The Effect Between Baicalein And Linezolid Against Methicillin-Resistant Staphylococcus Aureus Biofilm In Vivo

PART 1 ESTABLISHMENT OF MRSA BIOFILM INFECTION MODEL IN RATSObjective To establish two MRSA-BF infection models in SD rats.One model will be used to establish the MRSA-BF infection in the CMC pouch of the SD rats with different immune status(immune normal state and immunocompromised state),whereas the other model will be used to set up the MRSA-BF infection with vector on the back of SD rats with immune normal state.The mortality rates,white blood cell(WBC)counts and colony forming units(CFU)will be used to assess whether these models are established successfully.In addition,the morphology of the MRSA-BF will be observed by Scanning electron microscopy(SEM).These results will be used to assess the feasibility and stability of the two models.Methods To establish a model for MRSA-BF infection in immunosuppressed SD rats induced by CTX: The immunosuppressive state was induced in SD rats by CTX,and then WBC counts were counted by orbital venous blood sampling in SD rats to assess the degree of immunosuppression.MRSA was cultured in LB broth and air pouch was formed in the dorsal scapular area of ??the SD rat.Sterile 3%CMC was injected at 10 m L on the next day to form a CMC pouch.At the same time,MRSA at 107 CFU/m L was inoculated into CMC pouch to induce infection.The CMC pouch was harvested and homogenized for 5days after MRSA infection.CFU counts were performed by serial dilution plating method.The formation of BF in the CMC pouch was observed by SEM.To establish a model for MRSA-BF infection with vectors in normal immune status SD rats: air pouch was set up on the back scapular region of SD rats.The vectors were incubated with MRSA in LB broth for 3days,and then implanted into the air pouch of the SD rats.Or the vector was directly put into the air pouch of the SD rat for 3 days,and then inoculated with MRSA.CFU counts were performed by serial dilution plating method.The formation of BF on the surface of vector was observed by SEM.Results The immunosuppressive status of SD rat was well established by using the dose of CTX: 150(mg/kg/D1)+100(mg/kg/D4).WBC counts were began to decline on the 4th day of the experiment and began to rise on the 9th day.There was no difference in the survival rate of the immunosuppressed SD rats between in a clean environment and in a specific pathogen free environment.However,continued sample collection was affected the survival rate of SD rats.SEM was showed that MRSA-BF in the CMC pouch could be formed earlier in immunosuppressed SD rat than that in immunocompetent SD rat.After cultured the vectors three days in vitro,the bacteria colonies,the extracellular matrix and the early MRSA-BF were formed on the surface of the vectors.Over time,with different methods to infect the vectors,the CFU and MRSA-BF formation on the surface of the carrier were reduced.Compared to the method of inoculating MRSA into the air pouch after the vectors were implanted for 3 days,the other method,the vector was cultured 3 days in vitro before putted it into the air pouch,was more successful.The CFU load of MRSA and the MRSA-BF were more obvious than that.Conclusion It is successful to establishment two models with MRSA-BF infection in SD rats: immunosuppressed SD rats on the back of CMC pouch infection MRSA-BF model and implanted the vector,MRSA-BF infected 3 days in vitro,into the SD rat air pouch model.They can be carried out for different purposes in animals researches,to provide a choice of different animal models.PART ? THE EFFECTS OF BAICALEIN ON MRSA-BF INFECTION IN RATS AND THE PHARMACOKINETICS OF BAICALEIN IN RATSObjective To select the appropriate intervention dose of baicalein,observing the effect of baicalein on the formed MRSA-BF,and to further explore the baicalin metabolism in SD rats.Methods To establish the model of MRSA-BF infection on the CMC pouch in SD rat with normal immune status.SD rats were randomly divided into blank control group,high-dose baicalein group,medium dose group and low dose group.After BF was formed in vivo,different concentrations of baicalein were given to SD rats respectively.The changes of living conditions and survival rate of SD rats were observed after treatment 5 days.The CFU counts of MRSA were determined by serial dilution method.The CMC pouch tissues were analyzed by histopathology with haematoxylin and eosin,to observ the inflammation.The pharmacokinetics of baicalein in SD rats was determined by HPLC.Results The growth of SD rats in high-dose baicalein was poor,with the death of rats.The SD rats in middle-dose and low dose baicalein grew slightly better than those in the blank control group.In the baicalein group,the CFU counts of MRSA in the high-dose and the middle-dose groups were slightly lower than those in the blank control group,but not significantly decrease in the low dose group.With the increase of the baicalein concentration,there was the trend of reduction,but no significant difference.The histopathological image analysis of pouch tissues was showed that it can be seen more neutrophils,necrosis and abscess in blank control group.With the dose of the drug increased,the severity of inflammatory in the baicalein group was a decreasing trend.After a single intraperitoneal administration of baicalein to SD rats,a two-compartment open model was presented by HPLC.Its plasma concentration was peaked at about 1.5 hours and 8.0 hours,and the peak concentrations at the two