Intervention Of Baicalin Combined With Linezolid On Staphylococcu Saureus Biofilm In Vitro And In Vivo

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INTERVENTION OF BAICALIN COMBINED WITH LINEZOLID ON STAPHYLOCOCCUS AUREUS BIOFILM IN VITROObjective To test the ability to form biofilm by Staphylococcus aureus strains and establish a stable biofilm model in vitro.To test the efficacy of baicalin combined with linezolid on staphylococcus aureus biofilm in vitro.Methods Congo red assay was employed to test the ability to form biofilm by MRSA 17546.A medical grade silicon sheet was employed to establish biofilm in vitro.Silver stain and Scanning electron microscopy(SEM)were employed to observer the morphological structure of biofilm.MIC of baicalin and linezolid to MRSA 17546 was determined by double dilution method.Staphylococcus aureus biofilm were constructed in vitro for 3 days and 7 days.Drug intervention was added for 24 hours on the 3rd and 7th days.The experimental groups were as follows: control group,baicalin group,linezolid group,baicalin + linezolid group.Viable counting assay was performed to evaluate the bacteria load on the silicon sheet.Crystal violet was employed to evaluate the biomass.The morphology of biofilm was observed by scanningelectron microscopy.Results Staphylococcus aureus colony grew black on Congo red plate,and the colour of Congo red medium around the colony faded.MRSA 17546 could be identified as a positive strain which could form biofilm.After 3 days of continuous culture in the 24-well plate and treated with silver staining,ground glass and cloud flocculent biofilm could be seen with poor light transmission by optical microscopy.After 7 days of continuous culture,the denser biofim could be seen and the transmittance was further reduced.Early biofilm was formed after 3 days of culture,sticky extracellular matrix and scattered Staphylococcus aureus could be seen by SEM.More and more sticky extracellular matrix,including more Staphylococcus aureus could be seen on mature biofilm formed after 7 days of culture.The MIC of baicalin and linezolid for MRSA 17546 were2048 ?g/mL and 2 ?g/mL,respectively.For the 3rd and 7th days biofim,Viable counting assay and Crystal violet assay showed that both monotherapy groups can not reduce the bacterium and biomass when compared with the control group(P>0.05).The bacterial load and biomass showed a significant decrease in the baicalin combined with linezolid group compared with the control group and the both monotherapy groups(P < 0.05).Scanning electron microscopy showed that the extracellular matrix and Staphylococcus aureus within the biofilm of the carrier surface in baicalin combined with linezolid group decreased significantly.Conclusion The clinical isolated MRSA 17546 has biofilm-forming ability.Staphylococcus aureus biofilm can be established in vitro by using silicon sheet as carrier and LB broth as medium.Baicalin could destroy Staphylococcus aureus biofilms and exhibited synergistic effects with linezolid in vitro.PART ? ESTABLISHMENT OF STAPHYLOCOSSUS AUREUS BIOFILM IN AANNIMAL MODELObjective To establish staphylococcus aureus biofilm in vivo for the further drug intervention.Methods MRSA 17546 was employed to establish the staphylococcus aureus biofilm infection model in vivo.Carboxymethyl cellulose(CMC)air-pouch model and silicon sheet implanted on the dorsal area of healthy SD rat model were established respectively.Viable counting assay was performed to evaluate the bacteria load.The morphology of biofilm was observed by scanning electron microscopy.Results The number of bacteria within the biofilm of the CMC air-pouch model changed greatly,and the number of bacteria within the biofilm constructed by the silicon sheet implant-related infection remained relatively stable.Scanning electron microscopy showed that there were many viscous substances observed in the CMC air-pouch model,and it was difficult to distinguish them from bacterial biofilm.The typical Staphylococcus aureus biofilm can be seen on the surface of the silicon sheet.Conclusion CMC air-pounch infection model and Silicon sheet implant-related infection model can successfully simulate the animal infection model of Staphylococcus aureus biofilm.The latter model is more simple and stable.Therefore,the latter model is used for subsequent drug intervention.PART ? INTERVENTION OF BAICALIN COMBINED WITH LINEZOLID ON STAPHYLOCOCCUS AUREUS BIOFILM IN VIVOObjective To test the efficacy of baicalin combined with linezolid on staphylococcus aureus biofilm in vivo.Methods Silicon sheet implant-related infection model was establiesed.Drug intervention was given immediately on the day of modeling,which was divided into control group,baicalin group,linezolid group,baicalin combined with linezolid group.After treatment for 1,2 and 3 days,viable counting assay was performed to evaluate the bacteria load.The morphology of biofilm was observed by scanning electron microscopy.Inflammatory reaction in local tissue of back was examinated by gross pathology and histopathology assay.The blood was collected at the abdominal aorta for the detection of PCT and CRP levels.Results According to the result of viable counting assay,in the 1st day after treatment,the bacterial load on the implant silicon surface didn't showed a significant decrease in the baicalin monotherapy group and the linezolid monotherapy group compared with the control group(P > 0.05).But the bacterial load on the implant silicon surface showed a significant decrease in the baicalin plus linezolid group compared with the control group,the baicalin monotherapy group and the linezolid monotherapy group(P<0.05).In the 2nd day after treatment,bacterial load on the implant silicon surface showed a significant decrease in the linezolid monotherapy group compared with the control group and the baicalin monotherapy group(P < 0.05).Moreover,bacterial load showed a significant decrease in the baicalin plus linezolid group compared with the linezolid monotherapy group(P<0.05).In the 3rd day after treatment,bacterial load on the implant silicon surface showed a significant decrease in the baicalin plus linezolid group compared with the control group,the baicalin monotherapy group,and the linezolid monotherapy group(P<0.05).Observed by SEM,baicalin monotherapy didn't exert destroyed effect on the staphylococcus aureus biofilm structure after treatment for 3 days.Linezolid exerted wild destroyed effect on staphylococcus aureus biofilm structure after treatment 2 day.The formation of viscous biofilm was reduced,and the bacteria in the biofilm were obviously reduced when treatment with baicalin plus linezolid.The gross and histopathological examination demonstrated that bacailin plus linezolid therapy relieved the inflammatory response of the surrounding tissue since the 1st day.The PCT and CRP levels decreased significantly after the treatment of baicalin plus linezolid compared with the control group,the baicalin monotherapy group and the linezolid monotherapy group(P<0.05).Conclusion Baicalin could destroy staphylococcus aureus biofilms and exhibited synergistic effects with linezolid in vivo,further relieve the inflammation induced by biofilm-associated infections.