Biofilm Formation Of Foodborne Pathogens And Their Effects On Toxin Factor Expression Of Staphylococcus Aureus

In the research,The effect factors on formation of single and mixed bacterial biofilm,which was formed by foodborne pathogens(Staphylococcus aureus,Escherichia coli and Salmonella enterica),were studied.Moreover,the Real Time PCR method was used to detect the changes in the transcription levels of 28 toxin genes of Staphylococcus aureus in the biofilm of single bacteria and mixed bacteria.The following results were obtained:(1)It was proved that the mixed bacteria reached the maximum adhesion degree at 24 hours in culture by crystal violet staining method,silver staining method,and scanning electron microscopy method.The number of viable bacteria of the three bacteria in the mixed bacteria biofilm was lower than that of the single bacteria.Staphylococcus aureus is the dominant bacteria in the mixed bacteria biofilm.(2)The 96-well microplate was used as the adhesion carrier for single and mixed bacteria biofilms,and the factors affecting the formation of the biofilms were determined by the crystal violet staining method.? The optimum culture condition for mixed bacterial biofilm was pH 7.0,at 36?for 21 h.When the TSB medium with 1.6%mass fraction was used,the biofilm formed by the pathogen under the optimal conditions had the highest adhesion rate.The carbon source promoted the formation of biofilm.When the mass fraction of NaCl was more than 0.4%,the ability to form biofilms of foodbome pathogens was somewhat inhibited.? Chemical preservatives(EDTA)have inhibitory effect on the biofilm of mixed bacteria,and with the increase of concentration of EDTA,the adhesion degree of the mixed bacteria biofilm decreased gradually.When 0.25 mmol/L EDTA was added,the adhesion rate of the mixed bacteria biofilm decreased by 16.5%.The checking of AI-2 activity further confirmed that the adhesion degree was gradually reduced with the increase of EDTA concentration.The mixed bacteria biofilm was inhibited only during the adhesion phase(0 h-28 h).The stability of the mixed bacteria biofilm could be enhanced through addition of divalent metal ions(Mg2+,Ca2+,Fe2+).? Biological preservatives(antibacterial peptides produced by Lactobacillus ALAC-1,ALAC-2,ALAC-3,ALAC-4 and ALAC-5)had inhibitory effects on the biofilm and AI-2 molecular activity of single and mixed bacteria,but its inhibitory effect on single bacteria biofilm was better than the mixed bacteria biofilm.ALAC-5 and ALAC-1 had the highest inhibition rate on pathogenic biofilm,and they had inhibition rates of 78.95%and 69.47%on Staphylococcus aureus biofilm respectively.(3)The Real-time quantitative PCR revealed that 28 toxin genes were detected in Staphylococcus aureus,and expression level of 28 toxin factors were different in mixed bacteria and single bacteria biofilms.Comparing to the single bacteria biofilm,in the mixed bateria biofilm,the expression level of some genes were up regulated obviously(sep and ser gene),some were decreased significantly(hemolysin gene hla and the peeling toxin gene etd),and the enterotoxin gene seb was same.