Study On Lethal Effects And Molecular Mechanisms Of Escherichia Coli O157:H7 Under Ultrasonic Field

With the increasing demands for fresh,nutritious and safe foods,the application of non-thermal processing technologies has attracted much attention.As an effective non-thermal processing method,ultrasound can achieve the microbial safety and assure the food quality at the same time.In most studies,acoustic cavitation is widely accepted as the inactivation mechanism,but there is still lack of comprehensive and systematic research on ultrasonic action mechanisms against microorganisms.What’s more,the microbial stress responses under ultrasonic field have not been reported yet,which can increase the tolerance to environmental stress.This may lead to the occurrence of incomplete sterilization and limit the application of ultrasonic processing technology in microbial inactivation.In this study,foodborne pathogen Escherichia coli O157:H7 was selected as the model microbe.Molecular biotechnology,transcriptome and proteome were used to investigate ultrasonic lethal effects and molecular mechanisms.Differentially expressed genes and proteins of E.coli O157:H7 were analyzed to elucidate the relationship between biological characteristics and gene regulations,and microbial stress response mechanisms were revealed under ultrasonic filed.This work lays the theoretical foundation for the further optimization of ultrasonic processing technology,and contributes to ensure the microbial safety in food industry.The main contents and results are summarized as follows:(1)Inactivation effect of E.coli O157:H7 under ultrasonic fieldThe inactivation effect of ultrasound on E.coli O157:H7 was evaluated by different criteria of cell viability,including cell culturability,membrane integrity,esterase activity,and metabolic capacity.The results of plate counting and flow cytometry analysis showed that ultrasound had strong bactericidal effect against E.coli O157:H7,and it became more significant as the duration time and power intensity increased.In addition,flow cytometry combined with double-staining method were used for real-time,quantitative analysis of cell heterogeneity after ultrasound treatments,and special attention was paid to sublethally injured cells and viable but not culturable(VBNC)cells.The results showed that the inactivation effect of ultrasound on the microorganism was not a process of cumulative damage,but indeed an all-ornothing process.(2)Multitarget lethal effects of E.coli O157:H7 under ultrasonic fieldIn order to clarify the action mechanisms and the lethal targets of ultrasound on E.coli O157:H7 at the cellular level,the changes of biological characteristics under different conditions were investigated.The shock waves and microjets from external to internal action interacted with the microstreams acting from the inside to outside,eventually causing irreversible cell damage.In terms of cell membrane function,the shear forces produced by acoustic cavitation could alter cell membrane permeability,and the hyperpolarization of membrane might be related to the opening of potassium ion channels on the cell membrane.As for energy metabolism,the ATP contents decreased after ultrasound treatments,which affected the metabolic capacity and normal physiological function of bacteria.When it comes to DNA molecular structure,ultrasound was able to gradually damage the superhelix and double helix structure of DNA molecules,but it showed there was no obvious damage to the primary structure of DNA in cells.(3)Programmed cell death of E.coli O157:H7 under ultrasonic fieldThe distribution of phosphatidylserine on cell membrane and the activity of apoptotic protease in E.coli O157:H7 cells were investigated after ultrasound treatments.It was shown that ultrasonic field could trigger genetically encoded apoptosis in E.coli O157:H7,resulting in the exhibition of apoptotic biochemical hallmarks like exposed phosphatidylserine and activated caspases.Besides,multifunctional regulator Rec A was identified as a critical factor in the execution of apoptosis.Furthermore,we analyzed the role of cavitation during the apoptosis of E.coli O157:H7.The sonochemical effects could increase the intracellular reactive oxygen species level through multiple pathways.By cooperating with the calcium ion signal,it was involved in the activation of apoptotic protease,triggering cells to enter the apoptotic state.This phenomenon embodied that bacteria gave higher priority to interests of collective than individuals.(4)Gene expression analysis of E.coli O157:H7 under ultrasonic fieldThe gene regulation at the transcription level in E.coli O157:H7 after ultrasound treatment was investigated by RNA-sequencing,and the most enriched pathways were further analyzed in each category.Gene transcription related to a series of crucial cellular processes was down-regulated under ultrasonic field,including genes encoded phosphotransferase system and glycolytic catabolism,genes involved in transmembrane transport and respiratory chain,and genes related to AI-2 mediated quorum sensing system.However,several amino acids biosynthetic pathways were shown to be upregulated with the strengthen of ribosome synthesis and assembly maturation.It was indicated the capacity to transport and decompose carbohydrate was decreased,and the membrane function was affected with energy loss.Besides,the bacterial signal transduction was inhibited,while the protein translation processing was benefited under ultrasonic field.(5)Stress response mechanisms of E.coli O157:H7 under ultrasonic fieldProtein expression changes of E.coli O157:H7 under ultrasonic field were quantitatively analyzed by i TRAQ comined with HPLC-MS/MS method.The molecular regulatory mechanisms of E.coli O157:H7 in response to ultrasonic stress were further investigated,including general stress response,mechanical stress response,DNA damage response,heat shock response and oxidative stress response.It was shown that E.coli O157:H7 could take some strategies to counteract the ultrasonic stress,including the accumulation of global stress response regulator Rpo S,the opening of bacterial mechanosensitive channels and the activation of SOS response system to respair the DNA damage.In addition,we proposed a possible action mechanism that the free radicals produced by acoustic cavitation might enter into cells via the activated mechanosensitive channels,leading to the elevated intracellular ROS level and subsequent cell death.Last but not the least,we inferred the all-or-nothing phenomenon of power ultrasound might due to the destruction of crucial cell defensive systems,including heat shock proteins and oxidative response regulators.