| Approximately 1010–1011 tons of animal manure is produced globally each year,which is routinely applied to land as a crop fertilizer or soil amendment.Manure can harbor large number of zoonotic bacterial pathogens.If manure is inadequately treated before land application,then viable pathogens may survive or grow in field soils,which could result in the soil biopollution and cause the epidemiology of the various zoonoses.Enterohemorrhagic Escherichia coli O157:H7,the most prevalent manure-borne zoonotic pathogen,is of concern due to its low infective dose(as few as 10 cells)and severity of complications(haemolytic uremic syndrome).This pathogen can invade into soil primarily through manure application and survive in soil or manure environments for more than a year,affording the opportunity for reinfection by animals.Furthermore,E.coli O157:H7 present in agricultural soils has the potential to be transported to both surface and ground water,or attach to plant surfaces and become internalized into plant tissues,which represents a serious risk to public health.It has been widely implicated in several hundred outbreaks of E.coli O157:H7 in the past 30 years all over the world,with severe outbreaks at mortality rate as high as 15% for children and the older.Therefore,E.coli O157:H7 and various soil components(different soil types,soil particles of different sizes,and clay minerals)were selected in this study.The soil chemical and molecular biology methods such as soil characterization,bacterial attachment,flat colony counting method,crystal violet staining method,qRT-PCR were conducted.Furthermore,advanced techniques including atomic force microscopy,scanning electron microscopy,and fluorescence microscopy combined with cell staining were performed to comprehensively investigate the mechanisms of interfacial interactions and survival between E.coli O157:H7 and soil components,so as to prevent and control the ecosystem contamination and human infection by bacterial pathogens.The major results were summarized as follows:(1)The correlations and contribution rates between soil physicochemical properties and E.coli O157:H7 attachment behaviors were analyzed.Simple linear results revealed that soil organic matter(P<0.01)was significantly negatively correlated with partition coefficients(Kd)of E.coli O157:H7,while soil pH,electric conductivity,clay content,specific surface area,and cation exchange capacity were not significantly correlated with Kd(P>0.05).Partial correlation analysis excluded the indirect effects of other factors and showed that soil organic matter and clay were the most important factors in determining the attachment of E.coli O157:H7 in soils.Furthermore,stepwise multiple regression analysis was conducted and correlation regression equation was expressed as: Kd =-1.306×OM+0.536×CC+51.036(P<0.01).Soil organic matter was obviously the most important variable,with a more contribution(66.95%)to Kd than clay(33.05%).These statistical analysis results show that soil characteristics,such as organic matter and clay content,determine the E.coli O157:H7 attachment,which significantly affects the transport and distribution of bacterial pathogen in soil environments.(2)The effects of soil type(Red soil and Brown soil),particle size(sand,silt,and clay),the presence of natural organic matter(NOM)or Fe/Al(hydro)oxides on E.coli O157:H7 survival in soil environments were assessed through survival,attachment,metabolic activity,and q RT-PCR analyses.The abundance of inoculated E.coli O157:H7 in Brown soil particles increased 0.6 log10 CFU/g–1.4 log10 CFU/g within 3d(except for NOM-stripped clay),while that in Red soil particles decreased 0.52 log10 CFU/g–0.97 log10 CFU/g in the initial 6h,and then continued to die off rapidly in the following 8d.Additionally,survival of E.coli O157:H7 was significantly enhanced when Fe/Al(hydro)oxides had been removed from Red soil particles.In the sense of particle size,E.coli O157:H7 survived the longest in NOM-present clays for the two soils.Clays were more effective than silts and sands in binding cells and significantly changing the expressions of acetate pathway associated genes(pta and ackA).After exposure to clays,the expressions of the acetate pathway associated genes showed a 6.1-fold–9.3-fold increased expression for pta,but a decreasing expression for ackA when compared with sands and silts,which could promote the accumulation of acetyl phosphate and then the bacterial transformation from a planktonic to biofilm phenotype.Moreover,The adenosine 5′-triphosphate(ATP)levels per E.coli O157:H7 were also observed to be higher in clays(0.36×10-18 mol ATP/CFU