Synergistic Anti-bacterial Effects And Mechanism Of Theaflavin With β-lactam Antibiotics Against MRSA

At present,methicillin-resistant Staphylococcus aureus(MRSA)has become a worldwide problem.It has developed resistance to almost all available β-lactams.The emergence of drug resistance has greatly restricted the clinical use of antibiotics,and the development speed of new antibiotics is far less than that of drug resistance.Therefore,in addition to the discovery of new antibiotics,we must find new ways to change the process of drug resistance.Especially,we need to develop new antimicrobial strategies that can limit,redirect,and even reverse the evolution of resistance.Drug combination provides a promising strategy to overcome the mechanism of bacterial resistance and restore the effectiveness of antibiotics.The use of combination therapy can expand the range of antibacterial activity,reduce bacterial resistance,target new drugs,and reduce the toxicity and effective dose of these antimicrobial agents.In the development of anti MRSA drugs,the therapeutic potential of phytochemicals is increasingly recognized.The synergistic effect of theaflavin and β-lactam antibiotics against MRSA was studied by chessboard method.The results showed that theaflavin and 8 β-lactam antibiotics had obvious synergistic inhibitory effect against MRSA,among which theaflavin and ceftiofur had the best synergistic antibacterial effect and the FIC index was the lowest.We studied the synergistic antibacterial activity of theaflavin and ceftiofur in vivo by mouse model of S.aureus Pneumonia.The results showed that compared with the single antibiotic group,the combination group significantly reduced the amounts of bacteria in the lungs and improved the infection situation,and the survival rate increased by 20%.We studied the effects of theaflavin(64 μ g / ml)on the MRSA USA 300 proteins expression by TMT relative quantitative proteomics.In theaflavin treatment group,there were 333 protein differentially expressed,192 protein up-regulated,141 protein down regulated.Through bioinformatics analysis of the differentially expressed proteins,it was found that the synergistic antibacterial mechanism of theaflavin and β-lactam antibiotics against MRSA could be divided into the following four categories:1.The anti-bacterial effect of β-lactams were achieved by inhibiting penicillin binding protein(PBPs)and interfering the biosynthesis of cell wall.Theaflavin could reduce the synthesis of glutamate and indirectly affected the synthesis of cell wall.Therefore,the interference of cell wall synthesis might be one of the synergistic mechanisms of theaflavin and β-lactams.2.Theaflavin treatment induced the up regulation of two major autolytic enzymes of S.aureus,and the up regulation of two autolytic enzymes accelerated the bacterial autolysis induced by β-lactams.Therefore,accelerating bacterial autolysis may be another mechanism of the synergistic antibacterial activity of theaflavin and β-lactams.3.Theaflavin treatment resulted in the decrease of sodA and sodM expression and the increase of hydroxyl radicals,which promoted the cell death.Therefore,theaflavin could enhance the oxidative stress induced by β-lactams and accelerate the bacterial death.4.Theaflavin treatment down regulated the expression of key ribosomal proteins RimP and L 2,which affected the assembly of 70 s ribosome and inhibited the protein biosynthesis,including the synthesis of many drug-resistant proteins,leading to a decrease in bacterial growth and drug-resistant level.RT-PCR was used to study the effect of theaflavin(64 μ g / ml)on the transcription of mecA,lytM,nirD,sodA and rplB genes in USA300.The results showed that mecA gene had no differential expression,lytM gene was up-regulated,nirD,sodA,rplB genes were down-regulated,which was consistent with the results of proteomics.