| Recently,a larg number of outbreaks about non-O157 STEC,especially "big six",have been reported worldwide,which also belong to main food-borne pathogens.During the food processing and storage,the combination of various huddle factors can inhibit or even kill harmful bacteria,but more and more studies have found that they may have cross-protection and increase the survival rate of bacteria,which would increase risk of food safety.Non-0157 E.coli also has high acid resistance like 0157.At present,there are limited reports on the effects of heat shock-acid stress on the survival of non-O157 E.coli,especially the dynamic changes of stress genes and virulence genes expression during stress.Therefore,the objectives of this study were:Firsty,the contamination of non-0157 E.coli in fecal and beef samples was investigated,and then the most important serotype in "big six",i.e.026,was selected,to determine the resistance to lactic acid and hydrochloric acid.The strains with different tolerance to lactic acid,with or without virulence genes were selected.The survival and virulence genes expression of strains under the heat shock-acid stress were studied to explore the combined effects of heat shock/acid adaptation and acid stress.Furthermore,the stress response mechanism of E.coli 026 under heat shock-acid stress was studied from the changes of bacterial membrane fatty acid composition and stress genes.1?Isolation and Identification of bovine-derived non-0157 E.coli.Reference to the USDA detection method,after selective enrichment of beef and fecal samples,a multiplex PCR was used to determine the O-groups(0157,0121,0111,0103 and 026)as a prescreening method.The positive enriched fluid was streaked for isolation and purification on chromogenic Rainbow agar,and tested about the O-groups.The flagellar antigens were identified by PCR-restriction fragment length polymorphism(PCR-RFLP)and the positive isolates were further verified by means of serum agglutination test.The virulence genes(stx1,stx2,eae,hly)of positive isolates were detected by PCR.The results,a total of 153 fecal samples and 49 beef samples were collected.Overall,40 samples were tested positive for one or more O-groups,and the detection rate in fecal was higher than that in beef,19.3%of which was tested positive for non-0157,and 0.50%of which was tested positive for 0157.After isolation and purification,30 positive isolates were identified.026 was detected most often,and 026,O121,O103,O157 accounted for 73.3%,16.7%,6.7%and 3.3%,respectively.The results of virulence genes showed that 3 strains were detected virulence genes.An O26:H11 isolated from fecal carries the hly gene,two strains of O26:H11 isolated from beef,one stxl,hly positived,and the other hly positived.It suggested there is a potential risk of pollution of non-O157 E.coli in the retail beef market.2?Effects of heat shock-acid stress on the survival and virulence genes expression of E.coli O26.Based on the above experiment,E.coli O26,isolated most often,was selected as the experimental object.Accoring to the acid tolerance of E.coli O26,different strains with or without positive virulence genes were selected to investigate the effects of heat shock and acid adaptation on the survival and virulence genes expression of E.coli O26.(1)The lactic acid and hydrochloric acid tolerance test for E.coli O26 was carried out in hydrochloric acid-acidified TSB(pH=2.0)and lactic acid-acidified TSB(pH=3.5)at 37? for 2 h.The results of acid tolerance experiment showed that the survival counts of 23 strains were all significantly decreased after acid stress,and there were differences in all strains.After hydrochloric acid treatment,the survival rate of 133Z(hly+)strain was the highest(92.92%),while the survival rate of strain G13Z1(stx1+,hly+)was lowest(57.98%),and strain 43SZ had the highest survival rate of about 91.08%,strain 83Z3 had the lowest survival rate of about 62.54%after lactic acid treatment.(2)Seven strains of E.coli 026 were selected according to the acid tolerance test above.The experimental groups included acid stress group,heat shock-acid stress group,acid adaption-acid stress group and heat shock-acid adaptation-acid stress group.Heat-shocked cells were prepared in 48? water bath for 15 min,and acid-adapted cells were prepared under pH 5 lactic acid for 3 h.All treatment groups were stressed at 37?in lactic acid-acidified TSB(pH=3.5)for 2 or 8 h,and TSB group as the control.The results showed that all the treatment groups inhibited the bacteria.Among them,5 strains were significantly inhibited by lactic acid.Heat shock/acid adaptation improved the viability of 5 strains under acidic conditions.Combinition of heat shock and acid adaptation produced a stronger cross protection effect.The acid resistance of two other strains increased after heat shock treatment,which were not found after treatment of acid adaptation or heat shock-acid adaptation.The effect of heat shock-acid stress on bacterial survival was not related to whether the strain carried virulence genes.