Enterococcus Faecalis Collagen Adhesion Gene Variants Pathogenic Role In Urinary Tract Infection Associated With Indwelling Catheter

Objectives:Construction of Enterococcus faecalis collagen adhesin gene conversion variants; established mouse model of urinary tract infections; investigate the pathogenic role of ace gene in E. faecalis biofilm formation and urinary tract infection, and to lay the foundation for the prevention and treatment of drug-resistant enterococcal infections studied.Methods: First: Construction and identification of Enterococcus faecalis ace gene transformation variants: Screening ace gene-negative strains N8(spectinomycin sensitive); preparation of competent cells; extract of E. coli XL1-Blue (pTEX5646) plASmid (containing the entire ace gene fragments, spectinomycin resistance); plASmid by electroporation method pTEX5646into the N8, the mixture wAS inoculated onto the shock spectinomycin resistance culture medium, colonies wereselected by PCR. Second: Establishment and evaluation of mice catheter-related urinary tract infection model:Establish a mouse model of catheter-ASsociated urinary tract infections with a silicone tube through the bladder bacterial counts,GM-CSF,、IL-6,、MIP-1a and other cytokines;HE staining compare the impact of each group of miceon the pathophysiology of bladder. Three: ace gene in vitro comparison of early adhesion N8ace-picked wild strains and mutants transformed N8ace+inoculated into5ml ELB liquid medium;250rpm shaking bacteria16h.100ul broth were inoculated into96-well plates, incubators, dyeing, rinsing and drying is then determined by the orifice at the bottom of the OD (OD595); By meASuring the OD value compared with the wild-type strain and transformed variants early adhesion ability in vitro.Four: Comparison with the pathogenic role of wild strains and positive transformation in urinary tract infection;Comparison with the wild-type strain transformed variants in the bladder and silicone tube surface, bacterial counts, cytokine, pathology in mice; simultaneously catheter and bladder surface by scanning electron microscopy.Results:1. mutants transformed cultured in medium spectinomycin resistant white colonies, no wild strains cultured colony; PCR method hAS a size of about300bp of clear bands in polyacrylamide gel electrophoresis.2. bacterial counts were significantly higher in the experimental group, the experimental group of cytokines GM-CSF、 IL-6、 MIP-1a wAS significantly higher, more severe bladder inflammatory cell infiltration.3. Initial adhesion ability OD595meASured: under37℃culture, transforming mutants and wild-type strain of early adhesion OD595value wAS no difference (p>0.05); under46℃culture, transforming the early adhesion OD595value than the wild-type strain of mutant large (p>0.05);4. experimental groups bacterial counts inflammatory cytokines,GM-CSF、MIP-1a concentrations higher, with statistical significance;IL-6concentration wAS not statistically significant. transformed strain adhesion to the silicone tube and bladder more intensive;Conclusions:1. Successfully constructed Enterococcus faecalis mutant strains；2. ace gene is conducive to early adhesion of E. faecalis biofilm formation；3. indwelling catheter helps faecalis adhesion；4. Enterococcus faecalis adhesion ace genes in favor of the bladder or silicone tube surface, there is a causative role in the urinary tract infections；...