Effect And Mechanisms Of Eugenol Against Yersinia Enterocolitica

Yersinia enterocolitica（Y.enterocolitica）is an important foodborne pathogen that can cause gastrointestinal symptoms or systemic disease when infected by a host,and severe infections can cause sepsis or even death.Food and water are the main routes of contamination by Y.enterocolitica.Y.enterocolitica has a strong cold tolerance and can survive and multiply in low temperature environments for a long time.Therefore,the bacteria may also be present in refrigerated foods,and consumers may experience symptoms of infection after eating contaminated food.Currently,the misuse of antibiotics leads to varying degrees of resistance to foodborne pathogens.Plant-derived active substances have become a hot spot for the development of natural antibacterial agents due to their broad-spectrum and efficient bacteriostatic ability to foodborne pathogenic bacteria.Studies have shown that some natural chemicals in food can inhibit foodborne pathogenic bacteria and improve the structure of the intestinal flora to promote body health.Eugenol is an active substance widely found in plants that can be used as an edible flavor and food preservative.Studies have reported that eugenol has analgesic,anesthetic,antioxidant,anticancer,anti-insectic,antibacterial and antibacterial activities,but eugenol’s bacteriostatic and anti-infective effects on Y.enterocolitica and its mechanism have not been reported.In this study,eugenol was used as the active substance of plant origin and Y.enterocolitica as the subject to analyze the bacteriostatic mechanism of eugenol against Y.enterocolitica.Study of the inhibitory effect of eugenol on Y.enterocolitica in laboratory models（PBS solution and TSB broth）and food models（sterilized skimmed milk and pork chyme）.The mechanism of action of eugenol on immature and mature biofilm cover were studied.Then the effects of sub-inhibitory concentrations of eugenol on environmental tolerance,exercise ability,quorum induction,toxin production,infection with intestinal epithelial cells and related gene transcription were investigated.Finally,the effect of eugenol on Y.enterocolitica infection in mice and its effect on host intestinal flora was investigated.This study has important theoretical significance and application value for the control of Y.enterocolitica and its infection in food.The results of the specific study are as follows:（1）The antibacterial concentrations of 15 plant-derived active substances against Y.enterocolitica were determined by liquid dilution method.Based on the antibacterial effect and safety of plant-derived active substances,eugenol was selected as the follow-up research to explore its inhibitory and anti-infective effects and mechanisms on Y.enterocolitica.The minimum inhibitory concentrations of eugenol against Y.enterocolitica from different sources were determined by liquid dilution method,which were 0.2～0.8 mg/m L.Finally,the inhibitory effect of eugenol on Y.enterocolitica in laboratory conditions phosphate buffered saline（PBS）and tryptone soy broth（TSB）and in food model skimmed sterilized milk and ground pork were investigated.The results showed that eugenol could effectively control Y.enterocolitica in PBS,TSB,skimmed sterilized milk and ground pork.（2）The growth curve of Y.enterocolitica on eugenol treatment was determined by liquid dilution method,and the results showed that eugenol could reduce the growth rate of Y.enterocolitica.The study further explored the possible antibacterial mechanism of eugenol against Y.enterocolitica.The findings suggest that eugenol alters bacterial cell morphology,leading to cell wall damage（increased AKP（alkaline phosphatase）enzyme leakage）in Y.enterocolitica.In addition,eugenol can cause Y.enterocolitica cell membrane damage,which is manifested by changing intracellular and extracellular ATP（adenosine triphosphate）concentrations,increasing leakage of cellular contents(OD_{260 nm}),and changing the membrane potential.These were further demonstrated by NPN（N-Phenyl-1-naphthylamine）absorption experiments,confocal laser microscopy and flow cytometry observations.The increase of intracellular ROS（reactive oxygen species）and MDA（malondialdehyde）levels induced by eugenol may be due to the damage of the cell membrane caused by eugenol to lipid oxidation of Y.enterocolitica.The results of RT-q PCR assay showed that the transcription of genes such as psp A,FORC2＿RS20610,met K,pst A and acr A related to survival of Y.enterocolitica were decreased by eugenol.（3）The minimum biofilm inhibitory concentration and minimum biofilm eradication concentration of eugenol on Y.enterocolitica were determined by liquid dilution method,firstly.Subsequently,the inactivation effect of eugenol on the biofilm Y.enterocolitica was explored from two aspects:the inhibition of the formation of Y.enterocolitica biofilm and the elimination of the mature biofilm.The Observation of the changes of viable bacteria,extracellular matrix,biofilm components and morphological structure in Y.enterocolitica biofilm were detected by MTT（3-（4,5）-dimethylthiahiazo（-z-y1）-3,5-di-phenytetrazoliumromide）method,flourier transform infrared spectroscopy,confocal laser microscopy and field emission scanning electron microscopy to explore the possible mechanism of action of eugenol.The results showed that eugenol could inhibit the formation of Y.enterocolitica biofilm and change the composition of the biofilm.In addition,for mature biofilms,eugenol could effectively eliminate the biofilm formed on the stainless-steel surface.The above results provide a theoretical basis for the use of eugenol in food processing to control Y.enterocolitica.（4）The sub-inhibitory concentration of eugenol on Y.enterocolitica was determined by the growth curve of Y.enterocolitica against eugenol.Firstly,the effects of sub-inhibitory concentrations of eugenol on the cold,gastric acid and bile salt tolerance of Y.enterocolitica were investigated,and then the effects of sub-inhibitory concentrations of eugenol on the motility,adhesion and invasion ability of Caco-2 cell,the production of stable toxin of Y.enterocolitica were investigated.Finally,the effect of eugenol on pathogenicity-related genes of Y.enterocolitica were detected by RT-q PCR.The results showed that eugenol can reduce the cold,gastric acid and bile salt tolerance of Y.enterocolitica.The motility,adhesion and invasion of host cells,quorum sensing and toxin production of Y.enterocolitica were also inhibited by eugenol.At the genetic level,eugenol down-regulates the transcription level of virulence-related genes in Y.enterocolitica.（5）The BALB/c mouse model of Y.enterocolitica was constructed to explore the attenuating effect of eugenol on the infection caused by Y.enterocolitica.BALB/c mice were pretreated with eugenol（0.2 mg/g）for 5 days and then orally infected with Y.enterocolitica once at a dose of 10⁹ CFU,during which eugenol treatment continued until day 10,while monitoring changes in mouse body weight.The results show that Y.enterocolitica can cause damage to the intestine,induce the increase of inflammatory factors IL-6,IL-10 and TNF-αin various tissues and induce the activation of NF-κB signaling pathway in ileum tissue.Eugenol treatment attenuated the host inflammatory response caused by Y.enterocolitica.RT-q PCR results showed that eugenol could resist the invasion of Y.enterocolitica by enhancing the intestinal barrier,and at the same time inhibit Y.enterocolitica-induced the transcription level of inflammation-related genes.Through 16s r DNA sequencing technology,it was found that eugenol can regulate the of gut microbiome in mice,and improve the intestinal microbiological disorder caused by Y.enterocolitica.