Response Of Acid Stress To Biofilm Formaton And Elimination Of Escherichia Coli O157:H7

Escherichia coli O157:H7 is one of the globally widespread zoonotic foodborne pathogens in the field of food safety.Most foodborne pathogens can form biofilm on the surface of a variety of food processing equipments to resist hostile environments,and thus cause cross contamination,potentially serious hazards to public healthy and huge economic losses to food industry.Therefore,it is important to study the formation and control of foodborne pathogens biofilm.However,the current research about Escherichia coli O157:H7 biofilm mainly focused on the impact of all kinds of environmental factors(mainly including temperature,pH,nutrients and interface type)on the count of biofilm.Actually,in the modern food industry,many food processing systems such as fermented foods are in acidic environments,many organic and inorganic acids are widely used as food additives,preservatives and disinfectants in the food industry.Besides,our laboratory research found that Escherichia coli O157:H7 have a certain resistance to acid stress,but current study on the formation and elimination of Escherichia coli O157:H7 biofilm in an acidic environment has rarely been reported.Considering the above,investigating the effect of long-term acid stress to biofilm formation of Escherichia coli O157:H7 has more practical significance.Therefore,in this study,all Escherichia coli O157:H7 strains collected in our lab were classified by adhesion ability,then the biofilm formation and elimination,changes of spatial structure and extracellular polymeric substances under the long-term acid stress were clarified.The context and results were shown as following:1.Comparing adhesion among different strains of E.coli O157:H7A total of 22 E.coli O157:H7 strains collected in our laboratory were evaluated their adhesion using crystal violet staining method,and then all these strains were classified according to OD value after culturing(25?,and 72 h)and dyeing.The results showed that all of the 22 strains had adhesion ability,in which strain J29(isolated from cow fecal)was the greatest one,stain ATCC43895 was the second,and the other 20 strains showed weaker ashesion ability.Further,strain J29,ATCC43895 and other 4 weak adhesion strains were selected to investigate biofilm formation(2,4,8,12,16,24,36,48,60,72,120,and 168 h)using crystal violet staining method.The results showed that weak ashesion strains began to form biofilm at 2 h but no change was observed as time went on,while strong ashesion strains biofilm increased significantly with the extension of incubation time as long as the nutrition and growth interface was enough.2.Biofilm formation of E.coli O157:H7 under the long-term acid stress and the acid resistance of mature biofilmThree different E.coli O157:H7 strains(J29,ATCC43895 and CICC21530)were treated with acid media(adjusted to a pH value of 3.5,4.0,4.5 and 5.0 with 3 M of lactic acid and 1 M of hydrochloric acid)or control medium(TSB),and their biofilm formation on stainless steel surface were compared.The results show that the ability of biofilm formation was significantly influenced by strains,acid type,acid concentration and incubation time.Compared with ATCC43895,J29 and CICC21530 showed stronger ability of biofilm formation under acid environment,and the lower the pH value was,the less the amounts of biofilm were.The inhibition effect of lactic acid on the biofilm formation was significantly higher than that of hydrochloric acid.The results also showed with the extension of incubation time,biofilm formation gradually increased under the weak acid environment(pH 5.0,4.5 adjusted with lactic acid),while biofilm formed to a certain extent and began to eliminate under the stronger acid environment(pH 4.0 adjusted with lactic acid),and there was no biofilm formation under the stronger acid environment(pH 3.5 adjusted with lactic acid).The mature biofilm elimination effect was affected significantly by acid concentration,and physiological saline adjusted to a pH value of 2.0 by lactic acid showed stronger elimination ability compared with pH 3.5,and the amount of biofilm significantly decreased with the prolonging of acid treatment time under the strong acid of pH 2.0.3.Effects of acid stress on micro-structure and extracellular polymeric substances of E.O157:H7 biofilmThe micro-structure and extracellular polymeric substances of biofilm formed by weak ashesion strain CICC21530 and strong adhesion strain J29 under the acid environment(pH 5.0 ajusted with lactic acid)were determined in situ by CLSM,SEM and ATR-FTIR,compares with those under the neutual environment.The results showed that the two strains were able to form biofilms with a three-dimensional structure,but the biofilm forming process under acid environment was relatively slower than that under the neutral medium.The ATR-FTIR analysis revealed that polysaccharides and proteins were the main composition of EPS.Compared to the spectra of biofilm of the neutral group,the spectra of acid stress group did not show more or less noticeable peak but showed less amount of compounds.Meanwhile,great differences in micro-structure and EPS components were observed between the two strains,compared with CICC21530,the micro-structure of biofilm formed by J29 was more stable and its EPS components of biofilm was more in amounts.