Study On The Anti-biofilm Effect And Mechanism Of Chlorogenic Acid And Derivatives

Objective:Pseudomonas aeruginosa(PA)biofilm can protect bacteria themselves from the surrounding environment and hinder phagocytosis,thus conferring themselves with long-term viability hindering the treatment of infections.Chlorogenic acid(CA)is the active ingredient of many herbal medicines,which has broad-spectrum antibacterial effect and can increase the permeability of bacterial outer cell membrane and plasma membrane to disrupt the cell membrane to exert antibacterial activity.In this paper,CA and its derivative 3-O-(4-nitrocinnamoyl)quinic acid(CP5)were used for the comparative study of the biofilm growth inhibition and mature biofilm elimination of drug-resistant PA.Using transcriptomic techniques and bioinformatic analysis,we identified the gene targets and key pathways of CA and its derivative CP5 on biofilm at sub-inhibitory concentrations,and explored the inhibition of biofilm growth at the molecular level.Additionally,metabolomics techniques are used to characterize bacterial intracellular metabolites,study the mechanism of anti-biofilm action,and search for potential biomarkers to provide theoretical basis and new strategies for CA in the treatment of clinical drug-resistant bacterial infections.Methods:1.Screening of biofilms for drug-resistant PA strainsThe paper diffusion method was used to screen for resistance of clinical PA bacteria,and PA strains that were resistant to Imipenem,Meropenem and Levofloxacin were screened.The resistant PA strains were determined by crystalline violet staining,and the resistant strains with strong film-producing ability were obtained for subsequent tests.2.Biofilm inhibition and elimination of drug-resistant PA strains by CAThe minimum inhibitory concentration of chlorogenic acid against drug-resistant PA bacteria was determined by broth microdilution method.The clarification of the solution in the observation wells was the lowest inhibitory drug concentration for bacteria not growing.Also use crystal violet staining method,CA in sub-inhibitory concentration of drug-resistant PA bacteria growth inhibition and elimination of mature biofilm effect,to determine the sub-inhibitory concentration range.3.Synthesis of CP5 and its effect on biofilmThe CA derivative CP5 was obtained by a 5-step synthesis reaction using D-(-)-quinic acid as the starting material,and was used to inhibit the growth of biofilm and eliminate the mature biofilm of drug-resistant strains at sub-inhibitory concentrations.The anti-biofilm activity was also investigated using molecular docking results with las A,las I and las R key gene expression proteins in the PA biofilm formation pathway as receptors and CA and CP5 as ligands.4.Transcriptomic analysis of CA and target compound for biofilm inhibitionCA and target compounds were used to act on drug-resistant PA strains at subinhibitory concentrations,and RNA was extracted for transcriptomic analysis,while the genes related to biofilm formation,las A,las I,las R,rhl I and rhl R,were measured by QPCR.The measured gene expression data were statistically analyzed to obtain the differentially expressed genes,and bioinformatic analysis was used to enrich the differentially expressed genes and analyze the pathways to explore the mechanism of inhibiting the growth of drug-resistant PA bacteria biofilm at the molecular level.5.Metabolomic analysis of CA and target compound inhibition biofilmsThe intracellular metabolite assay of the strain was established and methodologically validated using n-decanoic acid as an internal standard substance and the metabolic components palmitic acid and stearic acid as controls with GC-MS.The bacterial intracellular metabolites were qualitatively identified and quantitatively analyzed using applicable assays,and pathway analysis of the differential metabolites was performed to investigate the mechanism of CA and CP5 anti-biofilm and to search for potential biomarkers.Results:1.The inhibition circle diameters of Imipenem,Meropenem and Levofloxacin paper tablets against the quality control bacteria ATCC27853 were 21.31,29.26 and25.83 mm respectively,which met the quality control criteria and the