The Preliminary Study On The Molecular Mechanism Of Licochalcone A Against Staphylococcus Aureus Biofilm

Staphylococcus aureus (S.aureus) is a leading cause of humuan and animal infections. Withthe abusing of antibiotics, methicillin-resistant Staphylococcus aureus (MRSA) is increasing yearby year with a wide range and severity of infection. It is one of the most common pathogens ofnosocomial diseases with a significant morbidity and mortality. It produces a variety of toxins andinvasive enzymes, which can cause serious infectious diseases. It also appears a variety ofresistance gene, which increased the difficulty of the treatment caused by S.aureus. S.aureus iseasy to form biofilm, and the biofilm enhances the pathogenicity and increases the resistance, sothe treatment of S.aureus infection is more difficult. Therefore, finding the effective drugs againstthe biofilm produced by S.aureus has been crunch time task.Due to the bacterial resistance and the adverse reaction of antibiotic drugs, it makes moredifficult to find effective drugs in chemical composition. With the development of newantibacterial drugs and drug targets, traditional Chinese medicine have become a valuable resourcefor new drug research. Herbal medicine and natural products, which contained a variety of smallmolecule chemical ingredients with a large number of the unique and varied structural types, hasprovided a broad space for finding a new drug to inhibit S.aureus.In this study, we used Congo red, crystal violet, silver staining and confocal laser to identifythe ability of ATCC29213and20clinical isolates MRSA in biofilm formation. The resultsshowed that, the selected21strains of S.aureus were able to form biofilms. In addition, wesuccessfully constructed model of biofilm in vitro. The minimum inhibitory concentrations (MICs)and minimum bactericidal concentrations (MBCs) against S.aureus ATCC29213and20clinicalisolates MRSA in suspension were determined by microbroth dilution and agar plate assays; theminimum biofilm inhibitory concentrations(MBICs) and minimum biofilm bactericidalconcentrations(MBBCs) in biofilm were determined by agar plate assays. The ability ofLicochalcone A which eliminated the biofilm of the tested isolates were determined by confocallaser scanning microscopy at24h and48h. In planktonic cultures, the minimum inhibitoryconcentrations (MICs) and the minimum bactericidal concentrations (MBCs) of LicochalconeAaganist S.aureus strains were1to8μg/mL and2to16μg/mL, respectively. The minimumbiofilm inhibition concentrations (MBICs) and the minimum biofilm bactericidal concentrations(MBBCs) of Licochalcone A against S.aureus strains were8to64μg/mL and≥1024μg/mL inbiofilms, respectively. Laser scanning confocal microscopy to quantify the biofilm characteristics,the results showed that the thickness of biofilm was reduced from9120nm to4880-2460nm after treatment with the drug at24h, while at48h,the thickness of biofilm was reduced from14820nmto5700-8840nm. The results demonstrated that Licochalcone A had a strong bactericidal activityagainst S. aureus.The inhibition curve of Licochalcone A against suspension and biofilm of S.aureus ATCC29213were determination by spectrophotometric and XTT method. According to previous results,we chose Licochalcone A with the concentration at4×MIBC against S.aureus biofilm in1h formicroarray transcriptome studies. In this study, the chips will provide a powerful tool for studyingthe global gene expression profile of S.aureus induced by Licochalcone A. Microarray dataanalysis revealed that a large number of genes (824) were differentially expressed in response toLicochalcone A treatment.355genes increased in expression and469genes were inhibited.The genes included autolysin and regulatory gene, biofilm morphogenesis-related protein,cell wall related protein, protein synthesis, capsular polysaccharide synthesis, toxins andcolonization factors were significantly regulated by Licochalcone A. In addition, quantitativereal-time RT-PCR was performed to verify the microarray results of the selected genes. There waspositive correlation between microarray data and real time RT-PCR data for autolysin genes inresponse to Licochalcone A.This study clarifies the molecular mechanism of Licochalcone A inhibits S.aureus biofilm,which provides the new treatment protocols to clinical treatment of bacteria infections; provides auseful theoretical basis for studying the action mechanisms of Licochalcone A against pathogenicbacteria; and lays the foundation for the development and utilization of Licochalcone A.