Effect Of Enterococcus Endocarditis Antigen EfaA On The Forming Process Of Enterococcus Faecalis Biofilms

Objective: In this work we investigate the relationship between enterococcus endocarditis antigen(efaA) and biofilm formation, in order to do the groundwork for further study of E.faecalis biofilm's pathogenicity and antibiotics resistance.Methods:1. Using ordinary optical microscope to observe the biofilm formation of efaA~+, efaA~- E.faecalis on cover glasses at different incubation time,we generally identify biofilm formation time.2. 1% crystal violet(CV) stain the adhesion of different stages'enterococci on the 96-well plate.CV was solubilized in ethanol-acetone, and optical density at 570 nm, in order to compare the biofilm formation ability of efaA~+, efaA~- E.faecalis.3. MTT method was used to test viable bacteria counts of efaA~+, efaA~- E.faecalis at different incubation time, indicated by OD492,then compare their bioactivity of E.faecalis biofilms.4. The efaA~+, efaA~- E.faecalis biofilms stained with AO/EB dye,put in under CLSM to observe, compare the biofilm biofilm thickness, the inner, intermediate and the outer layer bacterium density and bacterial activity of efaA~+, efaA~- E.faecalis,do statistics analysis.5. Using SYBR Greenâ… quantitative real-time PCR technology to test the efaA mRNA expression of efaA~-positive and efaA~-negative strain, compare the efaA expression of different incubation time, contrast with biofilm thickness.Results:1. Initially identify the adherence time of E.faecalis was 2 hours,basic formation time was 6 hours, mature time was 24 hours.2. The OD570 of mutant efaA~-positive strain were larger than mutant control strain and wild strain, indicate the biofilm formation ability of efaA~-positive strain are stronger.3. The bioactivity of efaA~-positive E.faecalis biofilm was higher than efaA~-negative stain.4. The biofilm thickness, the inner,intermediate,outer layer bacterium density and viable bacterium percentage of efaA~-negative strain were all lower than efaA~-positive strain. Analysis by SPSS13.0 show that the difference between them are significant.5. We haven't tested efaA mRNA in efaA~-negative strain, the variation tendency of efaA expression in efaA~-positive strain is similar to biofilms thickness's.Conclusions:efaA gene is beneficial for the enterococci's preliminary adherence and biofilm formation.