The Formation Of Staphylococcus Aureus Biofilm In The Dialysis Tube And The Intervention Role Of Minocycline On Its Biofilm

PART I Establish S.aureus biofilm standing model of dialysis tube in vitroObjective:To establish Staphylococcus aureus biofilm standing model in the peritoneal dialysis—peritoneal dialysis fluid system in vitro.Methods:(1) Congo red plate method, preliminary qualitative clinical strains of whether the formation of the BF.(2)Using peritoneal dialysis tube as a Carrier,using peritoneal dialysis fluid—50%of the LB as medium,put it aside for the establishment of S.aureus BF standing model in vitro.Respectively,the3days and7days,using scanning electron microscope(SEM)、silver staining observe the surface of vector formed BF.Also including the line tablet colony counting, crystal violet staining BF semi-quantitative.Results:(1) Hospital clinical isolates of MRSA strains34134colonies on Congo red plates become black, surrounding fade.(2) Cultured for3days, carrier of silver staining shows visible lumps, cotton-like BF, SEM observe carrier surface can be seen the early BF;Cultured for7days, shows that the volume is more large mass, cotton-like of the BF,and the three-dimensional structure of the maturity of the BF by SEM.(3)3,7-day plate culture count、crystal violet staining BF semi-quantitative both have statistically significant difference, and7days compared with3days was statistically significant (P<0.01).Conclusions:(1) The hospital clinical isolates of MRSA strains34134, can successful formate biofilm.(2) Peritoneal dialysis tube as a carrier, the use of peritoneal dialysis fluid-50%of the LB medium in24-well plates can be successfully established S.aureus BF standing model in vitro,3day of the early film,7day of the mature film.PART ⅡThe intervention role of minocycline on S.aureus biofilmObjective:To investigate the destructive effects of minocycline to S.aureus BF of the dialysis tube, as well as its bactericidal effect of BF within S.aureus.Methods:(1) First detection of the MIC、MBC of minocycline on S.aureus.(2) Divided into blank control group,1/2MIC minocycline group,1MIC minocycline group,2MIC minocycline group,Cultured3days,7days later,the above—mentioned drug are added the medium.24hours later,SEM and silver staining observe the surface of the carrier’s BF,and crystal violet staining BF semi-quantitative;drug for4、8、24hours,using flat-panel micro-quantitative method to count viable cells on the surface of the dialysis tube.Results:(1)The MIC、MBC of minocycline on S.aureus is8ug/ml,128ug/ml respectively.(2) Whether it is cultured for3days, or cultured for7days, after24hours of drug action, SEM、silver staining by light microscopy observation found that1MIC minocycline group、2MIC minocycline group dialysis tube surface BF structure and size were significantly damaged and reduced compared with blank control group.Further more,the2MIC minocycline group’s BF significantly were smaller and less then1MIC minocycline group.(3) Whether cultured3days, or7days,4、8、24hours after the flat-panel micro-quantitative method of drug action to count viable cells and for24hours underwent crystal violet staining BF semi-quantitative, results showed that1MIC minocycline group、2MIC minocycline group were decreased significantly compared with1/2MIC minocycline group and blank control group (P<0.01). Further more, the2MIC minocycline group compared with1MIC minocycline group was statistically significant (P<0.01), and the number of viable cells decrease with time, but viable cells in BF isn’t completely removed.Conclusions:Minocycline could destroy had already formed early or mature biofilm of S.aureus and played the destruction role on reduce the number of viable cells within S.aureus BF with time.