experimental system was reliable.A total of 20 strains of PA resistant to all three drug-sensitive papers were screened from the library of clinical pathogenic bacteria collected in the past 3 years.Among the 20 drug-resistant PA strains,the strains with good biofilm growth were PA1803,PA1902,PA1906,PA1907,PA1909,PA1910,PA9104,PA9105.strains PA1902,PA1906,PA1910,PA9105 showed impurities during the staining process,while strains PA1803,PA1909,PA1904 showed impurities during the staining process.PA1909,PA1904 strains had unstable biofilm growth.Therefore,drugresistant strain PA1907 was selected as the strain for subsequent experimental studies.2.The MIC of Levofloxacin against QC strain ATCC27853 was 1 μg/m L,which is in accordance with the QC range of 0.5-4 μg/m L in CLSI M100 S27,indicating that this experimental system is reliable.The MIC ranges of CA and levofloxacin against resistant PA strains were 8-16 mg/m L and 8-32 μg/m L,respectively.In biofilm growth inhibition,CA at subinhibitory concentrations of 4 mg/m L(1/2 MIC)and 2 mg/m L(1/4MIC)showed highly significant inhibition(P<0.01)of biofilm growth of drug-resistant PA1907 strain.The biofilm growth was significantly inhibited at a concentration of 1mg/m L(P<0.05).While in the elimination of mature biofilm,chlorogenic acid at subinhibitory concentration of 4 mg/m L(1/2 MIC)had a highly significant elimination of mature biofilm of resistant PA1907 strain(P<0.01).The growth of the mature biofilm was significantly inhibited at concentrations of 2 mg/m L and 1 mg/m L(P<0.05).3.The CA derivative CP5 was obtained by a five-step synthetic reaction using D-(-)-quinic acid as the starting material,with good yields of each intermediate in the reaction.CP5 showed highly significant inhibition of biofilm growth of drug-resistant PA1907 strain at subinhibitory concentrations of 1.6 mg/m L(1MIC)to 0.2 mg/m L(1/8MIC)(P<0.01);significant inhibition was observed at 0.1 mg/m L(P<0.05).The binding energies of eight structurally designed derivatives CP1～CP8 and CA with las A,las I and las R genes were calculated by molecular docking technique,and the results showed that the CP5 had the highest binding energies of-7.6,-7.5 and-11.2 kcal/mol for the three gene expression proteins,respectively,which verified the effect of CP5 on biofilm activity.4.After CA inhibition concentration of 1.0 mg/m L(1/8 MIC)solution was applied to the resistant PA1907 strain,588 differentially expressed genes were obtained after screening compared with the control group,of which 121 genes were significantly upregulated and 467 genes were significantly down-regulated.In contrast,1813 differentially expressed genes were obtained in the CP5 subinhibitory concentration 0.1mg/m L(1/16 MIC)solution of resistant PA1907 strain,of which 768 genes were significantly up-regulated and 1045 genes were significantly down-regulated when compared with the control group.Enrichment analysis of key genes obtained by screening in CA and CP5 groups yielded 109 and 742 GO enrichment terms,respectively.The results of KEGG pathway analysis showed that the pathways in both CA and CP5 groups were: biofilm formation,flagellar assembly,and ABC transport.Fluorescence real-time quantitative Q-PCR results showed that the expression of biofilm formation-related genes las A,las I,las R,rhl I and rhl R were down-regulated in CA and CP5-acting resistant PA1907 strains,which was consistent with the expression results of related down-regulated genes in transcriptomics.5.The biotransformed metabolic components were better separated in the chromatogram after pretreatment with BSTFA+TMCS(99:1)derivatization,with higher peak response values for each compound.The retention times of n-decanoic acid internal standard and palmitic acid and stearic acid control were 16.5,24.3 and 26.2min,respectively,in GC-MS total ion flow chromatogram species,and the internal standard and control did not interfere with each other under this chromatographic separation condition.The linear correlation coefficients of palmitic acid and stearic acid were above 0.999,indicating good linearity,and the detection ranges were 4.02～128.75μg/m L and 4.15～133.00 μg/m L,respectively.The recoveries of palmitic acid and stearic acid