The Formation Of Dormancy And The Killing Of Escherichia Coli O157:H7 In Farmland Soil

Currently,the frequent outbreaks of Escherichia coli O157:H7 from vegetable contamination has seriously threaten the food safety and public health in the worldwide,hence it is an emerging case to prevent and control the spread of foodborne pathogens in soil.E.coli O157:H7 can enter a long-term Viable but Non-Culturable(VBNC)state after transported to soil,and they could resuscitate under suitable conditions and result in the risk of infection.However,it is not well researched about the formation mechanisms of VBNC state and the sterilizing technologies of VBNC pathogens in soil.In this dissertation,we established a method for quantifying the VBNC E.coli O157:H7in soil,explored its characteristics of VBNC state formation in paddy soil,and clarified the sterilizing effect and mechanism by biochar addition and solarization in soil.The main findings are as follows.(1)A method was developed for quantifying viable E.coli O157:H7 in soil,which based on elution,density gradient centrifugation and PMA-q PCR.Coupled with culture counting method,VBNC state of E.coli O157:H7 could be quantified.Elution and cell screening could reduce the interference of high turbidity in bacterial extracts,and density gradient centrifugation could remove a high level of dead bacteria.The method could be applied in black soil,alluvial soil,red soil and paddy soil to accurately and rapidly quantify the VBNC subpopulation.(2)E.coli O157:H7 could enter the VBNC state after entry into paddy soil for 30-40 days.E.coli O157:H7 was negatively detected by the culture counting method.However,the method established in this paper confirmed the presence of 10~4-10~5 viable cells/g soil.The dormant cells could be resuscitated,which showed potential risk.(3)Biochar addition could significantly reduce the activity of E.coli O157:H7 in paddy soil.Rice hull biochar of 10%(w/w)had the best bactericidal effect.After 15days incubation,E.coli O157:H7 completely lost its culturability.The number of viable bacteria was as low as 2.09×10~2 cells/g soil,and these cells could not be resuscitated.Free radicals were detected in rice hull biochar,and it had direct killing effect on bacteria in the solution system,which was one of the important sterilization mechanism of rice hull biochar in paddy soil.The relative abundance of Geobacter,Lutispora,Bosea and Ruminiclostridium,the potential antagonists of E.coli O157:H7,in rice husk carbon group were significantly higher than those in other treatment groups,which might enhance the soil bacteriostasis.(4)Soil solarization could significantly kill E.coli O157:H7 in paddy soil.After8 h of soil solarization,the bacteria lost their culturability.The number of viable bacteria was as low as 2.09×10~2cells/g soil,and these cells could not be resuscitated.The high temperature(up to about 50℃)was be the main reason for effective sterilization.The relative abundance of Streptomyces,Clostridium and Burkholderia,potential antagonists of E.coli O157:H7,in the soil solarization with mulching group were significantly higher than those in the soil solarization without mulching group and the control group,which might enhance the soil bacteriostasis.(5)We illustrated the mechanism of the high temperature on VBNC E.coli O157:H7 in solution.Intracellular ROS accumulation caused by heat treatment was an important reason for the complete killing of VBNC bacteria.Protective protein aggregates and a certain level of ATP could help VBNC cells to resuscitation by depolymerized protein aggregates.The main enrichment proteins were related to metabolic pathways,biosynthesis of secondary metabolites,ribosomes and carbon metabolism.After continuous treatment,irreversible accumulation of protein aggregates and low ATP level were found to be insufficient to support depolymerization of protein aggregates,might cause dormant bacteria to fail to resuscitate.The above results are helpful to deepen the understanding of the formation of dormancy state and killing mechanism of foodborne pathogenic bacteria,such as E.coli O157:H7 in the farmland soil.Our research shed light on theoretical basis and technical support for the prevention and control of similar pathogenic bacteria in soil,which is of great significance for the safety of agricultural products and people’s health.