Studies On The Relationship Of Enterococci.Faecalis Biofilm Formation And Fsr System,gelE,sprE Genes Under The Condition Of Nitrogen And Phosphorus

PurposeEcosystems were established to study the nitrogen and phosphorus effect on antibiotic resistance in Enterococcus faecalis(ampicillinerythrosine,oxtetracycline,vancomycin,ciprofloxacin and chloramphenicol).While the biofilm and cell adhesion of E.faecalis were also studied in these systems.Thetwo-compound system fsr and downstream genes sprE and gelE expression in E.faecalis were studied;finally,the relationship among ecosystems with fsr two-compound/sprE,gelE expression were analyzed.MethodFour groups were established in the ecosystem and each group was replicated.Different concentration of nitrogen(0 mg/L,1.50 mg/L,4.50 mg/L,and 12.50 mg/L),and phosphorus(Omg/L?0.15 mg/L,0.15 mg/L,and 1.25 mg/L)were added to four groups respectively.E.faecalis were isolated at 0d,1d,7d,14d,21d,28d,40d,60d,and 95d.Antibiotic resistance to ampicillin,chloramphenicol,erythrosine,vancomycin,ox-tetracycline,vancomycin and ciprofloxacin were studied.Biofilm formation was determinated staining adhered bacteria with violet crystal dye and cell adhesion was determined by semi-quantitatively.Fsr two-compound and downstream genes of sprE and gelE expression were studied by fluorescence qualitative PCR.Total nitrogen and phosphorus in ecosystems were detected after each isolation time.Antibiotic-resistant E.faecalis were identified by CLSI method.Finally,the effect of nitrogen and phosphorus on the expression of fsr system and the downstream genes sprE and gelE were also studied.Results1.Nitrogen and phosphorus were added after establishing ecosystems.Nitrogen and phosphorus concentrations were different in groups.Antibiotic-resistant results show that antibiotic sensitive strain isolated from control group;low rates of ERY/OXY-resistance were appeared in low-does group,approaching 25%;high rates of OXY-resistant was appeared in moderate-dose group that was 68.6%,which was approaching to 75%at the end of ecosystem time and low rates of ERY-resistant was appeared;25%,100%rates of ERY and OXY-resistant respectively were appeared in high-does group.Above all,ERY/OXY-resistant in E.faecalis appeared in high does group;while multi-resistance to AMP,ERY,OXY and CHL were appeared in the low-does group.2.Biofilm formation was different depend on groups.Biofilm was formed greatest in moderate-dose groups.Biofilm formed in high-dose group obviously than in a low-dose group.Cell adhesion result shows that the adhesive force in E.faecalis appeared in high/moderate-groups obviously;and which was greatest in high does-group.Finally,we found that the biofilm formation was related to cell adhesive force in E.faecalis in the ecosystem.3.Fluorescent quantitative PCR results show that:fsrA and gelE genes were expressed in the control group;the expression of gelE gene and fsrA was suppressed and delayed in the low-dose group respectively.FsrA and gelE genes were induced in moderate/high-dose groups,those genes were induced more ten thousand times in expression than the control group.But the expression of gelE was decreased soon after it was in peak.4.In conclusions,the expression of fsr system and the downstream genes of sprE,gelE were affected by nitrogen and phosphorus.While nitrogen play a key important in the ecosystem for the concentration different more in groups than phosphorus dose.Conclusion1.Antibiotic-resistant were induced greatest in the moderate-dose group,and keeping longer time;while it was toke second place in the high-dose group.2.Strains isolated from the ecosystem with high resistance or multi-resistant were followed by greater biofilm formation.Biofilm was appeared in longest time by the moderate-dose group,which was toke second place in the high-dose group.Otherwise,biofilm could appear in E.faecalis by the normal condition.Cell adhesive force was related to biofilm formation in E.faecalis.3.The expression of fsrA gene was delayed by the low-dose group,which were suppressed strongly in the low-does group and suppressed greatest in the high-dose group.The expression of the gelE gene was delayed in low-dose group and induced greatly in the moderate-dose group,which appeared peak in a short time in the high-dose group.4.Genes selective expression in E.faecalis were induced by nitrogen and phosphorus,biofilm formation was greater and cell adhesion was decreased for suppressed of fsrA gene and induced of gelE gene respectively.