| Shiga toxin-producing Escherichia coli(STEC)is an important food-borne pathogen,including many serotypes of E.coli O157 and non-O157,which can cause many diseases after human infection.It has been reported that some outbreaks of STEC are related to the acidic foods,such as fermented sausages,suggesting that STEC can be resistant to various hurdle factors(such as organic acids,salt and antimicrobial substances metabolized by lactic acid bacteria)in fermented sausages,but the specific regulatory mechanism has not been clarified.At present,studies of combination of different stress factors on the inhibition of STEC mostly focused on the strain of E.coli O157:H7,while there are few studies about non-O157.And these studies mostly reported the effect of combination on bacterial survival,but rarely on the characteristics of virulence,especially the mechanism of synergistic and antagonistic effects of combination have not been elucidated.Therefore,the strains of O157:H7 and the most important serotype of non-O157,O26:H11,were utilized as the objects in this study.Firstly,the dynamic changes of survival and virulence in E.coli O157:H7 and O26:H11 were analyzed during the process of fermented sausage.Then,according to the major stress factors of fermented sausage,the effects of combination of lactic acid and salt on the survival,physiological characteristic and virulence of STEC,as well as its regulatory mechanism,were investigated.Based on this result,the effects of combination of lactic acid bacteria and salt on the survival,gene expression of virulence and stress of E.coli O157:H7 and O26:H11 were investigated,which could reveal the antibacterial effect and its possible mechanism.This experiment can provide a scientific thinking for the risk assessment and comprehensive prevention and control of STEC in fermented foods.The specific contents and results of this research are as follows:1.Changes of survival and virulence genes of Shiga toxin-producing Escherichia coli in fermented sausagesIn this experiment,E.coli O157:H7(ATCC 43895)and O26:H11(G13Z1)were respectively inoculated into the meat,and the meat was not inoculated with STEC used as the control group.These meats were conducted to sausage fermentation test according to the processing technology of salami fermented sausage in some company.The results showed that the pH value dropped rapidly,the concentration of lactic acid increased,the value of water value gradually decreased,as well as the number of lactic acid bacteria increased first and then decreased during the fermentation of sausages.And there was no significant difference between the group of inoculation and no-inoculation.During the whole fermentation process of sausage,there were different effects of inhibition on the strain of O157 and O26.The count of O157 and O26 respectively decreased by 1.0 and 0.28 log CFU/g within 5 days,and dropped by 0.5 and 1.1 log CFU/g within 5-26 days.However,the survival of STEC was still greater than 4.6 log CFU/g after sausage production,which indicated that both strains of O157 and O26 could tolerant to the stress condition of fermented sausage,as well as the strain of O26 had better tolerance than the strain of O157.The results of virulence genes showed that the expression of virulence genes of O157 and O26 increased during sausage fermentation,while there was no significant difference on some expression of virulence genes.It was indicated that there was difference between the two serotypes of STEC,but the expression of virulence genes per log survival significantly increased during the later stage of sausage process.These results indicated that there are differences on the inhibition effects of different serotypes of STEC during the process of sausage production.And the survival of STEC decreased while the expression of virulence genes increased.2.Effect of combination stress of lactic acid and sodium chloride on the survival,physiological characteristics,and expressions of related genes of Shiga toxin-producing Escherichia coliThe strains of Escherichia coli O157:H7(ATCC 43895,ATCC 35150 and CICC 21530)and O26:H11(S433,G13Z1 and G10Z1)were selected and used under the stress of combination of acid and salt.The experiment can be divided into the control group,the group of acid treatment(pH 3.5),and the group of salt supplementation(pH 3.5 + 3,6,12 and 15%Na Cl),all treatments are cultured at 25 ℃ for 48 h.The results showed that the survival of E.coli O157:H7 and O26:H11 were significantly inhibited after lactic acid stress,and the combination of various concentrations of salt showed different effects on the inhibition of survival during the later stage of stress.The results of transmission electron microscope and scanning electron microscope showed that the damage of STEC was not obvious under the early stage of lactic acid stress.When combination of lactic acid and salt,the damage of cell was obvious under the later stage of stress,and there were different effects of microstructure between the group of different salt addition.The results of confocal laser scanning microscope showed that combination stress of acid and salt had not significant damage on the membrane permeability of cell.Membrane fatty acid composition of O157 and O26 significantly