peaks were 7.63ug/m L and 10.27ug/m L,respectively.It suggested that baicalein be rapidly distributed in vivo,but slowly metabolized and can still be detected within 12 hours.Conclusion Baicalein has no obvious antibacterial effect on MRSA-BF on CMC pouch.Histopathological image is analyzed that baicalein could reduce the severity of inflammatory cells.The concentration of baicalein is effectively detected by HPLC.Combined with the CFU of the wall of CMC pouch,pathological image analysis and the plasma concentration-time curve of baicalein by HPLC,they are indicated that the appropriate intervention method of baicalein is100mg/Kg/q12 h,i.p.in vivo,which could be used to further research.PART ? ANTIBACTERIAL SYNERGY BETWEEN BAICALEIN AND LINEZOLID AGAINST MRSA-BF INFECTIONG IN A RAT MODELObjective To implant the vector into the air pouch on the back of SD rats after cultured with MRSA-BF for 3 days in vitro.And then to treat the SD rats with baicalein and(or)linezolid.To explore the effects of baicalein on MRSA-BF and to further explore the synergistic bactericidal effect of baicalein combined with linezolid on MRSA-BF in vivo.Methods To culture the implant to form MRSA-BF for 3 days,inserted it into the SD rat air pouch.Then the MRSA-BF was treated with baicalein(100mg/Kg/q12 h,i.p.)and linezolid(40mg/Kg/q12 h,i.p.)alone and in combination.The susceptibilities of MRSA biofilm to different treatment strategies was assessed by plate counting for bacterial colony forming units(CFU).It was advantageous to do histological staining of inflammatory process in the air pouch tissues by optical microscope and morphological examination of the biofilm structure on an implant by SEM.Results After treatment,the CFU counts of BF in the baicalein and linezolid monotherapeutic groups,baicalein combined with linezolid group were lower than those in the blank control group(P <0.05).Obviously,CFU counts were significantly lower in the combination group than those in other groups(P <0.05),which prolonged for two days or even longer.However,after treatment for 1 day,there was no significant decrease of CFU in the combination therapeutic group compared to the baicalein treatment group.Pouch histological hematoxylin and eosin staining showed that in the blank control group,neutrophils,necrosis,and abscesses were seen in the early stage,and more lymphocytes and foam cells were seen in the middle and late stages.The severity of inflammatory cell infiltration in the baicalein or linezolid monotherapy group was slightly less than that in the blank control group,which was the least severe in the combination treatment group,with little necrotic and abscess.The BF structure on the surface of the carrier was clearly sparse and more free colonies were seen by SEM in the combination treatment group.The BF morphology was observed on the1 th and 2th day after treatment in the combination treatment group,but no obvious BF on the 3th day after treatment.Conclusion Baicalein has a destructive and decreasing effect on MRSA-BF.Baicalein combined with linezolid have a synergistic bactericidal effect on MRSA-BF,which enhances the antibacterial activity of linezolid.The pathological image analysis shows that baicalein combined with linezolid can significantly reduce the count of inflammatory cells.It also shows the synergistic antibacterial effect of baicalein combination with linezolid by SEM.Baicalein could be used to treat MRSA-BF-related infections as an effective drug in the future.It is helpful to further discuss the possible mechanisms of the synergism and to study the virulence factors of MRSA and drug resistance mechanism in vivo.PART ? EFFECT OF BAICALEIN AND LINEZOLID ON THE VIRULENCE FACTOR AND INFLAMMATORY CYTOKINES EXPRESSION OF MRSAObjective To investigate the effect of baicalein and/or linezolid on MRSA virulence factors and inflammatory factors,such as: SEA,CRP and PCT.To discuss the anti-MRSA-BF infection mechanism of baicalein for further study.Methods To establish a model for MRSA-BF infection with vectors in normal immune status SD rats.The implants were cultured in LB broth for 3 days in vitro before implanted them into air pouches.The blank control group,linezolid group,baicalein group and baicalein plus linezolid group were set up,to treat for 3 days.The dosage of linezolid and baicalein was 40 mg/Kg/q12 h,i.p.and 100 mg/Kg/q12 h,i.p.,respectively.After treatment every day,the blood was collected at the abdominal aorta of SD rats in each group,and serum and plasma were collected by centrifugation.SEA,CRP and PCT in each group were detected by ELISA.Results After drug intervention,the concentration of SEA,CRP and PCT were lower in baicalein group than those in blank control group(P <0.05),which were decreased more significantly in baicalein plus linezolid group(P <0.05).In the linezolid group,the concentration of SEA was lower than that in the blank control group,but there was no significant difference between CRP(at 24 h and 48h)and PCT(at 48 h and72h)(P> 0.05).Conclusion The expression of SEA is inhibited in the baicalein group and the linezolid group,which is more pronounced in the baicalein combined with linezolid group.Since SEA is regulated by the Agr system.It is considered that Agr system could be inhibited by baicalein and baicalein combination linezolid,thereby downregulating the expression of SEA.The baicalein group and combined treatment group can reduce the level of CRP and PCT,but the level of CRP and PCT have no significant effect in linezolid group.