The Combined Efficacy And Mechanism Of Baicalin Plus Vancomycin Against Implant-Related Infections Caused By Methicillin-Resistant Staphylococcus Aureus Biofilms In A Rat Model

PART Ⅰ SCREENING FOR A SUITABLE TESTING STRAIN AND ESTABLISHING A RAT MODEL OF IMPLANT-RELATED INFECTIONS CAUSED BY METHICILLIN-RESISTANT STAPHYLOCOCCUS AUREUS BIOFILMSObjective To screen for a suitable MRSA testing strain and establish a ramodel of implant-related biofilm infection to further investigate the efficacy anmechanism of anti-biofilm agents against the MRSA biofilm in vivo.Methods A Congo red assay,polymerase chain reaction for icaA gene detection,crystal staining assay and viable counting method were employed for identifying and comparing the biofilm-forming ability of clinically isolated strain MRSA 17546 and the laboratory standard strain MRSA ATCC 29213 in vitro.A medical grade silicon sheet covered with mature biofilms of MRSA17546 or MRSA ATCC 29213 was subcutaneously embedded in the dorsal area of a healthy SD rat to establish an implant-related infection model.The silicon sheets and the surrounding tissues were collected at different time points.A viable count assay was performed to evaluate the bacterial loads on the silicon sheets and in the surrounding tissues.A scanning electron microscope was used to observe the morphological changes of MRSA biofilms on the implants.The survival conditions,survival rates and survival curves of the rats infected by two different MRSA strains were also compared.Results Both the clinically isolated strain MRSA 17546 and the laboratory standard strain MRSA ATCC 29213 had biofilm-forming ability and carried the ica A gene.Moreover,the biomass and viable counts in the biofilms of the clinically isolated strain were significantly higher than those of the standard strain(P<0.05)in vitro.In the first three days after implantation,there were no significant differences in the bacterial loads in the biofilms on the implant surfaces and in the surrounding tissues(P>0.05),no matter when they were infected by either kind of MRSA strain.However,significant reductions in the bacterial loads were observed from the 4th day to 7th day in the biofilms on the implant surfaces and in the surrounding tissues compared with the initial day(P<0.05).At any time point,bacterial loads on the silicon sheets and in the surrounding tissues of the clinically isolated strain infection group were significantly higher than those in the laboratory strain infection group.Typical biofilms that formed on the surfaces of the silicon sheets could be observed by scanning electron microscope during the first three days after implantation.At any time point,a more typical and compact biofilm structure was formed on the implant surface in the MRSA 17546 infection group compared to that in the MRSA 29213 group.The results of the survival analysis indicated that the model rats in the clinically isolated strain infection group had a higher survival rate than that of the laboratory standard strain infection group.Conclusions 1.The clinically isolated strain MRSA 17546 possesses a stronger biofilm-forming ability than the laboratory standard strain MRSA ATCC 29213 in vitro.2.The rat model of implant-related infection caused by a MRSA biofilm could be successfully established in the present study.At a certain period,a more typical and stable implant-related MRSA biofilm infection in the rat is developed by the clinically isolated strain MRSA 17546;therefore,this strain is suitable for establishing the rat model of an implant-related biofilm infection caused by a MRSA biofilm in the present study.PART Ⅱ THE COMBINED EFFICACY OF BAICALIN PLUS VANCOMYCIN AGAINST IMPLANT-RELATED INFECTION CAUSED BY METHICILLIN-RESISTANT STAPHYLOCOCCUS AUREUS BIOFILMSObjective To study the combined efficacy of baicalin plus vancomycin against implant-related infections caused by methicillin-resistant staphylococcus aureus biofilms in a rat model.Methods A medical grade silicon sheet covered with mature biofilms of MRSA 17546 was subcutaneously embedded in the dorsal area of a healthy SD rat to establish an implant-related infection model.The model rats were divided into the following