changed after lactic acid stress,and the ratio of unsaturated fatty acids/saturated fatty acids and intracellular pH significantly decreased.When compared with the group of acid treatment,there was no significant difference in the group of 3% salt addition and 12%salt addition.The results of the expression of stress genes showed that,the expression of rpoS,asr and gro EL genes of E.coli O157 and O26 significantly increased after acid stress for 2h,but there was no or down-regulated effect on the expression of genes with the time,indicating that STEC might maintain the cell activity by increasing the expression of acid resistance genes in the early stages of acid stress.When compared with the group of acid treatment,there was different effect on the expression of genes in the group of 3% salt addition,while the expression of asr and speF of O157 significantly up-regulated in the group of 12% salt addition,and there were no or down-regulated effects on the expression of genes of O26,which indicated that the changes of stress genes were different among different serotypes.And the expression of some genes of acid stress were significantly higher than the group of 3% salt addition of O157,which may be related with the acid resistance of the group of 12% salt addition.The results of expression of virulence genes showed that the expression of most virulence genes of O157 significantly increased after lactic acid stress,while the expression of most virulence genes of O26 significantly decreased or showed no difference,which indicated that there was difference on the expression of virulence genes between various serotypes after acid stress.When compared with the group of acid stress,the expression of virulence genes of O157 and O26 was different in the group of 3% salt addition with the various times of stress,while the expression of virulence genes of O157 significantly increased in the group of 12% salt addition.These results showed that there were different stress responses among different serotypes of STEC.The strains of STEC mainly exerts the acid resistance by changing the composition of membrane fatty acids and up-regulating the expression of acid stress genes during the early stage of lactic acid stress.Addition of different concentrations of salt had different combination effect on the survival of STEC under acid stress,it may be related to the regulation of stress genes.3.Effect of combination stress of lactic acid and sodium chloride on the transcriptomics analysis of Escherichia coli O157:H7The samples of the group of control,acid treatment,3% salt addition and 12% salt addition of E.coli O157:H7 were selected for the transcriptomics analysis.The results showed that there were 811 significantly differentially expressed genes(386 up-regulated and 425 down-regulated)in group of acid treatment when compared with the control group.When compared with the group of acid treatment,there were 51(42 up-regulated,9 downregulated)significantly differentially expressed genes in the group of 3% salt addition and23(1 up-regulated,22 down-regulated)genes in the group of 12% salt addition.There were139 significantly differentially expressed genes(31 up-regulated,108 down-regulated)in the group of 12% salt addition as compared with the group of 3% salt addition.The function analysis of different expressed genes showed that the pathways related to carbohydrate,amino acids and energy metabolism,as well as quorum sensing system increased in the strain of E.coli O157,while the bacterial chemotaxis and flagella assembly decreased after lactic acid treatment for 12 h,which indicated that E.coli O157 might activate the pathways of energy synthesis and inhibit the energy consumption,and then exert the effect of acid resistance.Compared with the group of acid treatment,the fatty acid metabolism increased in the group of 3% salt addition,while the propionic acid and the quorum sensing system decreased,as well as the bacterial infection and the type III secretion system decreased in the group of 12% salt addition.Compared with the group of 3% salt addition,the quorum sensing,amino acid synthesis and nucleic acid transport increased in the group of 12% salt addition,while the fatty acid metabolism and bacterial infection were decreased,which may be related with the antagonism effect under acid stress.These results indicated that the expression level of genes of E.coli O157 significantly changed under combination stress of lactic acid and salt.When compared with the group of acid treatment,the transcription level of E.coli O157 had significant difference under the addition of various concentrations of salt,this may be related to the different combination effect of salt under acid stress.4.Effect of combination stress of lactic acid and sodium chloride on the transcriptomics and proteomics analysis of Escherichia coli O26:H11The samples of the group of control,acid treatment,3% salt addition and 12% salt addition of E.coli O26:H11 were selected for the transcriptomics analysis.Transcriptomic results showed that there were 472 significantly differentially expressed genes(223 upregulated and 249 down-regulated)of E.coli O26:H11 in the group of when compared with the group of control.When compared with the group of acid