The Effect Of Quorum Sensing Systems On The Infection Of MRSA And The Intervention Of Erythromycin On It

Objective:(1)Determinate the growth curve of MRSA to guide the MRSA inoculation timing in later experiment and test the Minimal Bactericidal Concentration(BMC)of erythromycin on MRSA to guide the applied dose of erythromycin.(2)To establish the biofilm modle of MRSA.(3)To investigate the destructive effects of erythromycin on biofilm of MRSA and find the effection of erythromycin on QS related factors.Methods:(1)A single colony of S.aureus standard strain ATCC33591 was cultured in 5ml tryptone soy broth(TSB)medium at 37? with shaking for 16 hours.Then the bacterial suspension was diluted to1:1000 in TSB,and then test OD595 every two hours to get the growth curve of MRSA.Then adjust the concentration of bacterial suspension to 108CFU/ml.Add the above bacteria into 96 wells plate with erythromycin doubling dilution.Stay the plate at 37? overnight,then coating 50?l bacterial suspension on solid medium at 37? for 24 h.Observe the bacteria situation.(2)Establishing the model of biofilm.PVC 96-wells plates are used as the carrier,TSB is used as medium.Adjusting the concentration of bacteria to 108CFU/ml and inoculating it on the carrier,incubating in 37? without shaking.Observe the biofilm by crystal violet staining assay.(3)Erythromycin was added into the medium when bacteria were inoculated for 24 h.Test the biofilm and collect the supernatant or serum of rat at 6h,12 h,24h,48 h,72h and 7d after adding erythromycin to detect the concentration of autoinducing peptides(AIP)which is a signal transduction factor of QS.Results:(1)Bacteria stay at lag phase after inoculated into the liquid medium for 2-4h,then come to logarithmic phase form 4h to16 h subsequently bacteria pass into stationary phase.The MBC of erythromycin on MRSA is 256mg/L.(2)A layer of purple continuous laminar of biofilm can be observed by crystal violet stain on the wall of 96 wells plate.(3)There is a decreasing tendency of biofilm in a short time after adding erythromycin or TSB and then gradually increase until to a steady state.On the whole,biofilm of experimental group is less than control group.(4)Adding erythromycin or TSB to the mature biofilm,there are different results after 48 h.Biofilm broke up in the group with erythromycin.In spite of a decrease at first time,biofilm then increased in the TSB group.There is significant difference between the two groups.(5)Only adding erythromycin within 3 hours after inoculating bacteria can effectively kill the MRSA and prevent the biofilm from generation.(6)The development trend of AIP is similar with biofilm and it happens earlier than biofilm.(7)With the intervention of erythromycin,the synthesis or secretion of AIP is decreased.Conclusion:(1)Using PVC as carrier,biofilm models are successfully builded.(2)Adding erythromycin timely before biofilm formation can kill the bacteria effectively.Erythromycin can broke biofilm.(3)Biofilm is inhibited by AIP obviously.(4)To some extent,erythromycin can inhibit the synthesis or secretion of AIP.