Study On Prevention And Killing Efficacy Of Daptomycin-loaded β-tricalcium Phosphate/calcium Sulfate For MRSA Biofilms

Purpose:Comparison of the antimicrobial efficacy,prophylaxis,and killing of MRSA biofilms by calcium sulfate(CS)or β-tricalcium phosphate/CS(β-TCP/CS)loaded with daptomycin(D)and vancomycin(V),to verify the antibacterial effect of daptomycin against MRSA biofilms when loaded on CS or β-TCP/CS.Method:We incubated MRSA strain(ATCC43300)in trypticase soy broth at 37℃ under shaking conditions at 200 rpm overnight and determined the minimum inhibitory concentrations(MICs)of D,V and gentamicin(G)using the microfluidic dilution method.Antibiotic-loaded microbeads(ALB)were prepared in separate D,V and G groups,as well as in D+G/V+G groups,by mixing every 1000 mg of daptomycin or vancomycin and 240 mg of gentamicin with every 10 cc of Genex or Stimulan powder and applying moulds to produce 4.8 mm wide hemispherical sunken antibiotic microbeads.A modified Kirby Bauer test was used to determine the potency of ALB over time,converting to area units cm2 using the equation A=nr2 to record statistics.In the biofilm prevention assay,incubation was started by adding both microbeads and a concentration of approximately 106 CFU/mL MRS A bacterial solution,which was changed and freshly added every 24 h.For the killing experiment,2 mL of TSB 106 CFU/mL bacterial solution was inoculated per well and incubated at 37℃ and 50 rpm on a shaker for 72 hours until biofilm was established before adding 2 microbeads and exchanging 2 mL of fresh TSB medium daily.The effect of microspheres on the number of biofilms was evaluated by live-bacterial counting,after 24 or 72 hours of disturbance in the presence of antibiotic microbeads,using the(CFU)plate count method.Similarly,we also used the live-dead staining method(Invitrogen)in confocal laser scanning microscopy(CLSM)or fluorescence microscopy to observe the state of the bacterial biofilm.Result:This in vitro study compared the efficacy of daptomycin and vancomycin against MRSA biofilms.The elution kinetics of daptomycin and vancomycin,combined with gentamicin and loaded with either β-TCP/CS or CS,in the presence of MRSA infection,was assessed.In addition,the efficacy of daptomycin,vancomycin,and gentamicin in prophylaxis and eradication of MRSA biofilms was also evaluated.D+G and V+G displayed similar antimicrobial potency against MRSA,by either β-TCP/CS or CS.In the prevention assays,both D+G and V+G showed similar efficacy in preventing bacterial colony formation,with approximately 6 logs lower colony-forming units than those in the control group at both 1 and 3 days.The killing effect on pre-formed biofilms showed significant decreases of approximately 4 logs at 1 and 3 days following treatment with D+G and V+G.In addition,the confocal laser scanning microscopy results support the colony-forming unit data.Moreover,single use of daptomycin and vancomycin showed similar efficacies in preventing and killing MRSA biofilms,both of which were better than that of gentamicin.Conclusion:Daptomycin+gentamicin and vancomycin+gentamicin loaded with βtricalcium phosphate/calcium sulfate or calcium sulfate showed similar prophylactic and killing effects on MRSA biofilms,also daptomycin alone showed similar results to vancomycin.It implies a potential indication of local administration daptomycin for the treatment of MRSA-associated osteoarticular infections,especially if vancomycin administration presents limitation.