ranged from 98.83 to 113.91 % and 98.76 to 109.83 % with RSDs of 4.77 %and 3.50 %,respectively,indicating that the assay method was reliable.The results of the experiment identified 54 metabolites,and after statistical analysis,a total of 22 differential metabolites were screened in the chlorogenic acid group compared with the blank group,among which 7 products were up-regulated in expression,and the most obvious ones were compounds Spermine,L-Oxoproline,Tyramine and L-Ornithine.Among the 16 products with down-regulated expression,Inositol,L-isoleucine,Pyrrole-2-carboxylic acid and Glycine were the most obvious.In the CP5 group,19 differential metabolites were screened compared with the blank group,among which only one product,Tyramine,was up-regulated,while among the 18 products with down-regulated expression,Inositol,L-Valine,Pyrrole-2-carboxylic acid and Lisoleucine were the most obvious.Among the metabolites,Inositol(C00137),Pyrrole-2-carboxylic acid(C05942)and L-isoleucine(C00407)could be used as potential biomarkers in the PA biofilm.23 differential metabolites were obtained in the CA group compared with the blank group,and 16 of them were identified for metabolic pathway analysis.19 differential metabolites were obtained in the CP5 group compared with the blank group,and 14 of them were identified for metabolic pathway analysis.ABC transporter had the greatest effect on the biofilm metabolic pathway of drug-resistant PA1907 strain.Conclusion:1.The biofilm of drug-resistant PA1907 strain is stable and not easily eluted,and no other impurities interfere with the staining process;CA has the effect of inhibiting and eliminating the biofilm activity of drug-resistant PA1907 strain at a sub-inhibitory concentration of 1 mg/m L.2.CP5 sub-inhibitory concentration of 0.1 mg/m L showed significant inhibition and elimination of biofilm growth of drug-resistant PA1907 bacteria,and its active concentration was 1/10 of chlorogenic acid,indicating that the introduction of nitro substituents on the benzene ring in the CA structure could improve the anti-biofilm activity.3.The results of transcriptomic analysis showed that both CA and CP5 compounds were associated with biofilm formation pathway in the pathway analysis,verifying their pharmacological effects on biofilm growth inhibition.CA could inhibit biofilm formation by regulating the down-regulation of las and rhl gene expression in the QS system of biofilm formation pathway.The derivative CP5,on the other hand,regulated las,rhl and pqs family genes to inhibit biofilm formation.Meanwhile,both CP5 and CA can regulate the down-regulation of psl gene expression and block the extracellular polysaccharide synthesis pathway to inhibit biofilm synthesis.4.Establish a complete metabolite analysis method using GC-MS,and quality control the assay data through methodological validation.54 common metabolites were analyzed by data statistics and metabolic pathways,and the expression of 3 metabolites:Inositol,L-Valine,Pyrrole-2-carboxylic acid and L-isoleucine,was significantly downregulated.The expression of three metabolites,Inositol,L-Valine,Pyrrole-2-carboxylic acid and L-isoleucine,was significantly down-regulated and could be used as potential biomarkers.The enrichment of the two differential metabolite pathways revealed that ABC transporter had the greatest effect on the biofilm metabolic pathway of the drugresistant PA1907 strain.Since ABC transporter are involved in bacterial extracellular polysaccharide biosynthesis,the upstream transcriptomic analysis showed that CA and CP5 could inhibit biofilm growth by regulating ABC transporter and affecting extracellular polysaccharide production in drug-resistant PA1907 strain.In conclusion,CP5 had a stronger effect than CA on biofilm growth inhibition and mature tegument elimination of drug-resistant PA1907 bacteria,and CP5 was involved in regulating more gene expression and pathways and had better growth inhibitory activity than chlorogenic acid.At the same time,through the combined analysis of transcriptomics and metabolomics,CA and CP5 could affect the extracellular polysaccharide production of drug-resistant PA1907 strain by regulating ABC transporter,thus inhibiting biofilm growth.