Staphylococcus Aureus Biofilm Regulation Mechanisms: With Focus On Biological Process-based Transcription Analysis

Microorganism and its biofilm mediated food contamination is considered as the leading concerns among all the food safety issue.Microorganisms adopts two survival strategies in nature and artificial environment,which are plankton and forming biofilm.Also,biofilm also involving mono-biofilm and polymicrobial biofilms.Staphylococcus aureus(S.aureus)is the most prevalent foodborne pathogens,with capacity of drug resistance,biofilm formation and virulence.To now,investigations are not sufficient concerning mechanisms regulating S.aureus three survival strategies,while these mechanisms are the most important for preventing and eliminating S.aureus mediated contaminations.Concerning S.aureus mediated food contamination with three survival strategies,this project investigated universal and unique regulation mechanisms of S.aureus in planktonic,biofilm and S.aureus-Candida albicans(C.albicans)polymicrobial biofilms.(1)Establishment of dynamic model of S.aureus biofilm and analytical methods.A dynamic model for S.aureus mono-biofilm was set up.Biofilm formation was divided into three stages:initial adhesion(8 h),maturation(2 d)and dispersal stage(14 d).Also,S.aureus grew as planktonic cells were also divided into early log(2 h),late log(6 h)and stationary phase(2 d).Besides,a S.aureus-C.albicans static model was established in 6-well plate.Microarray was employed to investigate the transcription condition mentioned above.After that,genes was annotated and enriched into metabolism,proliferation,biofilm,resistance and virulence group for the following analyzation.(2)S.aureus biofilm features among different formation stages.In initial adhesion stage of S.aureus mono-biofilm,S.aureus absorbed glucose as main carbon source and decreased other carbohydrates’(ketonic acid,glyceraldehyde and fructose)uptake genes transcription to 30%approximately.Glutamate and alanine were accumulated,with increase of Fe and Na~+uptake.Meanwhile,lytSR operon was activated,mediating cell autolysis,which consequently increased the adhesion to substrates.S.aureus revealed its resistance to tetracycline and fosfomycin,and increased toxin synthesis,includingγ-hemolysin,coagulase,leukotoxin D and type A enterotoxin.In maturation stage of S.aureus mono-biofilm,S.aureus cells started to compete oxygen and employed glycolysis as carbon source catabolic strategy.Meanwhile,HrcA-DnaK regulator were activated to cope with exothermal catabolic reactions.With the cell proliferation within biofilm,agr and epi were activated to control cell density.S.aureus upregulated saeRS,srrAB and downregulated arlRS to increase biofilm adhesion ability,with clfB and cap were upregulated.Drug resistance and virulence were downregulated due to the protection of unbroken biofilm extracellular matrix.In dispersal stage of S.aureus mono-biofilm,S.aureus accumulate alanine,glutamate and aspartic acid,and increase peptide uptake to upregulate protein synthesis.Biofilm matrix was mainly composed of ClfB,capsular saccharide and PIA,while FnBPs were decreased over80%.Meanwhile,S.aureus revealed active integration and transposition reactions,indicating gene transfer within cells.Abortive infection protein,type I enterotoxin,type 1 and type 5exotoxin expression were upregulated.During the formation of S.aureus mono-biofilms,the compensation effect was observed in agr regulated FnBPs and LuxS regulated capsular polysaccharide,which stabilized biofilm structure.(3)S.aureus biofilm features in S.aureus-C.albicans polymicrobial biofilmsIn S.aureus-C.albicans polymicrobial biofilms,S.aureus absorbed metabolites,galactitol,N-Acetylmuramic acid and trehalose,as main carbon source via PEP:PTS system,which mainly due to the carbohydrates competition between S.aureus and C.albicans.Besides,S.aureus upregulate atl and cidAB(over 300%)to increase cell autolysis,while PIA synthesis was impeded,and S.aureus formed FnBPs-dependent biofilm.Resistance toβ-lactam antibiotics was downregulated over 70%while hemolysin,Panton-Valentine Leukocidin,enterotoxin and immunodominance protein was specifically upregulated.To sum up,this investigation indicated that in both S.aureus mono-biofilm and S.aureus-C.albicans polymicrobial biofilms,S.aureus initiative changed the species of uptake carbohydrates,regulated amino acid catabolism,activated various TCS system,regulated agr-LuxS mediated biofilm extracellular matrix components synthesis and adopt different antimicrobial resistance and virulence strategies.All the fluctuated transcription level of the regulation system mentioned above assisted S.aureus to form and maintain biofilm.