| Objective:In this study,microcosms were established to abserve the effect of the lower concentration of nitrogen and phosphorous on resistance phenotype and biofilm-forming ability phenotype,and the relative expression of E.coli biofilm formation related genes(luxS,mqsR,motility genes flbD,fliA,mot A and type 1 fimbriae gene fimH).The correlation between E.coli biofilm-forming and antibiotics resistance was set up as well.Methods:According to the results obtained by our Research Group before and the base value of total nitrogen(TN)and total phosphorus(TP)in the microcosms,the control group and the treatment groups were set up which including four dose groups.Each group has two repeats.TN and TP concentration and permanganate index(CODMn)were detected.Soil-water samples were collected for separated and isolated of E.coli.Micro-broth dilution method which was recommended by CLSI was used to detect the antibiotics resistance of E.coli to 7 kinds of antibiotics.Glass tube method and semi-quantitative detection were used to detect the samples biofilm-forming ability.Real-time PCR was used to detect the mRNA expression levels of six E.coli biofilm formation related genes to explore its correlation between expression levels and E.coli biofilm formation.Results:(1)After adding low concentration of nitrogen and phosphorus,there was no significant change of TN concentration.While the concentrations of TP reduced to base value shortly after nitrogen and phosphorus adding.CODMn value of each dose group changed irregularly.Antibiotic sensitivity results showed that,there were no resistant strains isolated from the blank control group,while the resistance rates and multi-drug resistance rates of each treatment group was in high level.E.coli isolated from the medium-dose group showed the highest level of resistance,followed by high-dose group,lowest-dose group and low-dose group.Most of the E.coli strains showed resistance to cefazolin and ampicillin(resistance rates mostly between 50%-100%),and cefotaxime(resistance rates mostly below 50%).E.coli strains which were isolated from the lowest-dose group,medium-dose group and high-dose group showed resistance to tetracycline.Strains of high dose group showed resistance to chloramphenicol.But resistance rate to these two antibiotics was relatively low.(2)After adding nitrogen and phosphorus,the results of qualitative detection revealed that E.coli strain isolated from medium-dose group showed the highest biofilm-forming ability,followed by low-dose group,high-dose group and lowest-dose group.The results of Semi-quantitative test indicated that E.coli from medium-dose group formed stronger ability of adhesion,followed by high-dose group,low-dose group and lowest-dose group.All the results indicated that resistant E.coli showed higher biofilm formation ability in our study.(3)Took the samples of 0 d and control group as the calibration samples,then calculated the test results obtained by real-time PCR gene.The results of real-time PCR showed that after adding nitrogen and phosphorus for l d,the expression of luxS,flhD,fliA,motA and fimH gene of each treatment group can be observed.The difference only lied in the level of expression and the duration of it.There was no expression of mqsR gene observed.In addition,the gene expression trends of flhD,fliA,motA and fimH were consistent.The expression levels of fliA and motA were in higher level,followed by fimH and flhD genes.Conclusion:(1)The addition of low concentration of nitrogen and phosphorous could also lead to E.coli strains isolated from microcosms formed and maintained high level resistance,and multi-drug resistance.The concentrations of nitrogen and phosphorus of medium-dose group(The final concentrations of TN and TP were 3.5 mg/L,0.35 mg/L respectively)seemed have the greatest impact on E.coli resistance.(2)There was obviously effect of low concentration nitrogen and phosphorous on the formation of biofilm.In our study,it had strong correlation between E.coli resistance and biofilm formation ability.(3)After adding low concentration of nitrogen and phosphorus,the biofilm formation ability ofE.coli isolated from microcosms enhanced,which had relationship to luxs,flhD,fliA motA and fimH genes.(4)After adding low concentration of nitrogen and phosphorus in the microcosms,there was positive correlation between E.coli strains showed resistant and multi-drugs resistant characters and the biofilm formation ability,while it had negative correlation between resistant character to the expression and maintained levels of flagella and pili genes.There was positive correlation between 3 flagella genes and pili gene fimH. |
