Study On The Effect Of Some Biology Characteristics Of Surface Protein EF0591 In Enterococcus Faecalis XJ05 Inducing Encephalitis In Lambs

Enterococcus is a kind of conditional pathogenic bacteria, especially in the digestive tract of humans and animals, enterococcus can cause infections in humans, and is infective to animals such as swine, cattle, sheep, birds, cats, dogs and so on. Furthermore it can transfer horizontally between human beings and animals. When the immunization is compromised or enterococcus translocated, it can cause disease in humans, such as bacteremia, urinary tract infection, liver and gallbladder sepsis, endocarditis, surgical wound infection, infection of the pulp, newborn pyemia even life-threatening. The pathogenicity of enterococcus is related to its virulence factors closely,and which has been proved is that when lacking some virulence factors, virulence of enterococcus will be greatly reduced. Therefore, the research of virulence factors is significant to investigate the mechanism of pathogenicity. EF0591 is a newly discovered virulence factor, and it has been proved that it is closely related to clinical isolates, but the knowledge about the toxicity trait and pathogenicity mechanism of this gene is poor. This study constructed EF0591 mutation from E.faecalis XJ05 to investigate the role of this gene in adherence to the He La cells, survival in the RAW264.7 cells, mice infection model, and the ability to biofilm formation.The main research contents and results are as follows:1.The construction of Enterococcus faecalis mutation: Amplifying the fragment which was used to disrupt the EF0591 gene with PCR, digested the amplified fragment with kpnⅠ and salⅠ then reclaim and connected it to the shuttle vector p TX4577. Transform E. faecalis XJ05 competent cells with the recombination plasmid by electroporation after enzyme identification. Screening recombination with kanamycin and identify it by PCR. The genetic stability was proved by serially subculture. The results showed that the mutation was construted.2.The role of EF0591 in the adherence to He La cell, raw264.7 survival test and bacteria loaded in murine organs: Joining bacteria to He La cells and coincubated in 1h and 2h. Lysing cells with0.1 % Triton-100. Viable bacteria were quantified by plating aliquots of serial dilutions on BHI agar; Joining bacteria into RAW264.7 cells, subincubated in 1h. Mycillin was added and incubated in 3h and 24 h, cells was lysed. Viable bacteria were quantified by plating aliquots of serial dilutions on BHI agar. Cultured bacteria were inoculated into mice, mice were dissected at different times and liver, kidney, spleen, heart and brain were harvested. The organs were lysed in a Tissuelyser. Viable bacteria were quantified by plating aliquots of serial dilutions on BHI agar.Results showed a decreased adherence to He La cells, with a significant difference(p < 0.05). Less mutant were survived in RAW264.7 cells compared to the wild type with a significant difference(p< 0.05). And the detected bacteria were reduced in liver, kidney, spleen, heart(p < 0.05), with a same level in brains, when compared to the wild type.3.The effect on biofilm formation of E. faecalis XJ05-△0591: Incubating E.faecalis XJ05Δ0591and E.faecalis XJ05 parent strain in 96-well plates under the same conditions at 37 ℃. Plates werewashed with PBS. Using the microplate reader determined the OD595 at different time points after the biofilms were stained. Incubating mutant and wild strain on coupons in 6-well plate at 37℃.Plates were washed with PBS and stained with crystal violet, FITC-con A and PI respectively.Observe the biofilms with microscope and confocal laser scanning microscope(CLSM) in each time point. According to the OD595 values of different time points, both strains’ biofilms were observed at 6 h, and the thickest biofilms occurred at 24 h. Both biofilms became to degrade after24 h, and the mutant biofilms was obviously less than the parent strain, with a significant difference(p < 0.05). The microscopy showed that the wild type biofilms were far more complicated and the embedded bacteria were much more compared to the mutant biofilms. The biofilms formed by E.faecalis XJ05 was much excessive and intensive, and much more embedded bacteria compared to E. faecalis XJ05-△0591 viewed by CLSM.