Effect Of Pili And Surface Properties On Adhesion Of Lactic Acid-Injured And Recovered Escherichia Coli O157:H7

Sublethal injury refers to the reversible cellular injury that caused by different chemical or physical treatments.In food-processing environment,sublethally injured(SI)bacteria can recover its virulence and other physiological functions under suitable conditions,which greatly increased the difficulty in the prevention and control of foodborne bacteria contamination.The contamination of foodborne bacteria begins with surface adhesion.The bacteria adhering on the surface are easier to absorb nutrients and complete recovery,thus adhesion was crucial for the recovery of SI bacteria.Escherichia coli O157:H7 is one of the important foodborne bacteria and can be induced into SI state by lactic acid.So far,there were reports regarding the adhesion of healthy or stressed E.coli O157:H7 to abiotic or biotic surfaces,but little is known about the adhesion of SI and recovered cells and the factors affecting it.Our previous research had shown that the upregulated genes of SI E.coli O157:H7induced by lactic acid were significantly enriched in the process of pili assembly at early recovery.Therefore,this study constructed different types of pili-deficient mutants of E.coli O157:H7 to investigate the changes of adhesion ability of the SI and recovered mutants on different surfaces.Then,we studied on the effect of mannose and surface hydrophobicity and charge density on the adhesion of SI and recovered E.coli O157:H7.Results showed that spinach always exhibited the larger adhesion population of E.coli O157:H7 than the other fresh produces.In addition,at least one key pili dominated adhesion on these surfaces,and curli was always included.The adhesion population and contribution of different types of pili was jointly affected by surface and physiological state.Among all the examined carbohydrates,addition of mannose could inhibit adhesion,which was affected by mannose concentration,bacterial physiological state,and surface.Overall,this study provided a theoretical basis for eliminating the repeated pollution of E.coli O157:H7in various foods and an effective reference for the prevention and control of other foodborne pathogens.The main research content and results are as follows:(1)Different types of pili-deficient mutants(O157ΔcsgB,O157Δfim A and O157Δppd D)were constructed through λ-red method.Primer pairs for gene knockout were designed with 20-nt 3′ ends homologous to the upstream and downstream regions of the FRT sites flanking the kanamycin resistance gene in p KD13 and 52-nt 5′ends homologous to the upstream and downstream sequence of the target genes.The primers were used for PCR amplification to obtain the targeting resistance fragments.Red recombinase of p KD46 in E.coli O157:H7 were expressed to replace the target gene with a targeting resistance fragment.Recombinant clones were obtained through colony PCR.Then,the FLP flippase on the p CP20 was used to eliminate the targeting resistance fragments in recombinant clones.Different types of pili-deficient strains without any antibiotic resistance were finally obtained.We also verified the influence of gene deletion on the bacterial sublethal injury,recovery and growth.Results showed that sublethal injury ratio,recovery ratio,and growth curve of all the constructed pili-deficient strains had no significant difference with the wild-type strain.This provided the experimental basis for the follow-up adhesion assay of SI and recovered E.coli O157:H7.(2)The effect of different types of pili(curli,type 1 fimbriae,and type Ⅳ pili)and surfaces(spinach,lettuce,arugula,red cabbage,stainless steel and glass)on the adhesion of SI and recovered(recovery for 20-180 min)E.coli O157:H7.Results showed the adhesion was affected by bacterial physiological states.The adhesion ability of SI cells significantly decreased,but with the gradual recovery,its adhesion ability can be partly or completely recovered.The adhesion ability of completely recovered cells can even surpass the untreated cells.The adhesion ability was different on different surfaces,no matter what the bacterial physiological state is,the adhesion population on spinach was always higher than that on other surfaces.The key pili dominating adhesion was different on different surfaces.The adhesion of E.coli O157:H7 to spinach only required curli,while the adhesion to lettuce and arugula required curli and type 1 fimbriae instead of type Ⅳ pili.The adhesion to red cabbage required all the three types of pili.Stainless steel and glass also required all the three types of pili,among the pili,curli and type 1 fimbriae are most important.Additionally,the contribution of key pili dominating adhesion was also dependent on physiological states.(3)Results obtained from scanning electron microscopy showed the aggregates of E.coli O157:H7 were found near the stomata of the spinach and red cabbage,while the cells were more dispersed away the stomata.Spinach surface was rugged,while red cabbage surface was flat,which may lead to the differences in adhesion between spinach and red cabbage.Co-occurrence of sublethal injury and pili deficiency has a synergistic inhibitory effect on the aggregate formation.(4)The relevance analysis between surface hydrophobicity and charge density of the fresh produces and the adhesion of E.coli O157:H7 was investigated.The surface charge density and hydrophobicity were positively correlated with the adhesion of untreated cells.For the SI and recovered cells,surface hydrophobicity was not correlated with the adhesion,while surface charge density was highly positively correlated with the adhesion.(5)The effect of the addition of exogenous mannose of different concentrations(0-1%)on the adhesion of SI and recovered E.coli O157 was investigated.Results showed that mannose can inhibit,which depended on mannose concentration,bacterial physiological state and surface.Under some conditions,mannose can significantly promote the adhesion.Therefore,it is indirectly speculated that the adhesion was also affected by the carbohydrates on food surfaces.Overall,this study showed that pili and properties of surfaces that bacteria adhere to was important in the adhesion of SI and recovered E.coli O157:H7.