Effects Of Tea Polyphenols On Virulence And Drug Resistance Of Klebsiella Pneumoniae Based On Quorum Sensing

Chapter 1 IntroductionKlebsiella pneumoniae is a common clinical pathogen that can cause severe infection.How to inhibit its high toxicity and high drug resistance is a hot and difficult point in clinical research.Based on the theory that quorum sensing system is an important regulatory mechanism of extracellular enzymes and toxins,biofilm formation and drug resistance of pathogenic bacteria.We try to provide a new way to solve the problem of global resistance against bacteria in severe form by using quenching bacterial quorum sensing system,which can reverse the sensitivity of antibiotic resistance and weaken the virulence of bacteria.It also provides experimental basis and theoretical basis for the combined use of quorum sensing inhibitors and traditional antimicrobial agents and the development of new drugs.Chapter 2 Effect of tea polyphenols on bacteriostasis and Drug Resistance of klebsiella pneumoniaeObjective: To prove that tea polyphenols can inhibit Klebsiella pneumoniae and reverse multidrug resistance of Klebsiella pneumoniae at sub-inhibitory concentration.Methods: The minimum inhibitory concentration of tea polyphenol on Klebsiella pneumoniae was detected by microplate bioassay.collected 44 strains of multidrug-resistant klebsiella pneumoniae isolated in the bacterial laboratory of a third class general hospital from January 2017 to december 2018 as the study strain.the strain homology analysis was carried out by pulse field gel electrophoresis to find out the different strains of drug-resistant species and to detect the bacterial ST typing,virulence and resistance genes.The ability of tea polyphenols to reverse multidrug resistance of Klebsiella pneumoniae was observed.Methods: The minimum inhibitory concentration of tea polyphenol on Klebsiella pneumoniae was detected by microplate bioassay.collected 44 strains of multidrug-resistant klebsiella pneumoniae isolated in the bacterial laboratory of a third class general hospital from january 2017 to december 2018 as the study strain.the strain homology analysis was carried out by pulse field gel electrophoresis to find out the different strains of drug-resistant species and to detect the bacterial ST typing,virulence and resistance genes.By observing the change of antibiotic K-B value after clinical use of tea polyphenols and other original drug-resistant antibiotics,the ability of tea polyphenols to reverse multidrug resistance of Klebsiella pneumoniae was observed at subantimicrobial concentration.Results: The inhibitory rate of tea polyphenols on Klebsiella pneumoniae increased with the increase of concentration,and when the concentration of tea polyphenols was 2048 ug/ml,the tested bacteria were completely inhibited,so the mic value was between 512-2048 ug/ml.The results showed that most of the strains were resistant to many kinds of antibiotics.In 512ug/ml,tea polyphenols were combined with other antibiotics.The results were determined by increasing the k-b value of antibiotics.Among them,imipenem,piperacillin/tazobactam and cefepime had 100% synergistic effect,cefotaxime had 85% synergistic effect and cefotaxime had 75% synergistic effect.Conclusion: Tea polyphenols combined with commonly used antibiotics in vitro can effectively reverse the antibiotic sensitivity of multidrug resistant Klebsiella pneumoniae and can be used in the adjuvant synergistic treatment of multidrug resistant Klebsiella pneumoniae.Chapter 3 Effects of tea polyphenols on virulence of Klebsiella pneumoniae populationObjective: To observe the inhibitory effect of tea polyphenols on the main virulence factors of Klebsiella pneumoniae.Methods: The effect of tea polyphenols on the mucoid of Klebsiella pneumoniae was observed by Congo red staining.The effect of tea polyphenols on the ability of producing polysaccharide from the main virulence factor of Klebsiella pneumoniae was observed.By observing the change of the size of clear circle produced by Klebsiella pneumoniae k7 strain after tea polyphenols in nutrient broth medium,the ability of inhibiting the secretion of bacterial extracellular protease was observed.By observing the ability of tea polyphenols to treat k4 strains of Klebsiella pneumoniae infected with Caenorhabditis elegans.Results: The colony on the plate without tea polyphenol was black,which indicated that the strain had liquid-like secretion.However,the strains with tea polyphenols showed light pink color,which indicated that tea polyphenols could obviously inhibit the secretion of dip liquid.tea polyphenols can significantly reduce the content of capsule polysaccharides,but 200μg/ml and 400μg/ml tea polyphenols have no significant difference in the inhibition of the production capacity of klebsiella pneumoniae capsule polysaccharides,indicating that the inhibition of tea polyphenols on the production capacity of klebsiella pneumoniae capsule polysaccharides is not concentration-dependent.Tea polyphenols significantly inhibited the protease activity of Klebsiella pneumoniae k7 strain,and the area of transparent circle was significantly reduced,but there was no significant difference inhibition between 200 and 400μg/ml,suggesting that tea polyphenols inhibited the secretion of protease or protease activity of Klebsiella pneumoniae k7 strain.C.elegans infection experiment,in the presence of tea polyphenols,feeding Klebsiella pneumoniae k4 strain of C.elegans survival significantly increased.tea polyphenols at concentrations200-400μg/ml did not affect klebsiella pneumoniae growth.OP50 and 400μg/ml tea polyphenols-fed nematodes had no difference in survival rate from nematodes fed only OP50,which could indicate that tea polyphenols had no adverse effect on the growth of nematodes.Conclusion: Tea polyphenols can significantly inhibit the production of Klebsiella pneumoniae related virulence factors,and its inhibitory ability is not concentration dependent.The survival rate of Klebsiella pneumoniae infected with Caenorhabditis elegans was significantly improved by using tea polyphenols.Chapter 4 Study on the Mechanism of Tea Polyphenols on Klebsiella pneumoniaeObjective: To investigate the effect of polyphenols on the expression of Klebsiella pneumoniae quorum sensing signal molecules luxs genes,and the ability to regulate the expression rmp A2、iuc A major virulence genes and biofilm formation.Methods: The total rna of Klebsiella pneumoniae treated with different concentrations of tea polyphenols were extracted,and the luxs 、 rmpa2 、 iuca gene expression was detected by fluorescence quantitative pcr after reverse transcription,and the effect of tea polyphenols on bacterial kpc resistance genes was detected.The biological membrane formation ability of Klebsiella pneumoniae treated with different concentrations of tea polyphenols was detected by crystal wood violet staining.Results: Tea polyphenols could inhibit the expression of quorum sensing regulatory factor luxs of Klebsiella pneumoniae,but there was no statistical difference between 200 ug/ml and 400 ug/ml tea polyphenols.Tea polyphenols could also inhibit the expression of key virulence gene iuca and mucus phenotype regulator rmpa2 of Klebsiella pneumoniae iron carrier.tea polyphenols did not affect the resistance genes kpc klebsiella pneumoniae.Tea polyphenols can significantly inhibit the production of non-concentration-dependent Klebsiella pneumoniae biofilm at sub-inhibitory concentration.Conclusion: Tea polyphenols can inhibit Klebsiella pneumoniae quorum sensing system and inhibit the expression of key virulence genes and biofilm formation.