The Effect Of Nitrate And Phosphate Ions On The Formation Of Antibiotics Resistance Of Enterococcus Faecalis In The Model Ecosystem

Objective：Model ecosystems were established to study the effect of nitrate andphosphate nutrient salt on the resistant phenotype of Enterococcus Faecalis(E.faecalis) tooxytetracycline(OXY), ampicillin(AMP), ciprofloxacin(CIP), vancomycin(VAN),chloramphenicol(CHL) and erythromycin(ERY). The mechanisms of antibiotic resistancegenes (ARGs) were studied as well.Method: Soil samples were collected from the forest park in Fuzhou and sterilized. TheE.faecalis (ATCC29212) saved in our laboratory was recoveried and the sensitivity of theE.faecalis to six kinds of antibiotics were detected. The result showed that it wassensitive to five out of six antibiotics and intermediated sensitive to ERY, so it could beused as the testes strain in the model ecosystems. The strain was amplified and then thebacterial liquid were added to the sterile soil samples and mixed. The mixture was putinto the experimental box and35L distilled water were added. Eight boxes wereestablished and then divided into four groups. Each group had two replications. NH4NO3and NaH2PO4were applied to the model ecosystems as the nitrate and phosphate ions.The nitrogen content was0mg/L,1.5mg/L,4.5mg/L and12.5mg/L and the phosphatecontent was0mg/L,0.15mg/L,0.45mg/L, and1.25mg/L respectively. The E.faecalisstrains were isolated from the model ecosystems at the time of0d,1d,7d,14d,21d,28d,40d,60d,95d and sensitivity of these strains to six kinds of antibiotics were detectedunder the recommend of CLSI. The water samples were collected to determinate the totalnitrogen(TN), total phosphorus(TP), permanganate index(CODMn), chlorophyll-a(Chla)and secchi depth(SD) and then calculated the TLI index. PCR mothods were used todetect the ERY resistance genes(ermB, msrC, mefA), OXY resistance genes(tetM, tetL,tetS), CHL resistance genes(cat) and enterococcus surface protein gene(esp).Results:(1) After the addition of nitrate and phosphate ions, TN showed gradient variationcorresponding to the additional level which led to the Comprehensive Trophic LevelIndex (TLI). high dose group, middle dose group and low dose group showed severe eutrophication, mesotrophication, light eutrophication respectively at7d according to theTLI. The imbalances of the algae bloom made the different replications of the same dosegroup emerge dissimilar Chla value from7d to21d, then CODMnchanged as well. Theresistance to the six kinds of antibiotics of E.faecalis strains appeared from7d to60d,which suggested that the resistance of E.faecalis to the antibiotics had a significantrelationship to the variation of TN, Chla and CODMn.(2) The effect of different dosage of nitrate and phosphate ions on the separation rate ofE.faecalis was different. Because of the addition of E.faecalis strain, all separation ratesreached to100%at0d. The low dose group and the No.3box of middle dose group andNo.1box of high dose group showed higher separation rate than the control group from1d to28d, then they declined quickly. The separation rate of No.5box of middle dosegroup and No.8box of high dose group were below or equal to the control group.(3) Different dosage of nitrate and phosphate ions had an effect on the resistantphenotype of OXY, CIP, AMP, CHL and ERY. The impact on the OXY resistance showeda positive relativity to the dosage of the nitrate and phosphate ions. No.1and No.8box ofhigh dose group emerged resistance rate to71.5%and40%at7d and21d respectively,then both of them rose up to the highest level at60d. No.2and No.7box of the low dosegroup appeared OXY resistance at21d and28d and the highest resistance rates reached to66.7%and100%respectively. Resistance rate to CHL increased with the increasing ofdosage. No.5box of middle dose group and No.8box of high dose group appearedresistance rate to CHL at7d, and the rate reached to40%and66.7%respectively at60d.The resistance rate to CHL only emerged at28d and60d in low dose group. Theresistance rate to ERY had a positive relativity to the dosages of nitrate and phosphateions. No.5and No.8box of appeared resistance rate to ERY at7d, and the rate reached to16.7%and14.3%respectively at7d, then the rate rose up, the highest rate emerged at60d. No.7box of the low dose group appeared50%of the resistance rate to ERY at21d,then it went down to25%at40d. Only high dose group had an obviously impact on theCIP resistance. No.8box of high dose group appeared resistance rate to CIP at7d and itrose up to50%at40d then went down quickly. The impact of the adding of nitrate andphosphate ions to the resistance rate to AMP was unconspicuous, only No.5box of the middle dose group and No.7box of the low dose group showed the resistance rate to50%and16.7%at21d. There was no detectable resistant phenotype of E.faecalis to VAN.(4) The positive rate of the tetM, tetL and tetS genes was97.3%,62.2%and18.9%in the37strains of E.faecalis resistant to OXY, while the rate were89.7%,7.7%and0%in39sensitive strains. The positive rate of the cat gene was69.2%in13strains of E.faecalisresistant to CHL, while the rate was2.9%in70sensitive strains. The positive rate of theermB gene was77.3%in22strains of E.faecalis resistant to ERY and the rate were77.4%and75.0%in the31and16strains of ERY intermediate sensitive and sensitiveE.faecalis. The positive rate of the msrC gene was18.2%in22strains of E.faecalisresistant to ERY and both of the rate were0%in the40and13strains of ERYintermediate sensitive and sensitive E.faecalis. There was no mefA gene emerged inE.faecalis. esp gene was detected in92strains and only one strain carried esp gene.(5) About57.4%of the total antibiotic resistant E.faecalis showed multi antibioticresistance (MAR). The number of twofold, triple and quadruple MAR E.faecalis were19,9and3strains respectively, with35.2%,16.7%and5.6%ratio respectively, and the mostcommon genotype were OXY-ERY, OXY-CHL-ERY o. Twelve ARGs types weredetected and tetM-ermB was the most common type and the next was tetM-tetL-ermB.Conclusion:(1) The addition of nitrate and phosphate ions had a significant influence on the resistantphenotype of E.faecalis to OXY, CHL and ERY. Only high dosage led to the CIPresistance. Higher TN concentrations have highly relativity to the OXY and CIPresistance and higher Chla concentrations have highly relativity to ERY and CHLresistance. The addition of nitrate and phosphate could induce the resistance to AMP andno detectable VAN resistant phenotype appeared.(2) Different dose of nitrate and phosphate ions showed dissimilar effects on theresistance of E.faecalis to OXY, CIP, CHL and ERY. There was a positive correlation ofnitrate and phosphate ions on the OXY resistance. High dose of nitrate and phosphateions had greater influence on the resistance of E.faecalis to CHL and ERY, but not fullycorrelated with the dosage. Only high dosage had an obviously effect to CIP resistance. (3) The tetM, tetL and ermB gene had already existed before the addition of nitrate andphosphate ions, these genes might have been induced to express. The strains containedmsrC gene displayed high level of ERY resistance and the high rate of the cat genedetected from the resistance of E.faecalis to CHL and the appearance of esp genesuggested that the addition of nitrate and phosphate ions might cause different types ofmechanism to affect the resistance of E.faecalis to different antibiotics.