| Escherichia coli O157:H7 is an important pathogenic bacteria that infecting both domestic animals and one of the main foodborne pathogenic bacteria.Since found in 1982,there has been a high incidence and mortality at home and abroad.As its strong acid resistance,low infection dose,multiple transmission routes and easy cultivation,it has become a global public health problem.In the process of food processing and storage,a variety of stress environments can inhibit or even kill harmful bacteria.However,the study found that sublethal stress may induce the survival pathogenic bacteria to produce stress tolerance response,and may also enhance its toxicity,and thus produce higher food safety risk.Lactic acid is the main stress factor of fermented food and acid food.During the process of processin g and storage,the gradual increase of lactic acid level and the existence of other stress factors may produce sublethal effects on pathogenic bacteria,which may induce the stress tolerance of the bacteria.At present,there are few reports on the variation in the survival and virulence gene expression of different strains of E.coli O157:H7 under acid stress and combined stress of lactic acid and salt,especially the dynamic changes in the stress process.Therefore,in this study,the lactic acid resistance of the E.coli O157:H7 isolated from the cow fecal and the strains collected in our lab was compared,and then the different acid resistance strains with positive virulence genes were selected,and the changes of the survival and virulence gene expression under lactic acid stress with lactic acid adaptation,the combined stress of lactic acid and salt was investigated,and the correlation between the survival and virulence gene expression was further analyzed.This study can provide a theoretical basis for the risk assessment of the food safety of E.coli O157:H7,and provide a scientific idea for the effective implementation of the integrated anticorrosion measures of E.coli O157:H7 in the practical production and processing.The specific research content and results are as follows:1?Isolation and identification of Escherichia coli O157 from cattle and comparison of their acid tolerance ability309 fecal and beef samples were collected,enriched with immunomagnetic beads and spread onto CHROM agar,suspected colonies were picked and tested the genes of rfbO157 and fliCH7 by PCR.Then PCR-positive isolates were further identified by means of biochemical identification,serological identification and rapid detection of immune colloid gold.The 4 major virulence genes(eae?hly?stx1?stx2)were tested by multiplex PCR.A total of 24 E.coli O157 strains including the isolates above were respectively treated with hydrochloric acid and lactic acid and their acid tolerance ability was investigated.The results showed that 7 isolates of E.coli O157:H7 and 1 isolate of E.coli O157:NM were identified and the positive detection rate of E.coli O157 was 2.59%.The results of virulence genes showed that stxl and stx2 genes were not detected and eae and hly genes were detected in 6 isolates.The results of acid tolerance experiment showed that the survival counts of 24 strains was significantly decreased after treatment with hydrochloric acid or lactic acid for 2 h,however,the reductions in 24 strains were variable,and the acid tolerance ability of the same strain to different acid treatment was different.2?Effects of lactic acid stress with lactic acid adaptation on the survival and virulence gene expression of E.coli O157:H7Based on the above experiments,6 E.coli O157:H7 strains with different lactic acid tolerance were selected for lactic acid stress with lactic acid adaptation.After adapted under pH 5 lactic acid for different time(1,2,3 and 4 h),pH 3.5 lactic acid stress was carried out for different time(30,60,90 and 120 min),and the effects of this kind stress on the survival of different strains were measured.The results showed that the survival counts of 6 strains all decreased significantly after lactic stress,but the degree of reduction was not the same.After acid adaptation,the changes of lactic acid resistance were different,lactic acid resistance of strain J42 did not change significantly,the lactic acid resistance of CICC21530 did not increase,and even decreased after acid adaptation for 1 h compared with the non-adapted group.The lactic acid resistance of the other 4 strains increased significantly,and the highest lactic acid resistance was obtained by the strain 89 adapted for 3 h,the strain 95 adapted for 4 h,the strain 109 and 112 adapted for 3 h and 4 h.Further,the change of virulence gene expression was quantitatively analyzed by means of Real-time PCR.The results showed that the variation of virulence gene expression among strains was observed,and the change of different virulence genes of the same strain varied.After lactic stress,the expression level of stxl of strain 95 increased,while the eae expression of strain 95 decreased.Other virulence genes of other strains decreased or had no significant difference.Acid adaptation increased the expression of sixl,stx2,eae of strain CICC21530 and eae and hly of strain 89 and strain J42 in some stress treatment groups,while acid adaptation reduced the expression of stx1 and eae of strain 95 and eae and hly of strain 109 and strain 112 in some stress treatment groups.Based on the results of bacterial survival and virulence gene expression,it was found that there was no correlation between them.3?Effects of the combined stress of lactic acid and salt on the survival and virulence gene expression of E.coli O157:H7On the basis of the above experiments,3 strains of E.coli O157:H7 with different lactic acid tolerance were selected to treated with the combined stress of lactic acid and salt.The combined application of pH 3.5 lactic acid and different concentration salts(0%,6%,12%,18%)was used to treat strains for 0,3,6,9,12,24,36,48 and 72 h respectively.The results of bacterial survival showed that the greatest inhibition effect on the survival of three strains(CICC21530,95 and 109)was lactic acid stress alone.When the pH 3.5 lactic acid was combined with three different concentrations of salt,it was found that the presence of salt significantly enhanced the acid resistance of 3 strains,which the salt concentrations of greatest enhancing effect for strain CICC21530,95 and 109 were 6%,12%and 18%,respectively.The inhibition effect of salt stress on 3 strains was different.Compared with salt stress alone,the survival bacteria decreased significantly treat with the combined stress of acid and salt of certain concentration,indicating that the inhibitory effect of combination of acid with salt was higher than that of salt alone.Further,the change of virulence gene expression was quantitatively analyzed by means of Real-time PCR.The results showed that the expression of virulence genes varied in the 3 strains during the whole stress process.Compared with those before stress,the expression of some virulence genes in 3 strains increased significantly after stress.After the combined stress for 24 h,the virulence gene expression of combined stress of lactic acid and 18%salt in strain CICC21530 and strain 95 was higher than most other stress groups,while in strain 109 was 6%salt.There was no correlation between the bacterial survival and virulence gene expression.The survival number of the 3 strains decreased while the expression of some virulence genes increased significantly. |
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