to 0.39×10-18 mol ATP/CFU)than in sands and silts(0.13×10-18 mol ATP/CFU to 0.21×10-18 mol ATP/CFU)at the end of the time period,indicating that clays could enable bacteria to maintain a relatively high activity for extended periods.For silts and sands,no obvious difference was observed in bacterial ATP levels between NOM-stripped and NOM-present particles,implying that the role of NOM in our study was not nutrient supply.Furthermore,E.coli O157:H7 decayed more rapidly in NOM-present particles than in NOM-stripped particles,and the removal of NOM resulted in the more attachment of bacterial pathogen to soil particles.Therefore,slightly soluble NOM functioned mainly through interfacial interaction to prevent the attached bacterial phenotype and ultimately affected the survival of bacterial pathogen.This study highlights the importance of surface-attached bacterial phenotype and associated metabolic pathways in generating strong natural selection for bacterial pathogens to persist for extended periods in soil environments.(3)Herein,growth,biofilm formation,and virulence gene expression of E.coli O157:H7 were monitored,following exposure to a variety of clay minerals(montmorillonite,kaolinite,and goethite).Negatively charged montmorillonite was favorable for E.coli O157:H7 growth,but inhibited the biofilm formation.Positive charges from the exposed edge surfaces of kaolinite caused a stronger electrostatic attraction force than the montmorillonite and contributed to the biofilm formation.Positively charged goethite significantly inhibited the bacterial growth,but obviously promoted the biofilm formation.Fluorescence microscopy images showed that goethite was detrimental to E.coli O157:H7 and resulted in the bacterial death.However,cells attached to montmorillonite or kaolinite remained mostly viable.Using atomic force microscopy and scanning electron microscopy,it was revealed the direct evidence of a range of different association mechanisms between E.coli O157:H7 and minerals.Montmorillonite did not appear to be bacterial membrane bound,but goethite was closely attached to cell surfaces and formed a unique mineral envelope.Additionally,E.coli O157:H7 attached predominantly to the edge surfaces of the kaolinite.Montmorillonite significantly decreased the viscosity of biofilm(10.38±1.29nN)(P<0.05),but the viscosity of biofilm formation in the presence of kaolinite had no significantly change(19.85±3.34nN)compared with control(17.05±2.41nN)(P>0.05).On the contrary,goethite resulted in a loss of bacterial size and irregular morphology in the biofilm and promoted the high viscosity of biofilm(29.00±4.84nN).Changes in biofilm formation were also supported by changes in gene expression in minerals-exposed E.coli O157:H7.The expression of fliC,flagella encoding gene,was increased while the expression of curli encoding gene such as csgA,responsible for the irreversible adhesion,was reduced.Moreover,the expression of wcaM,colanic acid biosynthesis protein encoding gene,was also reduced in the presence of montmorillonite,which could decrease the viscosity of biofilm and inhibit the formation of biofilm.Kaolinite decreased the expression of fliC,but increased the expressions of csgA and wcaM.Moreover,the expressions of the acetate pathway associated genes showed an increased expression for pta,but a decreasing expression for ackA,which could promote the accumulation of acetyl phosphate and then the bacterial transformation from a planktonic to biofilm phenotype.The expressions of rcsA,rcsB and rcsC,regulator of colonic acid biosynthesis encoding genes,wcaM,and luxS,the autoinducer-2(AI-2)quorum-signaling molecule encoding gene,were significantly increased following exposure to goethite.Therefore,the presence of goethite significantly promoted the formation of biofilm.In the early stage of biofilm formation,the biofilm formation of E.coli O157:H7 was significantly inhibited,while the expressions of virulence genes(stxA-1 and stxA-2)were increased in the presence of clay minerals.However,in the presence of kaolinite and goethite,the expressions of stxA-1 and stxA-2 were significantly decreased,but the biofilm biomasses were obviously higher than the control in the late stage of biofilm formation,implying the negative relation between virulence gene expression and biofilm formation.Different clay minerals have specific physiochemical properties(surface charge and morphology),which could control the adhesion forces and association types of minerals and E.coli O157:H7 and impact differentially on the fate of bacterial pathogens in soil environments. |
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