| Background:The emergence of multidrug-resistant bacteria has become a significant threat to public health.In aquatic environment,nutrient which causes nutrient pollution and also contributes to bacterial pathogenicity and resistance are poorly understood.The main objective of this research was to evaluate the antibiotic resistance capability of Enterococcus faecalis by adding nitrogen(N)and phosphorus(P)periodically with different concentrations into the model ecosystem(mesocosm).In addition,the effect of a nutrient on E.faecalis biofilm development and quorum-sensing was also of interest in the aquatic environment.The quorum-sensing system is the population density-dependent regulatory mechanism whereby bacteria communicate with each other via signaling molecules.E.faecalis quorum-sensing systems control major virulence determinants which causes nosocomial infections.The E.faecalis quorum-sensing systems include several virulence factors that are regulated by the cytolysin operon,which encodes the cytolysin toxin.The cytolysin and Fsr virulence factor systems are linked to enterococcal diseases that affect the health of humans and other host models.Therefore,there is substantial interest in understanding the role of quorum-sensing in the aquatic environment.Methods:Mesocosms were replicated to evaluate the ecological effects of nutrient influx.The mesocosms were divided into four different N and P regimens.These four regimens were(i)control(no nutrient added);(ii)NP1,total nitrogen(TN)amended with NH4NO3 to maintain the N concentration at 1.5 mg/L and total phosphorus(TP)amended with KH2PO4 to maintain the P concentration at 0.15 mg/L;(iii)NP2,addition of 4.5 mg/L N and 0.45 mg/L P;and(iv)NP3,addition of 12.5 mg/L N and 1.25 mg/L P.E.faecalis were isolated from the mesocosms at 8 sampling events during 96 days to evaluate sensitivity to oxytetracycline(OXY),erythromycin(ERY),ciprofloxacin(CIP),ampicillin(AMP),vancomycin(VAN),and chloramphenicol(CHL)using Broth microdilution method.Microtiter Dish Biofilm Formation Assay was used to assess the capability of E.faecalis biofilm development.Quantitative real-time PCR was used to compare mRNA levels of E.faecalis quorum-sensing related genes(cylLs,cylLL,cylB,cylA,cylI,cylM,cylR1,cylR2,and gelE)in Mueller-Hinton broth.Resistance genes for ERY(ermB,msrC,and mefA),OXY[tet(M),tet(L)and tet(S)]and CHL(cat)as well as the enterococcal surface protein gene(esp)were investigated using PCR.The TN,TP,chemical oxygen demand permanganate index(CODMn),chlorophyll-a,secchi depth and trophic level index(TLI)were also analyzed.Results:The TLI indices of control and nutrient-enriched mesocosms were significantly(p<0.05)different.On Day 1,the TLI of all nutrient-enriched(46.77 ± 2.36)and control(47.54 ± 2.38)mesocosms was in the mesotrophic state.However,on Day 7,in the NP3 mesocosm,the CODMn was 6.53 mg/L and the contents of TN,TP,and Chl-a in the water column were 15.86 mg/L,0.39 mg/L and 113.3 mg/m3 respectively,associated with the high eutrophic state(71.31 ± 13.51).E.faecalis,when exposed to mesocosms with different N and P concentrations,they exhibited resistance to OXY,CIP,AMP,CHL,and ERY.The biofilm development of E.faecalis isolates were also significantly increased(F11,24 = 42.36,p<0.01)over time.Antibiotic resistant genes such as tet(L)and tet(S)for OXY,msrC for ERY and cat for CHL were found to be associated with resistance in E.faecalis.The incidences of tet(L)and tet(S)resistances were 62.2%and 18.9%,respectively,in 37 E.faecalis strains.The expression of quorum-sensing-related genes was significantly up-regulated,about tenfold at the transcriptional level in nutrient-enriched treatments than control(unenriched of N and P).Conclusion:The overall objective of this study was to examine the response of E.faecalis upon exposure to different enriched concentrations of N and P nutrients in mesocosm environments.We added E.faecalis to mesocosms,having different concentrations of N and P to evaluate their survivability,antibiotic sensitivity,biofilm development and quorum-sensing-related genes expression on different occasions.We found that E.faecalis can survive approximately three months in tap water mesocosms.We also observed that,although the isolation rate of E.faecalis in control mesocosm was reached to 50%on day 7,however,this bacterium was likely to be isolated over a period of 96 days from all treatments including control.This work also demonstrated that nutrients not only induced antibiotic resistance but also increased biofilm-forming capability in E.faecalis over time.In addition,the transcriptional profiles of quorum-sensing-related genes were also comparatively high in the nutrient-enriched mesocosms.Taken together,these findings may yield insight into how E.faecalis evolve to become opportunistic pathogen within aquatic environments.Furthermore,the exposure of this pathogen(from the aquatic environment)to humans and other animal has still not well investigated,and future studies on the pathogenic response mechanisms of environmental E.faecalis are important. |
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