Study On The Formation Mechanism Of Retained Bacteria In Staphylococcus Aureus Biofilm

Staphylococcus aureus is the most common pathogen in hospital-and community-acquired infections.The bacteria can cause multiple infections,such as superficial skin,deep skin,wound sepsis,pneumonia,infectious arthritis,toxic shock syndrome,endocarditis,osteomyelitis,etc.After treatment with antimicrobials,morbidity and mortality are still high.Among infections caused by Staphylococcus aureus,endocarditis has the highest mortality rate,ranging from 20%to 40%.Therefore,bacterial infection caused by Staphylococcus aureus has become a problem that cannot be ignored.Biofilm is a way of survival of microorganisms in nature,and it has super drug resistance,which leads to the stubbornness and refractory of S.aureus infection.In 1994,BIGGER discovered that the phenotype-resistant but genetically susceptible subgroup of bacteria "retaining bacteria",they grow slowly,form during the maturation of biofilms,and are resistant to antibacterial drugs The existence of retained bacteria may be the main reason for its clinical treatment failure and recurring infections.Therefore,this topic explores the basic characteristics and formation mechanism of ?-polylysine for Staphylococcus aureus retainers,which is a retainer for other species The research and development provides a theoretical basisObjective1.To understand the antibacterial activity of ?-polylysine on Staphylococcus aureus-retaining bacteria and its effect on biofilm,and further explore the bacteriostatic mechanism of ?-polylysine on Staphylococcus aureus-retaining bacteria2.Aetiology monitors the formation of Staphylococcus aureus retention bacteria during biofilm formation,especially during the adhesion phase,to further explain the possible relationship between retention bacteria formation and early biofilm and adhesion phase3.Taking the early biofilm of the adhesion stage as the research object,the prokaryotic transcriptome sequencing technology of the biofilm cell population was used to analyze the gene expression changes,and the suspicious genes related to the formation of retained bacteria were initially screened,so as to further explore the retention of S.aureus The specific mechanism of the signal transduction pathways related to the formation of bacteria provides a basis,provides new targets for the research of new antibacterial drugs,and provides new ideas for clinical treatment of intractable recurrent bacterial infections and removal of retained bacteriaMethodsStaphylococcus aureus biofilm-retaining bacteria are sterilization curves that appear after impacting Staphylococcus aureus biofilm with high concentration of ?-polylysine.After the retention bacteria are discovered,the dose-dependent killing curve is the most effective method for retention bacteria isolation research.In this experiment,we used the method of micro-turbidimetry to determine the staphylococcus aureus biofilm-retaining bacteria by epsilon-polylysine(10MIC).MIC used the results of the previous research in the team.First,the surface of a 96-well cell culture plate was pre-covered with Staphylococcus aureus USA300 for 24h to form an early biofilm,and observation under a microscope confirmed that the attached surface was completely covered,and dynamically monitored the production of retained bacteria during the bioforming process.The determination of retention bacteria is based on the assessment of cell viability after the action of epsilon-polylysine.In addition,we also monitored the level of retained bacterial cells in the biofilm while adding ?-polylysine to treat the biofilm while destroying the attachment by scrapingFluorescence spectrophotometer was used to determine the content of ROS reactive oxygen species in Staphylococcus aureus bacteria before and after ?-polylysine treatment;ELISA was used to determine the effect of starvation on the formation of Staphylococcus aureus biofilm-retaining bacteria;Changes in protein profiles of Staphylococcus aureus before and after lysine treatment.Prokaryotic transcriptome sequencing screened differentially expressed genes to obtain related metabolic pathwaysResults1.The optimal drug concentration for screening retained bacteria after ?-PL acting on S aureus was 312.5mg/mL.?2.After ?-PL acts on S.aureus,there are retained bacteria3.The ormation of Staphylococcus aureus biofilm retention bacteria is significantly related to the maturity of the biofilm.The nutritional status has an impact on the production of S.aureus biofilm retention bacteria,and starvation can increase the production ratio of retention bacteria,revealing that the formation of S.aureus retention bacteria is related to energy4.Prokaryotic transcriptome sequencing analysis detected significant differences in the expression levels of tpiA,glpK,UreG,pgi,clpP and eno in Staphylococcus aureusbiofilm-retaining bacteria(P<0.05),not only determined by a single signaling pathway,but also regulated by many complex signaling pathwaysConclusion1.The formation of Staphylococcus aureus retention bacteria is closely related to its biofilm formation2.Prokaryotic transcriptome is the most effective method for rapid screening of differentially expressed genes.The data we obtained laid the foundation for the follow-up research on the mechanism of retention bacteria production and gene pathway function.The formation of Staphylococcus aureus biofilm-retaining bacteria is closely related to the genes tpiA,pgi and eno.