Effect Of Emodin And Lactobacillus Biosurfactant Against Staphylococcus Aureus Biofilm In Vitro

Staphylococcus aureus(S.aureus)is one of the most common pathogens,which can canse skin and soft tissue infections,pneumonia,pericarditis,pseudo membranous enteritis,and even systemic sepsis.An important feature of Staphylococcus aureus is the formation of bacterial biofilms(BF)at the site of infection.Bacteria inside biofilms display an increased resistance to antibiotics and disinfectants,resulting in an increasingly serious situation.Therefore,it has become a hot spot in the current research to find a new type of effective anti-biofilm drugs.Because of sub-MICs of antibiotics can lead to bacterial resistance problems,in new antibacterial and anti-biofilm drug development,the nature of the Chinese herbal medicine and natural products has become a valuable resource for drug development.In addition,Lactobacillus biosurfactant(BS)with excellent safety and antibacterial,anti-adhesion and other biological activity,in the development of biological medicine,new antibacterial,anti-biofilm agent has great application prospect.In this study,Staphylococcus aureus was used as the biofilm model in vitro.Firstly,the activities of 4 kinds of Chinese herbal medicines,such as emodin,luteolin,baicalin and berberine hydrochloride,were studied.The emodin with good antibacterial activity was selected as the key research object,We investigated the effect of emodin on the biofilm formation of Staphylococcus aureus in vitro and the possible mechanism.The results showed that emodin had good inhibitory effect on the growth of S.aureus(MIC: 4~8 g/m L)and the release of hemolysin,As quantified by crystal violet method,emodin significantly decreased S.aureus biofilm growth in a dose-dependent manner.The above findings were further supported by images obtained by scanning electron microscopy(SEM).Moreover,the present study demonstrated that sub-MICs emodin obviously intervened the release of extracellular DNA(e DNA)and inhibited the expression of the biofilm-related genes(cid A,ica A,dlt B,agr A,sortase A and sar A)by RTq-PCR.In addition,the concentration of emodin in sub-MIC also had an effect on the release of signal molecule AI-2 in QS of S.aureus.We continued to use S.aureus as a biofilm in vitro model,4 strains of probiotic Lactobacillus biosurfactant(BS)were obtained by fermentation.The antibacterial activity and anti-biofilm activity of crude BS were investigated and the possible mechanism is analyzed.The results showed that the MIC values of the 4 groups of Lactobacillus BS were higher than 100mg/ml,the intervention results on the growth curve of planktonic bacteria agent showed that 4 groups of Lactobacillus BS had some inhibitory effect on S.aureus,but the antibacterial effect is not strong.50mg/ml Lactobacillus BS significantly inhibited the release of hemolysin.The experimental results showed that the 4 groups of Lactobacillus BS could inhibit bacterial adhesion at the concentration of 12.5mg/m L~50mg/mL.The results of crystal violet staining showed that 4 groups of Lactobacillus BS had the ability to inhibit the formation of S.aureus biofilm.The above findings were further supported by images obtained by SEM.The effects of 4 strains of Lactobacillus BS on the expression of mRNA gene of S.aureus biofilm formation in 12.5mg/ml~50mg/ml were detected by RTq-PCR.Interestingly,the effects of Lactobacillus BS on the expression of genes related to biofilm formation were different,The agr A and sar A genes were down regulated by Lactobacillus helveticus 27058 BS,and the expression of agr A and cid A gene was down regulated by Lactobacillus helveticus 27170 BS,The expression of ica A gene was significantly inhibited by Pediococcus acidilactici 27167 BS and the expression of cid A gene was significantly inhibited by Lactobacillus plantarum 27172 BS.The results showed that 4 strains of Lactobacillus BS could regulate the expression of genes related to biofilm formation probably by regulating the cid operon,ica operon and agr regulatory system.In addition,the 4 groups of Lactobacillus BS all affect the release of S.aureus AI-2 signaling molecules.The SDS-PAGE protein electrophoresis analysis of 4 groups of Lactobacillus BS showed that they contained protein components,and the protein bands near 27167 BS 15 KDa was selected for protein mass spectrometry analysis,Through the comparison of the database and the analysis of the related software,two kinds of proteins were retrieved.These two proteins are: DNA starvation/stationary protection protein;ferritin-like DNA-binding protein.In view of the good anti-biofilm activity of emodin and Lactobacillus BS,this study tried to inhibit the formation of bacterial biofilm by the combination of traditional Chinese medicine monomer and Lactobacillus BS.By comparing the inhibitory rate of S.aureus biofilm,it can be concluded that the combination of both of them has a good synergistic effect at low emodin concentration(1/4MIC,1/8MIC)and low concentration of Lactobacillus BS(12.5mg/m L).On the contrary,emodin and Lactobacillus BS had no synergistic effect at high concentration.In this study,we confirmed that emodin and Lactobacillus BS had a good ability to interfere with the formation of S.aureus biofilm in vitro.The mechanism may be related to the inhibition of the expression of biofilm related genes and the inhibition of the release of signaling molecule AI-2 in the quorum sensing system of S.aureus.In addition,we tried to use the combination of traditional Chinese medicine monomer and Lactobacillus BS to resist bacterial biofilm.These results showed that a promising application for emodin and Lactobacillus BS as a therapeutic agent and an effective strategy to prevent bacterial biofilm-related infections.