Preliminary Study On The Effects Of Berberine Hydrochloride On The Enterococcus Feacails Biofilm Through The Pathway Of SortaseA And Esp

1. Observation on the biofilm development and genes expression during biofilm formation in Enterococcus faecalisObjective:To observate the dynamic changes in Enterococcus faecalis biofilm formation and study the relationship between early biofilm formation in Enterococcus faecalis and the expression of sortaseA and esp.Methods:(1) Screen and identificate the Enterococcus faecalis biofilm bacteria related to UTIs from the Clinic, divide into biofilm and planktonic bacteria groups.Crystal violet staining assay for quantification of biofilm volume.(2) The invitro model of Enterococcus faecalis EF06biofilm was formed by modified plating method, cultivating1,8,16,24,48,72hours respectivily.The fluorescence images of space changes were obtained by Confocal laser scanning microscope(CLSM) and fluorescence microsco-py.(3) Real-time quantitative PCR method for biofilm and planktonic Enterococcus faecalis groups of sortaseA and esp, two early biofilm formation genes whose expression were detected and analyzed statistically.Results:(1)36.3%(36/99)strains were screen as biofilm.27.8%(10/36) were Enterococcus faecalis, whose biofilm volum were detected.(2) Crystal violet staining assay show that bacteria biofilm was formating when cultivating for16hours.when cultivated to24hours, The volume of biofilm was up to peak.Later, the volume returned gradually. the biofilm volume decreased for48hours. at the72nd, volume decreased much more.(3) The invitro model showed that the micro-colonies were formed and certain thickened after16hours, means that the biofilm formated elementarily. Biofilm matured after24hours. Bacteria started disseminating when cultivated for48hours. With development of biofilm formation, the viable and dead bacteria increased gradually. When cultivated for48hours, the viable and dead bacteria started decreasing. The total amount of bacteria decreased progressively from adherence side to free side. The density of viable bacteria in middle layer was highest.(4) The sortaseA and esp were colsely related to the early biofilm formation in Enterococcus faecalis. The expression quantity of sortaseA and esp in biofilm group is7.9times and13.5times respectively compared with them in planktonic group.Conclusions:(1) Detection rate of biofilm bacteria related to UTIs was36.3%in our experiment. Enterococcus faecalis take up most (27.8%).This result was consistent with the result we had got3years ago. Which suggest that distribution of biofilm bacteria related to UTIs in xiangya3rd hospital changed unobviously.(2) CLSM and fluorescence microscopy showed that Enterococcus faecalis biofilm formation progress contain primal planting, reversible adhering, structure splitting,maturing and disaggregation.. The total amount of bacteria decreased progressively from adhereing side to free side. The density of viable bacteria in middle layer was highest.(3) The sortaseA and esp were colsely related to the biofilm formation, which promote the early biofilm formation in Enterococcus faecalis possibly.2. Effects of berberine hydrochloride on biofilm formation in Enteroco-ccus faecalisObjective:To research the Effects of berberine hydrochloride on biofilm forma-tion in Enterococcus faecalis and explore its mechanism. Methods:Take the EF06as the experimental strain,dividing into2groups.group, the strain with different concentrations of berberine incubated for24h. Group,after the strain cultured alone for24h,with different concentrations of berberine to cultivated together for24h. Fluorescence microscopy observed the variances between drug untreated and treated groups. Crystal violet staining assay for differences quantification among the groups and analyzed statistically.Results:(1)Microscopic images showed that,in group, at concentrations of20and40μg/mL, the density of bacteria of EF06was higher than those of the respective strains incubated without berberine hydrochloride. As the concentration of berberine hydrochloride increased, the density of bacteria of EF06became lower.In group,there were not apparent changes at concentrations of200,400and600μg/ml, the density of bacteria decreased a little at a concentration of800μg/ml, the density of bacteria reduce more at a the concentration of1000μg/ml.(2) biofilm volum showed significant difference between drug untre-ated and treated groups,among the group.Conclusions:(1) Berberine hydrochloride inhibited Enterococcus faecalis biofilm formation.Crystal violet staining assay showed that drug concentration has positive correlation with inhibital effect.(2) Berberine hydrochloride degraded Enterococcus faecalis biofilm formation amount.berberine could inhibit biofilm formation in a dose-dependent manner, the higher dose of the dug,the more decrease amount of the biofilm.