Study On The Mechanism Of Quinic Acid Against Drug-Resistant Pseudomonas Aeruginosa Biofilm

Pseudomonas aeruginosa(PA)is a Gram-negative bacterium that is peculiarly responsible for ventilator-associated pneumonia,cystic fibrosis,urinary tract infections and systemic infections in hospitals.PA is a common pathogenic pathogen in clinical reasearch.On account of its complicated drug resistance mechanism,it is more likely for PA to form biofilm and secrete a variety of virulence factors,which makes it difficult for traditional antibiotics to develop an ideal curative effect,causing a primary challenge in clinical treatment of infection.Traditional Chinese medicine and natural products have attracted to an increasing number of attentions in the field of anti-infection.Chlorogenic acid is the main antibacterial active ingredient of a variety of Heat-clearing and Toxin-resolving Chinese medical herbs.It is particularly instable after entrancing the organism,which will turn into various metabolites.The application foreground of its metabolites in anti-bacterial infection is wide enough,but the antibacterial activity of the metabolites has not been explored in depth.In this paper,quinic acid with preferable effect of combating biofilm of PA was screened out from the main metabolites of chlorogenic acid.The effects of quinic acid on restraining the formation of clinical drug-resistant PA biofilm and eliminating mature biofilm,virulence factors and bacterial motility were studied.What is more,RNA-Seq technology,fluorescence quantitative PCR,molecular docking and other analysis methods were applied to explore the target and mechanism of quinic acid on drug resistance PA.These results will provide theoretical basis and reference for quinic acid and chlorogenic acid related metabolites prove to be potential antiPA biofilm drugs and treatment of related infectious diseases.This paper is divided into the following three parts:Part Ⅰ: The effect of quinic acid on drug resistant PA in vitro.The minimum inhibitory concentration(MIC)of chlorogenic acid and its main 12 metabolites on PA1905 was determined,and the anti-PA biofilm test was also performed.The MIC50 value of quinic acid against drug-resistant PA1905 was proved to be 8 mg/m L by microbroth dilution method,which exhibited that quinic acid possessed the inhibitory effect of resisting clinical drug-resistant bacteria.According to crystal violet staining experienment,quinic acid reduced the biofilm formation of PA and eliminated mature biofilm when the sub-inhibitory concentration(subMIC)was 1/2MIC-1/32 MIC.Quinic acid with strong anti-PA1905 biofilm activity was finally selected for subsequent experiments.The morphology and structure of PA could be clearly observed by scanning electron microscope.The bacteria in the control group were surrounded by a large number of reticular biofilms,while the biofilm attachment around the bacteria in the administration group was reduced.The inhibitory effect of sub-MIC quinic acid on the biofilm formation of PA was enhanced with the increase of drug mass concentration.Combined quinic acid with clarithromycin,azithromycin and levofloxacin for anti-biofilm experiments,sub-MIC quinic acid,clarithromycin,azithromycin and levofloxacin all revealed obvious inhibitory effects on the formation of PA1905 biofilm,and quinic acid and levofloxacin showed synergic anti-BF effect under partial sub-MIC conditions.However,the synergistic effect of macrolides and quinic acid was limited.Rearching the effect of sub-MIC quinic acid on the secretion of virulence factors such as PA1905 pyocyanin and elastase,the result revealed that 1MIC-1/4MIC quinic acid could inhibit the secretion of PA1905 pyocyanin and elastase,and showed a doseeffect relationship.Part Ⅱ: The effect of sub-MIC quinic acid on the exercise capacity of drug resistant PA.The inhibitory ability of quinic acid on the swimming and twitching motion of PA1905 was analyzed at different sub-MIC concentrations.When quinic acid was 4 and 2 mg/m L(equivalent to 1/2MIC,1/4MIC),the swimming motion ability of PA1905 was weakened,and the measured colony growth diameter decreased significantly(P<0.001),showing significant inhibitory effect on PA1905 swimming motion.When the sub-MIC of quinic acid was 4 mg/m L(equivalent to 1/2MIC),the twitching motion of PA1905 was also notably decreased(P<0.001);When the sub-MIC was 2,1 mg/m L(equivalent to 1/4MIC,1/8MIC),the twitching motion of PA1905 was attenuated(P<0.01,P<0.05),which indicated that quinic acid could significantly inhibited the twitching motion of PA1905.Part Ⅱ: Study on the mechanism of sub-MIC quinic acid on clinical drug resistant PA.The transcriptomic study of sub-MIC quinic acid treated PA1905 was conducted by RNA sequencing.Compared with the control group,a total of 590 genes were significantly changed after quinic acid treatment of PA1905,among which 255 genes were notably down-regulated and 335 genes were notably up-regulated.GO bioenrichment and KEGG pathway analysis were performed,and the changed genes were mainly enriched in flagellar assembly,bacterial chemotaxis,oxidative phosphorylation,ribosome,motion-related pathway function,cyanoamino acid metabolism related pathways,biofilm formation of PA and quorum sensing.Fluorescence quantitative PCR revealed the changes of genes related to the QS system.Compared with the bacterial control group,sub-MIC quinic acid down-regulated the expressions of Rhl R,Las A,Las R,Rhl I and Las I genes in PA1905,which was consistent with the transcriptomic results.By molecular docking method,it was found that there were a large number of aromatic amino acid residues around quinic acid six-membered ring structure.Quinic acid formed complexes with the key proteins of quora-sensing pathway in PA(Rhl R,Rhl A and Rhl B)through hydrogen bonding and hydrophobic interaction,respectively,and competed with their endogenous ligands,therefore interfering with the quorum sensing system of PA.In conclusion,in this study,the anti-PA1905 biofilm activity of chlorogenic acid and its12 main metabolites was compared,and quinic acid with strong anti-PA biofilm activity was selected as the research object.The inhibitory effect of sub-MIC quinic acid on PA1905 biofilm was observed by scanning electron microscope.Quinic acid inhibited the secretion of PA1905 pyocyanin and elastase when the sub-MIC was 1MIC-1/4MIC.Effect of quinic acid on PA1905 swimming and twiching motion.Through transcriptomic analysis of the effect of sub-MIC quinic acid on PA1905,the flagellar assembly,bacterial chemotaxis,oxidative phosphorylation,ribosome,motion-related pathway function,amino acid metabolism(including tryptophan,alanine,aspartic acid,glutamic acid,etc.)related pathways,biofilm formation – Pseudomonas aeruginosa and quorum sensing related functions were inhibited.Quantitative PCR analysis exhibited that quinic acid down-regulated the expression of quorum sensing related genes in Las A,Las R,Las I,Rhl I,Rhl R.By molecular docking method,it was found that there were a large number of aromatic amino acid residues around the six-membered ring structure of quinic acid,and quinic acid could form multiple hydrogen bonds with aromatic amino acid residues on Rhl R,Rhl A and Rhl B structures.These groups limited the molecular mobility of quinic acid to a certain extent,enhanced the binding affinity between quinic acid and the three proteins,therefore further restricted the structure and function of the target proteins of the Rhl system.The expression levels of Flg B and Pil G that related to the second messenger cyclic diguanylic acid,were also analyzed.These results prove that these targets,pathways and biological processes are likely to be the mechanism of action of quinic acid against PA1905 biofilm,provideing a reference for quinic acid to become a potential anti-infective drug.