(3)The virulence genes expression was further quantitatively detected by real-time PCR.The results showed that the stxl expression of strain S433 and 110Z4 were upregulated after acid stress.The stxl expression of acid-stressed cells were downregulated after acid adaptation,while the expression of cells of acid-adapted acid stress were upregulated after heat shock treatement.The hly expressions of strains S433 and G13Z1 were downregulated after acid stress,and the expression of three strains of acid-adapted acid stress was upregulated after heat shock treatment.In summary,it was found that heat shock and/or acid adaptation increased the acid resistance of most strains,increased their survival,and prsented the cross-protection effect.Moreover,heat shock and/or acid adaptation caused upregulation of stx1 and hly genes expression of some acid-stressed strains,enhanced their virulence.The results indicated that heat shock/acid adaptation could increase the risk of acid-stressed bacteria.3?Effects of heat shock-acid stress on membrane fatty acid composition of E.coli 026.Based on the above survival experiments,3 E.coli 026 strains were selected to dettected the membrane fatty acid composition of bacterial under heat shock-acid stress for 4 h by means of gas chromatography.The results showed that the fatty acid composition of the three E.coli 026 after heat shock-acid stress changed.Except for acid stress group and heat shock acid stress group of strain S433,all other groups showed a decrease in USFA content,an increase in SFA content,and a significant decrease in U/S after acid stress.The U/S decrease of three strains of heat shock-acid adaptation-acid stress group was most obvious.The membrane fluidity decreased and proton influx was inhibited into the cell.It suggested that the mechanism of cross-protection under heat shock/acid adaptation,which could enhance bacterial survival,is related to the change of bacterial membrane fatty acid composition and the decrease of U/S.4?Effects of heat shock-acid stress on related genes expression of E.coli 026.Real-time PCR was used to quantitatively detect the gene's expression of 3 E.coli 026 strains under heat shock-acid stress for 2 h and 4 h,which included general stress gene(rpoS),acid stress-related genes(asr,ycfR and gadA)and heat stress-related genes(rpoH,DnaK,clpB and groEL),and to reveal the response mechanism.The results were as follows:(1)After heat shock treatment,an upregulation of rpoS gene was detected in all 3 acid-stressed strains of E.coli.The treatment of heat shock-acid adaptation/acid-adaptation changed the expression of rpoS gene,and there were differences in three strains.(2)In addition to the upregulation of gadA gene expression of strain S433 and G13Z1 at 2 h,a downregulation of acid related genes in all 3 strains under lactic acid stress.The expressions of asr and ycfR genes were upregulated in all 3 acid-stressed strains after heat shock/acid-adapted treatment,while heat shock treatment downregulated the expression of gadA gene in all 3 acid-stressed strains at 4 h.Acid adaptation resulted in the downregulation of asr gene expression in heat shock acid-stressed cells of all 3 strains.Heat shock-acid adaptation resulted in downregulation of gadA gene expression in all 3 strains of acid-adapted acid stress,and upregulation of ycfR gene expression in all 3 strains of acid-adapted acid stress at 4 h.(3)A significantly downregulation of expression of rpoH,groEL and clpB genes in all 3 acid-stressed E.coli 026 stains,but the expression of dnaK gene were differences in all 3 strains.The expressions of rpoH and groEL genes were upregulated in all 3 acid-stressed strains after heat shock,acid-adapted or heat shock-acid adapted treatment,but the the expression of clpB and dnaK gene were differences in all 3 strains.Acid adaptation caused differences in heat stress gene expression strains of 3 heat shock acid-stressed strains.Heat shock resulted in upregulation of rpoH gene expression in all 3 strains of acid adaptaed acid stresss at 4 h,and the expression of clpB and dnaK genes in strains S433 and G13Z1 were significantly upregulated.There were differences among other gene expression strains.In summary,the results suggested that the mechanism of cross-protection of heat shock and/or acid adaptation to acid-stressed cells were related to the expression of genes such as rpoS,asr,ycfR,rpoH and groEL. |
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