The Research Of The Synthetic Peptide IDR-1018 On The Effects Of MRSA Biofilm

Objective:(1)To 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 explore the synergistic effect of IDR-1018 with erythromycin of the damaging effects of MRSA biofilm.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)IDR-1018/Erythromycin was added into the medium when bacteria were inoculated for 24h/48 h,incubating in 37? without shaking.Observe the biofilm by crystal violet staining assay.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 250mg/L.(2)A layer ofpurple continuous laminar of biofilm can be observed by crystal violet stain on the wall of 96 wells plate.(3)IDR-1018/Erythromycin was added into the medium when bacteria were inoculated for 48 h,incubating in 37? for 24 h,we can observe that IDR-1018 can do a lot of damage to the biofilm.For the erythromycin,it can destory the biofilm at the high concentration(1g/L~250mg/L),with the concentration decrease,the effect of the erythromycin are not obvious(<250mg/L).(4)IDR-1018/Erythromycin was added into the medium when bacteria were inoculated for 24 h,incubating in 37?for 24 h,we can observed that IDR-1018 can inhibition the grouth of the biofilm obviously.For the erythromycin,it can destory the biofilm at the high concentration(1g/L~250mg/L),with the concentration decrease,the effect of the erythromycin are not obvious(<250mg/L).(5)When combin the IDR-1018 with erythromycin,the two drugs produced strong synergies,while under low concentration(15mg/L,both),the mixed drug had a stronger effect on biofilm when compared with IDR-1018 or erythromycin.Conclusion:(1)Using PVC as carrier,biofilm models are successfully builded.(2)IDR-1018 can destroy MRSA formed biofilm,erythromycin also have the same effect,but not significant.(3)IDR-1018 can inhibit biofilm formation in MRSA,erythromycin also has the same effect,but the effect is not so obvious.(4)When combin the IDR-1018 with erythromycin,the two drugs produced strong synergies,while under low concentration,the mixed drug had a stronger effect on biofilm when compared with IDR-1018 or erythromycin.