treatment,there were 273 differentially expressed genes(103 up-regulated,170 down-regulated)in the group of 3%salt addition,and 39 significantly differentially expressed genes(19 up-regulated and 20down-regulated)in the group of 12% salt addition.There were 456 differentially expressed genes(249 up-regulated,207 down-regulated)in the group of 12% salt addition as compared with the group of 3% salt addition.The functional analysis of different expressed genes showed that the pathways related to carbohydrate metabolism,amino acid metabolism,energy metabolism,fatty acid degradation,thiamine metabolism and quorum sensing of E.coli O26 increased after lactic acid stress for 12 h,but the pyrimidine metabolism decreased,which indicated that E.coli O26 exerted the acid resistance by activating the pathways of energy synthesis.When compared with the group of acid treatment,the nitrogen metabolism and phage protein response improved in the group of 3% salt addition,while the TCA cycle,carbohydrate metabolism,amino acid synthesis,fatty acid degradation and pyrimidine metabolism decreased,which indicated that the pathways of energy synthesis were inhibited when the addition of 3% salt.The purine metabolism increased in the group of 12% salt addition as compared with the group of acid stress,but the pyrimidine metabolism decreased,which maybe related with the energy conservation.Compared with the group of 3% salt addition,the TCA cycle,propionic acid and glyoxylic acid metabolism,arginine synthesis,and lysine degradation increased in the group of 12% salt addition,which indicated that the pathways of energy synthesis of E.coli O26 was activated when the addition of 12% salt.Proteomics results showed that there were 80 different expressed proteins(43 up-regulated and 37 down-regulated)in the group of 3% salt addition as compared with the group of acid treatment.Functional analysis of different expressed proteins showed that the transport and metabolism of carbohydrates changed,while the expression of proteins of DNA recombination and repair,as well as some transcriptional factors increased in the group of 3%salt addition when compared with the group of acid treatment.These results indicated that the expression level of genes of E.coli O26 significantly changed under the combination stress of lactic acid and salt.When compared with the group of acid treatment,the transcriptional level of genes was significant difference when the addition of various concentrations of salt,which may be related with different effects of combination of salt under acid stress.In addition,the changes of expression level of protein might be related to the enhancing antibacterial activity of acid under the addition of 3% salt.5.The effect of salt addition on the inhibition of lactic acid bacteria on the survival of Escherichia coli O157:H7 and O26:H11In this experiment,three strains of lactic acid bacteria(Lactobacillus sakei,Lactobacillus plantarum and Pediococcus pentosaceus)were isolated from the sausage starter.And these lactic acid bacteria were co-cultured with E.coli O157:H7(ATCC 43895)or O26:H11(G13Z1)in the liquid medium of LB:MRS(2:1)or supplemented with 3% salt.There were two experiments of co-culture,including mixed culture and separated culture.The results showed that the growth of E.coli O157 and O26 increased some in the mixed culture at the initial stage,and the survival gradually decreased with the increased metabolic production of lactic acid bacteria,and the inhibition effect of 3% salt supplement was better.When E.coli O157 and O26 were separately cultured with P.pent,the inhibition effect increased as the time prolonged,and the inhibition effect was more obvious when supplementation of 3% salt.The results of expression of stress genes showed that the expression of acid stress-related genes of cad A and ycf R in E.coli O157 significantly increased when P.pent separately cultured with E.coli,while the expression of salt stressrelated genes significantly inhibited.And the expression of acid stress-related genes of rpoS and cad A and the salt stress-related genes of proV and proW significantly increased when the addition of 3% salt.The expression of acid stress-related gene of ycf R in E.coli O26 significantly increased when P.pent separately cultured with E.coli,while the expression of salt stress-related genes significantly decreased.And the expression of acid stress-related gene of rpoS and salt stress-related genes of ompC,proV and proW significantly increased when supplementation of 3% salt.The results of virulence genes showed that the expression of virulence genes in E.coli O157 significantly decreased when P.pent separately cultured with E.coli in the medium with or without 3% salt,while the expression of stx1 gene in E.coli O26 increased.These results showed that the survival of E.coli was inhibited when E.coli O157 or O26 was co-cultured or separately cultured with lactic acid bacteria,and the inhibition effect was better when the addition of 3% salt.E.coli O157 or O26 may resist the response of P.pent and salt stress by up-regulating the expression of acid stress-related and salt stress-related genes.There were different expressions of virulence genes among different serotypes of STEC under stress conditions. |
