Effect Of Lactic Acid Stress On Biofilm Formation Of Escherichia Coli O26

Food-borne pathogens can attach to food processing machines,equipment,food and other surfaces,grow on them,and further form biofilm,which can cause cross-contamination,and greatly increase food safety risks.Biofilm formed by food-borne pathogens has been an important problem that needs to be solved urgently in food industry.In recent years,outbreaks of non-O157 Eecherichia coli,especially the "big six",have gradually increased,which have been considered as one of the important foodborne pathogens.At present,researches on biofilm formed by non-O157 E.coli are limited.Non-O157 E.coli,like E.coli O157:H7,has certain acid resistance.The acidic environment in food processing can induce stress response of bacteria.Different temperatures may affect its stress response and further aftect the formation of biofilm.Therefore,in this study,the biofilm formed by the most important serotype of non-0157 E.coli,i.e.026,was investigated.Firstly,the adhesion characteristics and bacterial surface characteristics of 22 strains of E.coli 026 collected in our laboratory were analyzed.Secondly,two strains with different adhesion properties were selected to investigate the effects of different temperature and lactic acid concentrations on the formation of biofilnis,and the dynamic changes of the microscopic structure of the biofilm were further revealed by CLSM and SEM techniques.This study will provide a theoretical reference for the risk assessment of E.coli 026 in the actual production and processing of acidic foods,and provide a scientific basis for further research on effective methods for removing E.coli 026 biofilm.The context and results were shown as following:1.Adhesion properties and surface characteristics of different strains of E.coli O26The adhesion of 22 strains of E.coli O26 collected in the laboratory was measured simultaneously by means of micro-plate crystal violet method and stainless steel tablet counting method.After incubation at 25? for 72 hours,the adhesion of the strain was compared according to the OD values and culture counts.The results of micro-plate crystal violet method showed that 22 strains of E.coli O26 had adhesion,and there were significant differences in strains(P<0.05).Among them,strain 105H2 had the weakest adhesion while strain 126Z was the strongest.The results of steel sheet counting method showed that 22 strains of E.coli 026 formed adherent biofilm on the surface of stainless steel sheet,and there were significant differences in strains(P<0.05).The adhesion results obtained form two methods were not completely consistent.The soft agar plates were used to evaluate the mobility of the strains.The results showed that there was a difference in the mobility among the strains.The diameter of strain 123Z2 was the largest in the test of swimming 24 h,while strain G13Z2 was the smallest.The diameter of strain 83Z2 was the largest in the test of swarming 20 h,while strain 57Z1 was the smallest.The surface hydrophobicity and electron-acquisition ability of E.coli 026 were evaluated by their affinity with xylene,chloroform and ethyl acetate solutions.It was found that there were significant differences in the afifinity of the three substances,and strain 40Z2 was more hydrophobic.Strain 8921 had strong electron supply capability,and strain F7Z2 had strong electronic ability.Further correlation analysis showed that there was a significant positive correlation(P<0.05)between the adhesion of bacteria on the stainless steel sheet and the adhesion on the microplate,and there was a very significant positive correlation(P<0.05)between the ability of swimming and swarming.But there was no correlation between mobility,hydrophobicity and adhesion of bacteria.2.Biofllm formation process of E.coli 026 under lactic acid stress at different temperatureAccording to the results above,two strains with different adhesion were selected for biofilm formation,including strong adhesion strain 126Z and weak adhesion strain G13Z2.The acidic TSB(pH 4.0,5.0,adjusted with lactic acid)and the normal TSB were used as the culture media,and the effects of lactic acid on the biofilm formation at 15? and 25?were studied.The results showed that biofilm formation of two strains in normal TSB at 15? was less than at 25?,and the biofilm formation of strain 126Z was more than that of strain G13Z2 at both temperatures.Lactic acid inhibited the biofilm formation of E.coli 026,and the more the concentration of lactic acid was,the less the number of planktonic cells and the amount of biofilm were.The effect of temperature on the number of planktonic bacteria and biofilm was related to the pH value of lactic acid.At a lower concentration of lactic acid,the higher the temperature was,the faster the number of planktonic bacteria and the amount of biofilm increased.The number of planktonic cells and the amount of biofilm of two strains were inhibited,and decreased in the later stage after treatlent of a higher concentration of lactic acid at 15?.There was significant difference in dynamic changes of the number of planktonic cells and biofilm formation in two strains at 25?,and the numbers of strain 126Z fluctuated,while the numbers of strain G13Z2 decreased significantly.The results indicated that the biofilm formation of E.coli 026 under lactic acid stress was related to temperature and adhesion of bacteria.3.Effect of acid stress on the microstructure of E.coli O26 biofilmOn the basis of the above experiments,acidic TSB(pH 5.0,adjusted with lactic acid)and the normal TSB were used as the culture media,and the microstructure of biofilm formed by strain 126Z and strain G13Z2 was observed by confocal laser scanning microscopy(CLSM)and scanning electron microscopy(SEM).Dynamic changes of microstructure of biofilm formed by two strains with different adhesion in two different media at different temperature at 1,3,5,7 d were compared.The results showed that E.coli O26 formed biofilm with a certain spatial structure under acid stress,and the biofilm formation in the acidic environment was slower than in the neutral environment.The microstructure of biofilm formed by the two strains with different adhesion was affected by time,temperature,and pH value.The biofiln formed by the strong adhesion strain 126Z was denser.