| Subletahl injury is a reversible injury of bacteria under stress.Sublethally injured cells cannot form single colony on selective medium that usually used in food detection.And these injured cells can recover all physiological activity and toxicity under suitable conditions.E.coli O157:H7,a common foodborne pathogen,can survive as the state of sublethal injury under lactic acid stress.At present,the study of sublethally injured bacteria mainly focuses on the condition of sublethal injury formation and recovery.However,it is limited that the study of effects of food ingredients on injury formation and recovery.Meanwhile,there are few studies focused on the functional changes of sublethally injured and recovered cells.Therefore,this study investigated the effects of some food ingredients and characteristics on formation and recovery of lactic acid-injured E.coli O157:H7,and the resistance of recovered cells to lactic acid.In addition,the changes and effect factors of motility and adhesion of E.coli O157:H7during lactic acid-sublethal injury and recovery are also investigated.It provides a theoretical basis for futher understanding the formation and recovery of sublethally injured bacteria.The mainly studies and results are listed as follows:（1）E.coli O157:H7 was treated by lactic acid with different concentration（0.5-8m M）,and the optimum preparation condition of sublethally injured E.coli O157:H7was obtained to investigate the effect of carbohydrate（glucose,fructose,mannose,lactose,maltose and sucrose）,cation(Fe3+,Fe2+,Mg2+,Ca2+,Zn2+and Mn2+)and Na Cl（0.85%,3%and 7%）on sublethal injury formation.Our results showed that E.coli O157:H7 treated by 4 m M lactic acid for 60 min,the sublethal ration was more than 99%and had no dead cells.The addition of carbohydrate reduced injured cells formation.Fe3+and Fe2+aggravated cells injury,but Mg2+,Ca2+and Zn2+reduced injured cells formation and the addition of Mn2+had no significantly effect on sublethal injury.In addition,the concentrations changes of Na Cl decreased sublethal injury formation,and E.coli O157:H7 died under high salt conditions.（2）The effects of carbohydrate and cation on initial recovery of lactic acid-injured E.coli O157:H7 were studied,and the resistance of recovered cells to lactic acid was also investigated.Results showed that sublethally injured E.coli O157:H7 could be recovered in minimum medium min A（min A）.The injured cells recovered by min A for20 min were initial recovered cells and for 180 min were completely recovered cells.Completely recovered cells were sensitive to lactic acid.Glucose,fructose,mannose and maltose increased the recovery ratio,but lactose and sucrose had no significantly effect on recovery of injured E.coli O157:H7.Fe3+,Fe2+and Zn2+inhibited recovery,but Ca2+promoted the recovery and Mg2+or Mn2+had no significantly effect on recovery of injured E.coli O157:H7.（3）Gene changes of untreated and lactic acid-sublethally injured E.coli O157:H7were analyzed by RNA-seq.Compared with untreated cells,151 differentially expressed genes were found in sublethally injured E.coli O157:H7,including 53 up-regulated and98 down-regulated genes.These genes were related to many cell functions,such as carbohydrate transport and metabolism,cation transport,response to stress,cell division,motility and adhesion.It was confirmed that carbohydrate,cation and osmotic affected the sublethal injury formation,and the virulence properties of lactic acid-injured E.coli O157:H7 were changed.（4）Gene changes of lactic acid-sublethally injured and initial recovered E.coli O157:H7 were analyzed by RNA-seq.Compared with sublethally injured cells,2480differentially expressed genes were found in initial recovered E.coli O157:H7,including 1110 up-regulated and 1370 down-regulated genes.These genes were significantly enriched in many GO term,such as biological adhesion,membrane,oxidation-reduction process and so on.Cell membrane was primarily repaired during recovery.The ability of carbohydrate and cation transport increased but oxidation-reduction decreased in initial recovered cells.Meanwhile,flagella and pili related genes were up-regulated and enriched in motility and biological adhesion GO term,and the T3SS genes related to virulence were also up-regulated.It was confirmed that carbohydrate and cation affected the injured cells recovery and the virulence of recovered E.coli O157:H7 was recovered even enhanced.（5）The changes and effects factor of motility and adhesion of E.coli O157:H7during lactic acid-sublethal injury formation and recovery were investigated.Our results showed that the motility and adhesion of E.coli O157:H7 were inhibited during lactic acid sublethal injury,but they were recovered and enhanced during recovery.The q RT-PCR results indicated that the motility in these processes might be mediated by flagella and the adhesion was controlled by pili.Meanwhile,carbohydrate and cation affected the motility and adhesion of sublethally injured and recovered cells,but the effects trend was different from that of sublethal formation and recovery. |
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