The Antibacterial Activity Of Ursolic Acid Combined With Frequently-used Antimicrobials Aganist MRSA

Objective:At present,most of the disease with “heat toxin symptom” are regarded as bacterial infection,and the traditional Chinese medicine(TCM)with “heat-clearing and detoxifying”function is often used to treat these kinds of diseases,especially in the drug-resistant bacteria,such as methicillin-resistant Staphylococcus aureus(MRSA),induced infection disease This study aims to observe the antibacterial activity of monomer from TCM with“heat-clearing and detoxifying” function,and explore the possible synergistic effect of the monomer combined with the frequently-used antimicrobials aganist MRSA.It will provide new therapeutic drugs for the treatment of drug-resistant bacteria induced infection disease,and provide the new strategy for the integration of traditional Chinese and western medicine against MRSA.Methods:First of all,we selected 35 kinds of active monomers from “heat-clearing and detoxifying” TCM which may have antibacterial activity according to the literature.Then the antibacterial activity of these monomers was evaluated by the minimal inhibitory concentration(MIC)assay on S.aureus and E.coli.Next,in vitro antibacterial activity evaluation were carried out for the selected monomer of Chinese medicine using the determination of antibacterial spectrum,the effects on bacterial growth curve,the synergistic effects combined with antibiotics frequently-used in clinic,the drug resistance assay,and the observation of their effects on bacteria morphology under electron microscopy.Finally,in the MRSA induced skin infection and lung infection animal model of mice,the anti-infection efficacy of the selected monomer in combination with antibiotics frequently-used in clinic were evaluated.Results:1)MIC measurement showed that ursolic acid(UA)from Hedyotis diffusa Willd had the most potent antibacterial activity against both S.aureus(ATCC29213)and MRSA(USA300),with the MIC at 32μg/ml.2)In MRSA(USA300),the fractional inhibitory concentration index(FICI)of ursolic acid combination with oxacillin,piperacillin,ampicillin or ceftazidime was equal or less than 0.5(≤0.5),it indicated that these two drugs had synergistic effect.When ursolic acid was used in combination with azithromycin,clarithromycin,roxithromycin,doxycycline,chloramphenicol,linezolid and vancomycin respectively,FICI was between 0.5 and 1,it indicated that these two drugs had partial synergistic effect.When ursolic acid was used in combination with minocycline,rifampicin or teicoplanin respectively,FICI was equal to 1,it indicated that these two drugs had additive effect.When ursolic acid was used in combination with imipenem,lincomycin or clindamycin,respectively,FICI was between 1and 4,it indicated that these two drugs had no interaction.3)The treatment of ursolic acid combined with oxacillin reduced the resistance of MRSA to oxacillin or ursolic acid significantly.The combination of clarithromycin and ursolic acid also slowed the delayed development of MRSA resistance to clarithromycin.And the resistance of MRSA to ursolic acid was also reduced after the treatment of ursolic acid combined with ampicillin,azithromycin or doxycycline.4)Ursolic acid roughened the surface of MRSA,caused abnormal bacterial cell division,creased the cell membrane,damaged the bacteria,and reduced the number of bacteria significantly.However,the bacterial cell surface roughness,abnormal division and cell membrane shrinkage became more obvious after treated with MRSA in combination with ampicillin,it suggested that ursolic acid could increase the sensitivity of the resistant bacteria to ampicillin.5)On the MRSA induced skin infection or lung infection mice model,UA combined with oxacillin or azithromycin showed a better treatment effect than alone,which could effectively reduce the inflammatory response and the bacterial number at the infected site and shorten the recovery time.Conclusion:1)Ursolic acid had potent antibacterial activity against gram-positive bacteria,even on some resistant gram-positive bacteria,but had no obvious effect on gram-negative bacteria.The antibacterial effect of ursolic acid was possible through inhibition of bacterial division.2)Ursolic acid had synergistic effects with most antibiotics which act on bacterial cell walls or ribosomes,such as oxacillin,piperacillin,ampicillin,ceftazidime,azithromycin,clarithromycin,roxithromycin,doxycycline,chloramphenicol,vancomycin,linezolid,etc.But when ursolic acid was combined with teicoplanin,imipenem,lincomycin,clindamycin,the synergistic effect was not obvious.3)Ursolic acid combined with antibacterial drugs could effectively slow down the generation of bacterial resistance,and the degree of slowing down was related to the intensity of the synergistic effect and the characteristics of the drug itself.The development of the drug resistance was dependent on the synergy degree.namely,the stronger synergistic effect of the combination of drugs showed the less occurred resistance to the combination of drugs.4)Ursolic acid combined with oxacillin or azithromycin had better antibacterial activity than them alone in the treatment of MRSA induced skin infection or lung infection in vivo,which could effectively reduce the symptoms of infection and relieve the pathological changes on infection site,and shorten the recover course of the infection.In conclusion,our results provided an important theoretical basis for treating infectious diseases caused by MRSA with the combination use of ursolic acid and frequently-used antibacterial drugs,and expanded the range of application of traditional Chinese medicine and western medicine for treating bacterial induced infectious diseases.