The Preliminary Study Of Characters Of Symbiotic Biofilm Formed By Prevotella Intermedia And Enterococcus Faecalis

1.Background and Objective In our life,common oral diseases such as dental caries,periapical periodontitis,pulpitis and periodontitis are caused by the invasion,colonization and infection of bacteria.The oral pathogenic bacteria are not scattering in the mouth with floating state,but with plaque biofilm state which can attach to the tooth surface,periapical zone,root canal and periodontal pocket in long term.The studies about pathogenic bacteria of oral biofilm are increasing now,and it became to a trend in recent years too.Prevotella intermedia and Enterococcus faecalis are common oral pathogenic anaerobic bacteria.These bacteria exist in periapical pathological tissue extensively,and also are the main pathogenic bacteria of the periapical periodontitis.The biofilm from these two kinds of symbiotic bacteria exists extensively in the diseased tissues.However,it is rarely to find the report of these symbiotic biofilm.The aim of this experiment is to study some characters of these symbiotic biofilm through comparing the single and symbiotic biofilm of these two kinds of bacteria,and to provide a new way of thinking about pathogenesis of periapical periodontitis.2.Methods 2.1 Crystal violet dyeing technology was used to deal with samples of biofilm.Then the optical density value(OD value)was measured by Microplate Reader.2.2 Live and Dead biofilm staining kit was used to dye the samples of biofilm,and then analyzed the distribution of live and dead bacteria through Confocal Lase-Scanning Microscopy.2.3 The genes of Prevotella intermedia and Enterococcus faecalis in single and symbiotic bacteria biofilm were measured and analyzed by RT-PCR and Real-Time RTPCR technology.3.Results 3.1 The OD mean value of Prevotella intermedia biofilm was minimum.The OD mean value of symbiotic biofilm was maximum.The OD mean value of Enterococcus faecalis biofilm was among them.3.2 The Confocal Lase-Scanning Microscopy scanned the biofilm samples through longitudinal axis(the Z-axis).The symbiotic biofilm was the thickest,while single bacteria biofilm was thinner.The Prevotella intermedia biofilm was the thinnest.The red dead bacteria distributed in the sparse bottom layer,and the green live bacteria distributed in the intensive upper bottom layer.3.3 The result of PCR 3.3.1 RT-PCR: The ahpc,PIN17 A1058,clpb genes from Prevotella intermedia and gel E gene from Enterococcus faecalis showed the clear single stripes in Gel electrophoresis technology.The reference genes of two bacteria were 16 sr RNA.3.3.2 Real-Time RT-PCR: The software of Real-Time RT-PCR product the amplification plots,dissociation curve and plate sample values of every gene.The plate sample values of ahpc,PIN17 A1058,clpb gene from Prevotella intermedia and gel E gene from Enterococcus faecalis in symbiotic biofilm were smaller than in single bacteria biofilm.The plate sample values of genes from Prevotella intermedia had greater different.4.Conclusion 4.1 The bacteria in symbiotic biofilm appears stronger ability of forming biofilm than in single bacteria biofilm.4.2 The expression of adhesive genes,ahpc,clpb,PIN17 A1058 and gel E show upregulation in symbiotic bacteria biofilm.4.3 The symbiotic bacteria biofilm has greater influence on adhesive character of Prevotella intermedia.