Study On The Pharmacodynamics And Mechanism Of Anti-drug-resistant Pseudomonas Aeruginosa Biofilm Of Qingrejiedu Traditional Chinese Medicine

Pseudomonas aeruginosa?PA?is a common clinical pathogen.According to the World Health Organization,PA has multiple resistance to a variety of antibiotics,especially carbapenem and the third generation cephalosporin.The biofilm formation is one of the main reasons for drug resistance.The biofilm can resist the host's immune response and antibiotic treatment.When the biofilm is formed,the relative impermeable nature of the membrane and the volatile physiological state of the microorganism reduce the sensitivity of the bacteria to antibiotics,leading to the development of drug resistance.Traditional Chinese medicine is a unique cultural treasure in our country,and also a treasure house of new drug sources.It is one of the important ways to find new antibiotics by discovering antibacterial active substances from traditional Chinese medicine,especially from the traditional Chinese medicine of clearing away heat and detoxifying.The traditional Chinese medicine of clearing heat and detoxifying can reduce or eliminate the fever syndrome.The theory of traditional Chinese medicine believes that clearing heat and detoxifying traditional Chinese medicine,which is cold in nature,can alleviate or eliminate the symptoms of fever syndrome caused by infection of pathogenic microorganisms such as bacteria and viruses.In this work,abased on clearing heat and detoxifying traditional Chinese medicine,and selecting recently clinically collected PAs to conduct research on anti-biofilms at subinhibitory concentrations,with a view finding a material basis with anticlinical drugresistant PA biofilm activity or reducing the secretion of PA virulence factors and related research mechanism research provides theoretical support for clinical antiinfective treatment.The thesis could be divided into four parts.Part I:The selection of research objects.Firstly,28 clinical PA strains collected recently were double screened,the strains with the strongest resistance and membrane production ability were selected out and selected as the research strains.Secondly,honeysuckle and forsythia with strong biofilm-clearing effect were selected as key research drugs from the 13 most commonly used heat-clearing and detoxifying traditional Chinese medicines through biofilm models established by the selected research strains.Part II:The effects of honeysuckle and forsythia on PA virulence factors.First,the minimal inhibitory concentrations of PA on honeysuckle and forsythia were experimentally studied.Secondly,the growth curve was studied,and it was found that honeysuckle and forsythia had a certain effect on the growth of PA within 24 h at a subbacterial inhibitor concentration of 1/2MIC-1/16 MIC.The effect of honeysuckle on PA is obvious,and there is a certain dose-effect relationship.Further experiments showed that honeysuckle can simultaneously inhibit the secretion of pyocyanine and elastase from PA strains at subinhibitory concentrations,and forsythia has no inhibitory effect on these two virulence factors.The mechanism and material basis of biofilms are studied.Hence,this topic will focus on the mechanism and material basis of honeysuckle antibiofilm.Part III:The relationship between antibiofilm and quorum sensing?QS?system of honeysuckle.The formation of PA biofilms and the secretion of many virulence factors are related to the regulation of the QS system.In this study,we selected two pairs of genes,lasr/lasi and rhlr/rhli,which are the dominant genes in QS system,and used fluorescence quantitative PCR to observe whether honeysuckle can affect the above genes in this system.It was found that honeysuckle can down-regulate the three genes LasR,RhlR and RhlI at subbacteriostatic concentrations.Part IV:Material basis of honeysuckle inhibition of biofilm.It has been reported in a large number of literatures that phenolic acids,such as chlorogenic acid,protocatechuic acid and quinic acid,play an important role in the antibacterial activities of honeysuckle.Firstly,the growth curve,biofilm,virulence factors and related genes of these phenolic acids were measured in vitro.It was found that chlorogenic acid inhibited the biofilm,virulence factors and major genes in the QS system of PA at the sub bacteriostatic concentration,while protocatechuic acid and quinic acid had weaker effects than chlorogenic acid.In vivo,chlorogenic acid is unstable in chemical properties and quickly metabolized,which is not the ultimate antibacterial substance basis.Based on this reason,the subject has studied and traced the antibacterial effect of 13 major metabolites metabolized by intestinal mucosa and liver in chlorogenic acid in clinically common pathogenic strains:Pseudomonas aeruginosa,Staphylococcus aureus,Escherichia coli,Klebsiella pneumonia.The results show that most of the tested strains,the antibacterial activities of some major metabolites were enhanced,ranging from 4 to 66 times higher than chlorogenic acid.Benzoic acid,phydroxybenzoic acid,pcoumaric acid,dihydrocaffeic acid and gallic acid were stronger than the other metabolites.Moreover,the antibacterial actions of dihydrocaffeic acid and gallic acid against MSSA and MRSA was increased 33⁶6 times respectively,but weak antibacterial activity against PA.Among the 13 metabolites of chlorogenic acid,except for quinic acid?MIC¹/64 MIC?and dihydrocaffeic acid?1/2 MIC?,which had elimination effect on PA biofilm,the other metabolites were obviously no effect,the other metabolites had no significant effect.In summary,this topic found that honeysuckle,a traditional Chinese medicine which is applicated in clearing heat and detoxifies,downregulates key genes such as LasR,RhlR,and RhlI in the QS system,and has the effect of eliminating biofilms and inhibiting the secretion of virulence factors.Its main phenolic component,chlorogenic acid,may be one of the material basis of its anti-biofilm.Moverever,due to the chlorogenic acid is easy to be metabolized,the material basis of the final anti-biofilm effect may be its metabolite quinic acid?also the active ingredient in honeysuckle?or dihydrocaffeic acid.At the same time,it is also found that dihydrocaffeic acid,a metabolite of chlorogenic acid,has a strong inhibitory effect on Staphylococcus aureus in vitro.Combined with the results of previous LD₅₀ preliminary experiments,dihydrocaffeic acid has very low toxicity in mice.Therefore,the research on the antibacterial effect of dihydrocaffeic acid on Staphylococcus aureus needs to be further studied in the later stage.