five groups: the sham-operation group,the model group,the baicalin monotherapy group,the vancomycin monotherapy group and the baicalin plus vancomycin combined-treatment group.After treatment for 1,2and 3 days,the silicon sheets and the surrounding tissues were collected.A viable count assay was performed to evaluate the bacterial load on the silicon sheets and in the surrounding tissues.A scanning electron microscope was used to observe the morphological changes of MRSA biofilms on the implants.The inflammatory reactions of the infected lesions were examined by gross pathology and histopathology assays.Peripheral blood samples were collected for evaluating white blood cell counts and C-reaction protein levels.Results During the 2nd day after treatment,the bacterial loads on the implant surfaces showed a significant decrease in the baicalin and vancomycin combined group,compared with the model group and the baicalin monotherapy group(P<0.05).The reduction of bacterial loads in the surrounding tissues was also observed in the combined treatment group when compared with both monotherapy groups and the model group(P<0.05).In the 3rd day after treatment,the bacterial loads of the combined treatment group were apparently reduced,compared with both monotherapy groups and the model group,whether on the silicon sheets or in the surrounding tissues(P<0.05).Under the electron scanning microscope,both baicalin and vancomycin monotherapy exerted a very mild destructive effect on the MRSA biofilm structure.However,the biofilm structure and the inflammatory cells adhering to the implant and the biofilm surfaces were eliminated even more when treated with baicalin plus vancomycin,as very few planktonic cells remained after three days of the combined treatment.The gross and histopathological examinations demonstrated that baicalin monotherapy could,to some extent,relieve the inflammatory response of the surrounding tissues from the 1st day.Notably,the baicalin and vancomycin combined treatment further relieved the hyperemia and edema of the surrounding tissues,alleviated the inflammatory cell infiltration,and promoted the healing of the tissues.Moreover,a lower white blood cell count was observed in the combined treatment group than in both monotherapy groups and the model group(P<0.05).Conclusions The combined therapy of baicalin plus vancomycin could exert a more distinct elimination effect than monotherapy against the MRSA biofilm in vivo,and it further relieves the inflammatory response and the pathological damage in the surrounding tissues.Taken together,combined therapy is effective in treating the implant-related infections caused by MRSA biofilms.PART Ⅲ THE MECHANISM OF BAICALIN IN COMBINATION WITH VANCOMYCIN AGAINST IMPLANT-RELATED INFECTIONS CAUSED BY METHICILLIN-RESISTANT STAPHYLOCOCCUS AUREUS BIOFILMSObjective To investigate the immunomodulated and anti-inflammatory mechanism of baicalin,in combination with vancomycin,against the implant-related infections caused by MRSA biofilms in vivo.Methods A medical grade silicon sheet covered with mature biofilms of MRSA was subcutaneously embedded in the dorsal area of a healthy SD rat to establish an implant-related infection model.The model rats were divided into the following five groups: the sham-operation group,the model group,the baicalin monotherapy group,the vancomycin monotherapy group and the baicalin plus vancomycin combined treatment group.After treatment for 3 days,the infected tissues around the silicon sheets in different groups were collected.An enzyme-linked immunosorbent assay(Elisa)was used to evaluate the expression levels of IL-1β,IL-4,IL-6,IL-10,TNF-α and IFN-γ in the surrounding tissues.q RT-PCR was employed to detect the expression levels of TLR2,My D88,NF-κB and IκB m RNA involved in the TLR2/My D88/NF-κB signaling pathway.The alterations of donated proteins in the TLR2/My D88/NF-κB signaling pathway were detected by western blot assays.Results Sharp increases in the expression levels of IL-1β,IL-6,TNF-α,IL-10,IL-4 and IFN-γ in the surrounding tissues were observed when the rats were subjected to implant-related infections caused by MRSA biofilms.Moreover,the IFN-γ/IL-4 ratio;the