)and Interleukin-6(IL-6)in the supernatant of retinal pigment epithelial cells infected by Klebsiella pneumonia(KP)strains of these two groups were detected separately through ELISA.1.Bacterial virulent test:? String test After the bacteria were collected,the tested bacteria were inoculated on 5%sheep blood agar plates,incubated overnight at 37?.Then the length of the viscous string generated by inoculation loop or needle was measured.? Gene rmpA test The rmpA gene of KP were amplified by PCR and the gene expression level of two groups were detected respectively.? Bacterial antiserum test The concentration of bacteria was adjusted to 107CFU/ml.Then strains of two groups were mixed with serum 1:1 separately and inoculated for 2h.After the mixed culture medium gradient dilution,the number of survived bacteria was counted and the survival rate of two groups were calculated respectively.? Survival within macrophages experiment The bacteria of two groups were incubated with a dose of 107CFU/ml and multiplied by MOI 100:1 ratio mixed with mouse macrophages Raw264.7 separately and were incubated for 1 h.Then after the mixation was diluted and the number of survived bacteria was counted,and the survival rate of anti-macrophages bacteria of two groups were calculated respectively.2.En2yme-linked immunosorbent assay(ELISA)After the RPE cells were infected by the bacteria of two groups separately for 10h,the expression level of IL-6 and MCP-1 in supernatant of RPE cells were detected through ELISA.Results1.Bacterial virulence testing:? The strains of hvKP group showed positive in the string test,while the strains of cKP group showed negative.? The results of gene rmpA experiment indicated that the strains of hvKP group showed expression of rmpA gene 543BP,but rmpA gene 543BP was not expressed in the strains of cKP group.? The results of antiserum experiment showed that the survival rate of hvKP group(18.2%)was significantly higher than that of cKP group(5.3%),and the difference was statistically significant(t = 1.872,P<0.05).? The results of survival within macrophages experiment manifested that the intracellular bacterial survival rate of hvKP group(1.1%)was statistically significant lower than that of cKP group(7.5%)(t = 5.434,P<0.05).2.ELISA The secretion level of MCP-1(1177pg/mL)and IL-6(119pg/mL)in the supernatant of RPE cells were both higher statistic significantly in hvKP group,whereas the level of MCP-1(670pg/mL)and IL-6(54pg/mL)secreted in the supernatant of RPE cells were lower in the cKP group.Comparatively,the level of MCP-land IL-6 expresssion by RPE cells were 1.76 and 2.20 times higher in hvKP group respectively than those of the cKP group,and the difference was statistically significantConclusions Compared with the cKP,the virulent strength of hvKP were stronger than the cKP and the hvKP could induce more serious inflammatory reaction on RPE cells.