Study On Mechanisms Of Persister Formation Of Helicobacter Pylori And The Bacteriostasis Of The Compounds Of Kronopolites Svenhedini And Chinese Traditional Medicine Against H.pylori

Object: 1.In this paper,the formation persister laws and mechanisms of Helicobacter pylori will be measured.It could provide a theoretical base of the development of new drugs to treat chronic and recurrent infections cause by H.pylori.2.To study the effect of the compounds of Kronopolites svenhedini with Hedysarum polybotrys,Rhodiola rosea,Rhizoma coptidis,Portulaca olerace,and Taraxacum mongolicum respectively against H.pylori.Methods: 1.The growth curve of H.pylori was constructed by the estabilished liquid model.The minimal inhibitory concentration（MIC）and minimal bactericidal concentration（MBC）of amoxicillin,clarithromycin,metronidazole and levofloxacin against H.pylori were detected by serial dilution method.The antibiotic of amoxicillin（2.50 μg·m L-1 and 0.63 μg·m L-1）,clarithromycin（0.16 μg·m L-1）,metronidazole（20.00 μg·m L-1 and 4.00 μg·m L-1）and levofloxacin（5.00 μg·m L-1）exposure experiments were used to select and measure the persister formation of H.pylori at the cultural time of 4 h,14 h,40 h,72 h.2.The total RNA of H.pylori cultured at 14 h and 40 h time points were extracted.The expression different level of m RNA were mearsured by transcriptome sequencing and the results were validated and analyzed by q RCR and bioinformatics.3.The effective components of the compounds of Kronopolites svenhedini with Hedysarum polybotrys,Rhodiola rosea,Rhizoma coptidis,Portulaca olerace,and Taraxacum mongolicum respectively,and single Kronopolites svenhedini,Rhodiola rosea,Rhizoma coptidis were extracted by water and ethanol.The effects of the extracted components on the inhibitory of against H.pylori were screened by the agar diffusion method.The MIC of single Kronopolites svenhedini,Rhizoma coptidis,and Rhodiola rosea and the compounds against H.pylori were determined by dilution method,and the synergistic effect after compatibility between drugs were compared.Results: 1.According to the growth curve of H.pylori,0-4 h during the culture is the lag phase;5-30 h is the logarithmic period.After 30 h,the cultures enter stationary phase.2.The MIC and the MBC ranges of amoxicillin against H.pylori were 0.06-0.13 μg·m L-1 and 0.13-0.25 μg·m L-1 respectively.The MIC and the MBC ranges of clarithromycin against H.pylori were 0.01-0.02 μg·m L-1 and 0.02-0.03 μg·m L-1 respectively.The MIC and the MBC ranges of metronidazole against H.pylori were 1.00-2.00 μg·m L-1 and 4.00-8.00 μg·m L-1 respectively.The MIC and the MBC ranges of levofloxacin against H.pylori were 0.25-0.50 μg·m L-1 and 1.00-2.00 μg·m L-1 respectively.3.H.pylori could form persisters when they were exposured with lethal concentrations.The quantities of persister were less at the lag phase and logarithmic phase while they were more at stationary phase.This phenomenon was independent with drug concentration.Metronidazole had a certain bactericidal effect on H.pylori persister.The changes of viable H.pylori were characterized as decreased trends at the first stage and increased trends at the second stage while they were exposured with lethal concentration of clarithromycin and levofloxacin.4.Compared with culture of H.pylori at 40 h to 14 h,there are 39 genes down-regulated and 17 genes up-regulated.The differences genes in transcription levels mainly focus on ribosome（include rpm C,rpm F,rpm J,rpm I,rpl R,rpl T,rpl O,rpl Q,rpl Y,rpl X,rpl E,rpl J,rpl L,rpl F,rps J,rps K,rps F,rps E,rps H,rps R,rps N,rps M,rps B,rps U）and metabolism（include tfs,rpo A,map,nuo A,tpx,gdh A,ure A,jhp₀586,jhp₀585）pathway.5.The compounds of Kronopolites svenhedini with Hedysarum polybotrys,Rhodiola rosea,Rhizoma coptidis,Portulaca olerace,Taraxacum mongolicum respectively all exhibited bacteriostasis against H.pylori and the most powerful compound among them is Kronopolites svenhedini accompanied with Rhizoma coptidis.The MIC ranges of Kronopolites svenhedini,Rhizoma coptidis,Rhodiola rosea,Kronopolites svenhedini-rhizoma coptidis,Kronopolites svenhedini-rhodiola rosea is 20-40,0.63-1.25,1.25-2.50,10-20 mg·m L-1 respectively.The MIC against H.pylori of Rhizoma coptidis and Rhodiola rosea were half lower than the single herbs when they were combined with Kronopolites svenhedini.Conclusions: 1.H.pylori can form persister against amoxicillin under cultured condition.The quantities of persister were less at the lag phase and logarithmic phase while they were more at stationary phase. Themechanisms of H.pylori persister formation may be associated with ribosome and metabolism pathway.2.Compounds of Kronopolites svenhedini with Hedysarum polybotrys,Rhodiola rosea,Rhizoma coptidis,Portulaca oleracea,Taraxacum mongolicum respectively all have bacteriostasis against H.pylori.Kronopolites svenhedini companied with Rhizoma coptidis or Rhodiola rosea can produce synergy when they are used to eliminate H.pylori.