Mechanism Of Synergy Between Amoxicillin And Baicalein Against Resistant Staphylococcus Aureus And Development Of The Compound Suspension

Bovine mastitis is a main costly disease with high incidence in cows,which seriously threatens the development of dairy industry.Staphylococcus aureus(S.aureus)is the predominant causative agent of bovine mastitis,and the most effective treatment for S.aureus infection is antibiotics in clinical.However,the antimicrobial resistance of aureus has been recognized as a serious global concen with the widespread use of antibiotics in clinical,for instance,Penicillin resistant Staphylococcus aureus(PRSA)and methicillin resistant Staphylococcus aureus(MRSA)have diminished greatly the efficacy of common antibiotics such as amoxicillin(AMO).Chinese medicine is our extraordinary national treasures with abundant natural products.The aim of this study is to screen candidate substance from Chinese herbs with intensify the activity of AMO against PRSA and MRSA,investigate the mechanism of the synergism,and develop a compound suspension for the treatment of bovine mastitis caused by S.aureus infections.A collection of 21 Chinese herbs was screened for candidate substance determined by combined effects.The MICs(Minimum Inhibitory Concentration)of AMO against ATCC33591 decreased to ?4?g/mL in the presence of baicalein(BAI,?2 ?g/mL),indicating that BAI(?2?g/mL)reverses the resistance of AMO against ATCC33591.Then,the combination of AMO and BAI(AMO+BAI)showed synergy against clinical MRSA(mecA+)and PRSA(blaZ+)isolates.The Minimum Bactericidal Concentration(MBC)of AMO against ATCC33591 decreased from 32 to 16 ?g/mL when combined with BAI at 16?g/mL;Time-kill assay showed all bacteria was killed at 24 h in the presence of AMO(32?g/mL)+BAI(64 ?g/mL).BAI enhanced the Post-antimicrobial effect(PAE)of AMO against ATCC33591 from 1 h to 3 h and decreased the Mutant Prevention Concentration(MPC),indicating that the occurrence of S.aureus resistance may be reduced by the combination.In addition,the effect of AMO+BAI in vivo was studied in neutropenic murine thigh infection model,and the synergy between them was observed in mice.Subsequently,the mechanism of the synergism between BAI and AMO against MRSA was investigated in the study.BAI protected AMO from penicillinase and increased the susceptibility of penicillinase-supplemented S.aureus ATCC 29213 in a dose-dependent manner and the inhibition of penicillinase activity by BAI should be responsible for the synergism and protective effect.The synergy between AMO and BAI was also observed in the recombinant strains RN4220+pAM401-mecA,which was constructed by molecular cloning technique to transfer the gene mecA to methicillin sensitive S.aureus.The effect of penicillin-binding protein 2a(PBP2a)protein expression by BAI was detected by Western Blot,and it revealed that BAI did not suppress PBP2a protein expression significantly.The fluorescence polarization method and fluorescence imaging technique were used to test the activity of PBPsinfluenced by BAI.Surprisingly,the activity of PBP3 instead of PBP2a could be inhibited significantly by BAI.After screening excipients such as disperse medium,the optimized formulation and process were gained through the orthogonal design experiment according to the total score of settling volume ratio after standing 24 h and redispersibility as the indices and 3 batches of pilot samples were produced in the pharmaceutical factory.Two HPLC methods were developed for determination of AMO and BAI in the suspension with high recovery(>99%),good linearity,specificity,precision,repeatability and durability.The content of AMO and BAI exceeded 97%in the 3 batch of pilot suspensions.The suspension remained stable>18 months at room temperature away from light.The clinical efficacy of AMO and BAI compound suspension was evaluated in lacting bovine mastitis.38 cows with clinically mastitis quarters were selected,which were divided into test group(25 quarters)and control group(25 quarters,Synulox(?)LC).The bacteriological efficacies of the test and control formulations against S.auerus were 50.00%and 45.00%,respectively,whereas the efficacy of test suspension against MRSA was higher than that of control(60.0%vs.20.0%).The results demonstrated that the two formulations presented good clinical efficacy for the treatment of bovine mastitis with the high similar cure ratesagainst all kinds of bacteria(60.94 vs.69.09%).In conclusion,the combination of AMO+BAI was observed synergy against resistance S.auerus,and the MPC of AMO was also decreased by BAI,indicating that the occurrence of S.aureus resistance may be reduced by the combination.The inhibition of activity of penicillinase and PBP3 by BAI should be responsible for the synergism.The suspension of AMO in combination with BAI was developed and it is effective for the treatment of bovine mastitis infected with S.auerus.The development of formulation combining antibiotics with Chinese herbs will offer a new sight for new drug research and development in veterinary and also provide us essential data and scientific support for the new formulation of bovine mastitis treatment.