expression levels of TLR2,My D88,NF-κB and IκB m RNA;and the related proteins involved in the TLR2/My D88/NF-κB signaling pathway were also significantly increased(P<0.05).Vancomycin monotherapy had almost no effect on the expression level of cytokines,genes and proteins as mention above(P>0.05).Baicalin monotherapy could lower the level of IL-1β,IL-6,TNF-α,IL-4 and IFN-γ;promote the synthesis of IL-10;and further elevated the IFN-γ/IL-4 ratio(P<0.05)compared with the model group.In addition,Baicalin monotherapy also significantly reduced the expression levels of TLR2,My D88 m RNA and proteins and declined the phosphorylation levels of NF-κB p65 and IκB(P<0.05).Notably,baicalin and vancomycin combination therapy showed a more obvious inhibitory effect on the release of IL-1β,IL-6,TNF-α,IL-4 and IFN-γ;further,the therapy promoted the IFN--γ/IL-4 ratio;lowered the expression levels of TLR2,My D88 m RNAs and proteins;and decreased the phosphorylation level of NF-κB p65 and IκB when compared with the baicalin monotherapy group(P<0.05).Conclusions Baicalin,in combination with vancomycin,could reduce the secretion of inflammatory cytokines caused by MRSA biofilm infections via the inhibition of the activation of the TLR2/My D88/NF-κB signaling pathway on the molecular and genetic levels.In addition,combination therapy could also modulate the balance of Th1/Th2 and shift it to the Th1-dominant immune response,thereby facilitating the elimination of the MRSA biofilm in vivo.PART Ⅳ DETERMINATION OF THE PLASMA CONCENTRATION OF BAICALIN AND STUDYING THE EFFECT OF BAICALIN ON THE PHARMACOKINETICS OF VANCOMYCIN IN RATSObjective To determine the plasma concentration of baicalin and to investigate the effect of baicalin on the pharmacokinetics of vancomycin in rats,thereby addressing the rationality of the combination therapy.Methods 1.Rats were randomly divided into three groups and intraperitoneally injected with a single bolus dose of baicalin at 25 mg/kg,50mg/kg and 100 mg/kg.Peripheral blood samples were collected from the angular vein at several time points,and the concentrations of baicalin in the plasma were measured by HPLC.The drug concentration-time curves and pharmacokinetic parameters of baicalin were analyzed by DAS 2.0 software.2.Rats were randomly divided into two groups and intraperitoneally injected with a single bolus dose of vancomycin at 100 mg/kg or co-administered with baicalin at 100 mg/kg plus vancomycin at 100 mg/kg.Peripheral blood samples were collected from the angular vein at several time points,and the concentrations of vancomycin in the plasma were measured by HPLC.The drug concentration-time curves and pharmacokinetic parameters of vancomycin were analyzed by DAS 2.0 software.Results 1.The peak concentrations(Cmax)of baicalin in the rat plasma were 65.38 ± 9.88 μg/m L,93.8 ± 22.63 μg/m L and 229.02 ± 31.17 μg/m L;the half-lives(t1/2)were 3.57 ± 0.19,3.19 ± 0.37 and 3.03 ± 0.42 h;and the areas under the curve(AUC(0→∞))were 80.06 ± 12.52,120.77 ± 10.05 and 219.20 ±16.42 when the rats were intraperitoneally injected with a single bolus dose of baicalin at 25 mg/kg,50 mg/kg and 100 mg/kg,respectively.The times to peak were 0.5 h for all three groups.2.The main pharmacokinetic parameters of vancomycin intraperitoneally administered alone were as follows: Cmax = 30.95± 2.51,Tmax = 0.5 h,t1/2 = 5.18 ± 0.72 h,AUC(0→∞)= 96.77 ± 2.29 μg/m L*h and CL= 1.03 ± 0.05 L/h/kg.The pharmacokinetic parameters of vancomycin intraperitoneally co-administered with baicalin were as follows: Cmax = 28.03 ±2.42,Tmax= 0.91 ± 0.20 h,t1/2 = 8.39 ± 0.95 h,AUC(0→∞)= 94.28 ± 5.71 μg/m L*h,CL = 1.06 ± 0.11 L/h/kg.The parameters of Tmax and t1/2 were significantly prolonged when co-administered with baicalin(P<0.05).Conclusions 1.The pharmacokinetic parameters of baicalin obtained from the current study could be taken as a basis for further analyzing the mechanism of baicalin in combination with vancomycin against the implant-related infections caused by MRSA biofilms.2.Baicalin prolongs the action time of vancomycin in the rat by delaying its elimination,as indicated in combined therapy,and,to some extent,is rational for the treatment of MRSA biofilm infections.