Antibacterial Mechanism Of Escherichia Coli O157:H7 And Its Biofilm By Ultrasonic Sterilization Technology

Enterohemorrhagic Escherichia coli,as one of the main food-borne pathogens,is very easy to form a biofilm on the surface of the food,which can enhance its pathogenic ability and resistance to bad environment.Ultrasonic technology,as a novel sterilization method with the advantages of high efficiency,cleanliness and no pollution,but its unclear antibacterial mechanism limits its further application in the field of food antibacterial.Therefore,this paper focused on exploring the antibacterial mechanism of ultrasonic sterilization on EHEC O157 and its biofilm.Firstly,we used transmission electron microscopy to observe the changes in bacterial morphology after ultrasonication.Then compared the changes of bacterial conductivity,?-galactosidase activity and intracellular biomolecules before and after ultrasonication to analyze the effect of ultrasonication on cell membrane permeability.The results of electron paramagnetic resonance and intracellular reactive oxygen species verified that the ultrasonication will generate free radicals,which will cause the rise of intracellular oxidative pressure.On this basis,we analyzed the effect of ultrasonication on bacterial physiological metabolism through the determination of key intracellular enzymes and the inhibition test of respiratory metabolism.In addition,in order to explore the inhibition mechanism of ultrasonication on EHEC O157: H7 biofilm,the effects of ultrasonication on bacterial flagella and fimbriae were verified through fluidity experiments and phenotype experiments.Subsequently,by comparing the content of extracellular polymer before and after ultrasonication treatment,the effect of ultrasonication on the synthesis of extracellular polymer in biofilm was revealed.Further,we studied the effects of ultrasonication on the expression of biofilm formation-related genes csgB and csgD at the molecular level.Finally,ultrasonication was applied to 3 kinds of fruits and vegetables and 3 kinds of fruits and vegetables juice to evaluate its actual antibacterial ability.The main research results are listed as follows:(1)The best treatment conditions of ultrasonication for EHEC O157: H7 were P: 100 W,T: 7 min,I: 50 W/cm2.Under this condition,the sterilization effect reached 99.99%.(2)After ultrasonication,the bacterial membrane structure was destroyed,with the increase of electrical conductivity and ?-galactosidase activity,as well as causing bacterial lysis revealed by transmission electron microscopy results.After ultrasonication treatment,the content of EHEC O157: H7 intracellular biomolecules including soluble protein,DNA,and ATP decreased significantly.In addition,ultrasonication resulted in the rise of the intracellular oxidative pressure through the reaction between the generated hydroxyl radicals with intracellular biomolecules to form secondary free radicals.Besides,the production of bacterial ATPase,AKP enzyme,and enzyme activity were greatly reduced after ultrasonication treatment.At the same time,ultrasonication mainly inhibited the HMP respiratory metabolism pathway of EHEC O157: H7 by decreasing the activity of the key enzyme G6 PDH in this pathway.(3)Crystal violet staining experiments showed that as the ultrasonication intensity increased,the eliminating ability for biofilm got better.Scanning electron microscopy showed that a dispersed biofilm was obtained after sonication treatment.Metabolic experiment showed that the metabolism of bacteria in the biofilm decreased after ultrasonication.(4)The fluidity test showed that the motility of bacteria decreased after ultrasonication treatment.In addition,the formation of curly fimbriae and cellulose were reduced after ultrasonication treatment as revealed by the phenotypic experiment.The extracellular polymers of EHEC O157: H7 were mainly composed of extracellular polysaccharides,extracellular proteins,and eDNA.After ultrasonication,the three extracellular polymers were significantly reduced.At the same time,the expression level of csgB and csgD decreased by ultrasonication treatment.(5)When applying ultrasonication in the treatment of fruit and vegetable juices,the results showed that it can reduce the number of bacteria by 3-4 Log.While treating biofilms on fruits and vegetables,only 1-2 Log reduction of bacteria population was achieved after ultrasonication treatmentcan,without the adverse impact on food quality as revealed by sensory evaluation results.