Impact Of Low-concentration Of Nitrogen And Phosphorous Nutrient On Quorum Sensing Of Escherichia Coli

Objective:Model pond ecosystems were established to study the effect of low-concentration of nitrogen and phosphorous on the resistant phenotype of E.coli to ampicillin(AMP),cefazolin(CFZ),cefotaxime(CTX),gentamicin(GM),tetracycline(TE),ciprofloxacin(CIP)and chloramphenicol(CHL).The aim of this study was to comprehend the relationship between the antibiotic resistance of E.coli and the low-dose of nitrogen and phosphorous.Multi-drugs resistance of E.coli strain were selected to study the correlation between the level of drug resistance and the expression levels of 6 genes sdiA,lsrR,lsrADB.ftsE,ftsQ,hflX,which belong to the quorum sensing system of E.coli.Methods:Model pond ecosystems were divided into 5 groups which included a control group and 4 different dosage group where NH4NO3 and NaH2PO4 were added to gain the concentration of 1.50 mg/L,0.15 mg/L;2.50 mg/L,0.25 mg/L;3.50 mg/L,0.35 mg/L;4.50 mg/L,0.45 mg/L,respectively.Each group consisted of two duplications.Water samples were collected to measure the TN,TP,CODMn and Chla,and samples collected from the interface of mud and water were used for the isolation of E.coli at the time of 0 d,1 d,8 d,15 d,22 d,29 d,36 d,54 d,85 d.Micro-broth dilution method was used to test the sensitivity of 7 kinds of antibiotics.Real-time quantitative(Real-time PCR)technique was applied in the study of the expression levels of 6 genes.Pearson correlation analysis was applied in analysis of the correlation between the expression levels of 6 genes and the high levels of antibiotics resistance of E.coli to AMP and CFZ.Result:(1)Different doses of nitrogen and phosphorus can lead to formation of antibiotic resistant of E.coli to AMP and CFZ,and the antibiotic resistant character changing similarly.In lowest-dose group,the E.coli strains isolated were resistant to AMP and CFZ with high-level except isolation from 15 d and 22 d samples.In low-dose group,the E.coli strains isolated were resistant to AMP and CFZ with high-level except isolation from 1 d and 8 d,which was sensitive to AMP and showed low level resistant to CFZ.In middle-dose group,E.coli strains isolated were resistant to AMP and CFZ with high-level.In high-dose group,E.coli strains isolated were resistant toAMP and CFZ with high-level except isolation from 8 d and 15 d.A few of E.coli isolated from different group were resistant to CTX.The E.coli strains were resistant to GM with low-level which isolate from 8 d and 15 d samples,and were resistant to TE with high-level isolated from 22 d in high-dose group.All of E.coli strains isolated were sensitive to CIP and CHL.(2)Pearson analysis showed that,the correlation coefficient between the TN,TP,CODMn,Chla of the water and the levels of antibiotic resistant is relatively low.But the correlation between the resistant level of AMP,CFZ and GM with the TN was 0.447?0.38 and 0.388 respectively,which was showing low correlation;the correlation with the Chla was 0.605,0.507 and 0.566 respectively,which was showing low correlation.(3)Pearson analysis showed that,there was negative correlation between the E.coli resistant level of AMP and CFZ to the expression of ftsQ gene,while there was positive correlation between the resistant phenotype to the other 5 genes which were included into QS of E.coli.In lowest-dose group,there was high and low correlation between the expression of lsrR?lsrADB?ftsE?hflX genes and AMP and CFZ resistant level,respectively.In low-dose group,there was low correlation between the expression of hflX gene to AMP resistant level,while there was low correlation between the expression of sdiA?lsrR?ftsE?hflX genes to CFZ resistant level.In middle-dose group,there was high correlation between the expression of lsrR?ftsE genes and the AMP resistant level while there was high correlation between the expression of lsrR gene and the CFZ resistant level.In high-dose group,there was high correlation between the expression of lsrR?lsrADB genes and the AMP resistant level while there was high correlation between the expression of ftsE?hflX genes and the CFZ resistant level.Conclusion:(1)Low concentration of nitrogen and phosphorous could induce the formation of antibiotics resistance to AMP and CFZ.There was no significant dosage-effect relationship in this study.(2)The resistant level of AMP and CFZ of E.coli indicated the correlation with TN and CODMn,but did not significantly.(3)There was correlation between high level antibiotics resistance to AMP and CFZ and the reduced expression of ftsQ gene and increased expression of sdiA,IsrR,IsrADB.ftsE,hflX genes in E.coli.(4)The E.coli can regulation the expression of genes of the Quorum Sensing system to adapt the environment changing caused by the adding of nitrogen and phosphorus,which led to the phenotype of resistant to some antibiotic in E.coli.