Interaction Between Escherichia Coli O157:H7 And Meat-borne Pseudomonas In The Process Of Mixed-species Biofilm Formation

Escherichia coli O157:H7 is a zoonotic food-borne pathogen,and Pseudomonas is the dominant spoilage bacteria in chilled fresh meat.Many reports indicate that spoilage and pathogenic bacteria can adhere to the surfaces of food raw materials and processing equipment to form biofilms,which will further contaminate foods,and cause harm to consumer health,cause economic loss to the food processing industry.Previously,most studies on biofilms were based on pure culture conditions of a single strain,but mixed biofilms of multiple bacteria more exit in the actual food production environment.The interaction between different bacteria may form mixed biofilms with different structures and properties due to differences in bacterial species,strains,culture conditions,and so on.Based on the contamination and spoilage of meat,E.coli O157:H7 and the meat-borne dominant spoilage bacteria Pseudomonas sp.were taken as the object in this thesis.Firstly,the biofilm formation ability(BFA)of different strains were compared,and then the representative strains were selected to study the interaction in the mixed biofilm formation.The representative combination of the two bacteria was further selected to study the changes of the bacterial amount of biofilm,EPS composition,spatial structure,and the expression of E.coli O157:H7 virulence genes,in the process of mixed biofilm formation.This study can provide a scientific basis for revealing the rule of mixed biofilm formation of harmful bacteria in meat slaughter and processing,and controlling meat pollution,and ensuring meat safety.The context and results were shown as following:1.Comparison of the biofilm formation ability of different strains of E.coli O157:H7and meat-borne PseudomonasThree strains of E.coli O157:H7(CICC23150,ATCC43895,95)and a standard strain ATCC13525 of Pseudomonas were selected to compare methanol-fixed and non-methanol-fixed microplate crystal violet staining method for determining BFA,and then the better method was used to determine BFA of 31 strains of meat-borne Pseudomonas.The results showed the method based on methanol-fixed was better due to higher OD value,lower standard deviation,smaller coefficient of variation,and shorter time of operation.Both methods found that the BFA of three E.coli O157:H7strains and Pseudomonas ATCC13525 were classified to weak,weak,medium,and stronger,respectively.Furthermore,the methanol-fixed method was used to compare the BFA of 31 Pseudomonas strains,which was found that their BFA showed obvious strain differences.2.The interaction between E.coli O157:H7 and Pseudomonas in mixed biofilm formationThree strains of E.coli O157:H7(CICC23150,ATCC43895,95)and 4 strains of Pseudomonas(Y_1-11,J_1-111,Y_2-17,ATCC13525)were selected,the cell mobility and the biofilm formation of the mixed culture of the two kinds of bacteria on the stainless-steel sheet with and without gas-liquid interface(25°C,72 h)were investigated.The results of cell mobility showed that the selected strains had a certain degree of mobility,and the mobility of E.coli O157:H7 strains were lower than Pseudomonas strains.The mobility diameter of the mixed bacterial culture was lower than the diameter sum of the two single bacteria,which indicated that the mobility of the two bacteria was mutually inhibited in the mixed culture.When mixed biofilm of 72 h was formed by two bacteria,the amount of biofilm formed without gas-liquid interface was less than that with gas-liquid interface.Under the two biofilms forming conditions,the interactive effects were basically consistent,that is,the biofilm formation of the 3strains of E.coli O157:H7 was not affected or was inhibited by Pseudomonas,while the biofilm formation of the 4 strains of Pseudomonas were all inhibited by E.coli O157:H7.3.Study of the process of mixed biofilm formationBased on the results of the above experiment,2 strains of E.coli O157:H7(ATCC43895,CICC23150)and 2 strains of meat-borne Pseudomonas(Y_2-17,J_1-111)with antagonistic effect were selected to investigate the process of mixed biofilm formation.The changes of bacterial amount of biofilms,polysaccharide and protein contents of EPS,the spatial structure of biofilms by means of scanning electron microscopy(SEM)and confocal laser scanning electron microscopy(CLSM)were analyzed.The results showed that the biofilm formation process of single E.coli O157:H7 or Pseudomonas was different.The interaction of two kinds of bacteria in the biofilm formation was mutual antagonistic except that ATCC43895 promoted the biofilm formation of Y_2-17.With the increase of time,based on the analysis of per unit area and per cell count,the polysaccharide and protein contents of EPS(L and B type)were increased in single biofilm formed by E.coli O157:H7 ATCC43895 or Pseudomonas J_1-111.The polysaccharides and protein contents of EPS in mixed biofilm was less than the sum of the corresponding content of two single bacterial biofilms,which indicated that the secretion of polysaccharide and protein of the two bacteria in the mixed biofilm was mutually inhibited.The SEM and CLSM showed that the microstructure changes of biofilm were consistent with the plate count results,and the amount of E.coli O157:H7 ATCC43895 or Pseudomonas J_1-111 in the mixed biofilm was significantly less than that in its single biofilm,especially ATCC43895 reduction in the mixed biofilm,which indicated that there was mutual inhibition between the two strains,and ATCC43895 was more significantly inhibited by J_1-111.The results suggested that there is an antagonistic effect between both bacteria during the process of mixed biofilm formation.4.Changes in the expression of E.coli O157:H7 virulence genes during the formation of biofilmBased on the results of the above experiment,the changes of virulence genes expression(stx1,stx2,hly and eae)of E.coli O157:H7 ATCC43895 were determined by means of RT-q PCR in the process of mixed biofilm formation of ATCC43895 and meat-borne Pseudomonas J_1-111.First,the standard detection plasmids were constructed,and the standard curves were established using real-time PCR.Then the expression of four virulence genes in single and mixed biofilms(72 h and 120 h)was determined using the absolute quantitative method.The results showed that compared with the expression of three virulence genes in ATCC43895 single biofilm at 72 h,genes expression per unit area at 120 h showed no significant difference,while genes expression per cell count at 120 h decreased significantly(P<0.05).Compared with ATCC43895 single biofilm,the expression of three virulence genes per unit area in the mixed biofilm showed no significant difference,while expression of three virulence genes per cell count in the mixed biofilm increased significantly(P<0.05),indicating that the expression of virulence of E.coli O157:H7 was inhibited by meat-borne Pseudomonas strain during the formation of their mixed biofilm.