Anti-biofilm Mechanism Of Cyclic Dipeptide On Staphylococcus Aureus And Its Application

Staphylococcus aureus(S.aureus)is a typical food-borne pathogen.It can adhere to the surface of food processing equipment such as milk and meat products and form biofilm.However,traditional biofilm scavengers have the disadvantages of strong irritation and strong corrosiveness.Cyclo L-Phe-L-Pro(cFP)has attracted wide attention from the food industry because of its safety,non-toxicity,and non-irritating properties.Previous studies have shown that cFP has a good inhibitory effect on S.aureus biofilm,but its inhibitory mechanism is not clear.This study aims to explore the inhibitory mechanism of cFP on S.aureus biofilm and provide a theoretical basis for its application in food field.Firstly,the effects of cFP on early biofilm formation and extracellular polymer were studied.On this basis,the effects of cFP on the expression of ica operon and agr system were studied at the molecular level.Furthermore,the binding sites of cFP and Agr C were studied by molecular docking.Finally,cFP was applied to the surface of four container materials,and its removal effect was evaluated.The main results are as follows:(1)The minimal biofilm inhibitory concentration(MBIC)and minimum biofilm removal concentration(MBEC)of cFP to S.aureus biofilm were 6.0 mg/m L and 12.0mg/m L,respectively.The results of plate colony counting showed that cFP could effectively remove S.aureus biofilm,and the removal rate reached 99.86% after 2MBIC concentration of cFP treated for 12 h.Laser scanning confocal microscope(CLSM)and scanning electron microscope(SEM)results showed that the three-dimensional structure of bacteria was destroyed,the biofilm became sparse,and the bacteria were dispersed into free bacteria and then inactivated by cyclic dipeptide.cFP also inhibited the metabolic activity of S.aureus.The metabolic activity of the bacteria decreased by 78.9% after 2MBIC concentration of cFP.In addition,the ability of biofilm formation was decreased after treatment with CFP,and the synthesis of extracellular proteins,extracellular polysaccharides and e DNA were inhibited by cFP.(2)The expression of ica R increased by 2.67 times,while the expression of ica A,ica C and ica D decreased to 78.1%,77.3% and 93.4%,respectively,indicating that cFP treatment inhibited the synthesis of polysaccharide intercellular adhesin,decreased intercellular adhesion and reduced the ability of biofilm formation.The expression of agr A,agr C and agr D decreased to 32.7%,78.4% and 86.1% respectively after treatment with cFP of MBIC.Phosphorylated Agr A can further activate the activity of the P3 promoter and reduce the transcription level of RNA ?,thereby affecting cell-to-cell communication.Molecular docking results showed that cFP could bind to Agr C,and the binding region was most likely between Loop1 and Loop2.At the same time,the histidine kinase domain of Agr C also played a role in the binding process.(3)The results showed that cFP could remove S.aureus biofilm on the surface of four materials(stainless steel,glass,plastic and ceramic)in milk and broth simulation system.In the milk simulation system,the removal rate of the four materials was all above 99.83% after treating with 2 MBIC concentration at 4 ? for 4 d;the removal rate of the four materials were all above 99.86% after treating with 2 MBIC concentration at 25 ? for 3 d.In the broth simulation system,the removal rate of the four materials was all above 99.79% after treating with 2 MBIC concentration at 4 ? for 4 d;the removal rate of the four materials were all above 99.87% after treating with 2 MBIC concentration at 25 ? for 3 d.