Inhibitory Effect Of Different Antibiotics On Helicobacter Hepaticus And Preventive Effect Of Probiotics On Helicobacter Hepaticus Reinfection Post-erid Icate Therapy

Objective: To analyze the inhibitory effect of different antibiotics on H.hepaticus in vitro and the curative effect of some sensitive antibiotics togetheron H.hepaticus to further screen clinical sensitive antibiotics for eradicating H.hepaticus and to provide the experimental basis for eradication therapy inexperimental animals infected with H.hepaticus.Methods: H.hepaticus standard strain ATCC51450and separated strainswere screened by using E-test method and Kirby-Bauer paper AGAR diffusionmethod to test minimum inhibitory concentration(MIC) and bacteriostaticannulus diameters to discuss the inhibitory effect of different antibiotics on H.hepaticus in vitro. Then by reoccupy board dilution, to calculate fractionalinhibitory concentration (FIC) when sensitive antibiotics on H.hepaticuscombined, further to screen combination program with apparently synergistisand cumulative effect.Results: E-test method and Kirby-Bauer paper AGAR diffusion methodresult showed H.hepaticus standard strains ATCC51450and separate strainswere also sensitive to levofloxacin, moxifloxacin, amoxicillin, levofloxacinclarithromycin, tinidazole, metronidazole and tetracycline, in whichlevofloxacin and moxifloxacin had most inhibitory effect on H. hepaticus. Andstrains were not inhibited by cephalosporin, sugar peptide, chloramphenicol,and gentamicin antibiotics. Reoccupy board result showed two differentantibiotics appeared synergy or accumulative effect,FIC was≤0.5or0.5～1, inwhich ofloxacin combined amoxicillin/metronidazole had most inhibitoryeffect on H.hepaticus, followed by moxifloxacin combined tinidazole whichboth had synergistic inhibitory effect.The other two-antibiotic-combinated programs also showed different degrees of cumulative suppression bacteriaeffect.Conclusion:1. Amoxicillin of the Penicillins, clarithromycin of the macrolides,metronidazole and tinidazole of the nitroimidazoles, ofloxacin andmoxifloxacin of the fluoroquinolones, tetracycline could significant inhibit H.hepaticus in vitro, in which levofloxacin and moxifloxacin had most inhibitoryeffect on H.hepaticus. And strains were not inhibited by cephalosporin,sugarpeptide, chloramphenicol, and gentamicin antibiotics.2. Two kinds of antibiotics each of which appeared obviously inhibitoryeffect to H.hepaticus appeared synergistic and cumulative inhibitory effect, inwhich levofloxacin combined amoxicillin/metronidazole had most inhibitoryeffect on H.hepaticus, followed by moxifloxacin combined tinidazole hadsynergistic inhibitory effect; The other two-antibiotic-combinated progromsalso showed different degrees of cumulative inhibitory effect. Objective: H.hepaticus and H.pylori both belong to Helicobacter species,our group were on the basis that Lactobacillus acidophilus was screenedsignificant inhibitory effects on H.pylori to further observe the inhibitory effectof Lactobacillus acidophilus on H.hepaticus standard strain ATCC51450andseparated strain, to lay the foundation for further prevention and treatment ofH.hepaticus infection with eco bacteria.Methods: First, to observe inhibitory effect of L4and L6culturesupernatant of Lactobacillus acidophilus which has significant inhibitoryeffects on H.pylori on H.hepaticus standard strain and separated strain by solidtraining punch method. Followed co-culture with Lactobacillus acidophilus andH.hepaticus in the liquid culture. After6,12,24,36,48h, the inhibitory effect ofanti-HP Lactobacillus acidophilus on H.hepaticus growth and urease activityassay were observed.Results: The pre-screened Lactobacillus acidophilus L4and L6culturesupernatant which has significant inhibitory effects on H.pylori had strongestinhibitory effect on H.hepaticus standard strain and separated strain, theinhibition zone higher than that of lactic acid control group and Brucella brothgroup(P<0.01),still had a significant inhibitory effect after the correction pHvalue. In liquid medium, under the condition of the L4strain of Lactobacillusacidophilus and H.hepaticus standarded strain ATCC51450and separatedstrains co-cultured,the counts of H.hepaticus colony and urease activity wereboth lower than Lactobacillus acidophilus standard control group and simpleH.hepaticus culture group(P<0.05), and the colony counts and the ureaseactivity gradually decreased with prolonging of culture time. There was nodifference in standard strain and separated group. Conclusions:1. The pre-screened two Lactobacillus acidophilus stains whose culturesupernatant had significant inhibitory effects on H.pylori had a similarinhibitory effect on H.hepaticus standard strain and separated strain in the solidculture conditions, and the inhibitory effect was not related with the secretionof lactic acid.2. The pre-screened two Lactobacillus acidophilus stains culturesupernatant whose culture supernatant had significant inhibitory effect onH.pylori had inhibitory effect on the growth and urease activity of H.hepaticusstandard strain and separated strain in the liquid culture conditions. Objective: Observe the inhibitory effect of single and combinedantibiotics on H.hepaticus in vitro. And based on apparent inhibitory effect ofLactobacillus acidophilus on H.hepaticus, further, the eradicated rates ofBALB/cCr mice infected with H.hepaticus using different single or combinedantibiotics were observed and the preventive effect of Lactobacillus acidophiluson H. hepaticus recurrence in BALB/c Cr mice to supply information to furtherclinical treatment.Methods: After feeding H.hepaticus8weeks,135SPF male BALB c Crmice, H. hepaticus was isolated and cultured from mice feces to get infectedsituation of H. hepaticus; randomly8mice whose H.hepaticus was positivewere executed and caecum, respectively, jejunum, ileum colon, and liverspecimens were cultured micro viable aerobics, and specimens were evaluatedhistologic situation and H.hepaticus specific16SrRNA gene was amplificated.Then110mice infected H.hepaticus were divided into amoxicillinmonotherapy group, metronidazole monotherapy group, clarithromycinmonotherapy group, levofloxacin monotherapy group, tetracycline mono-therapy group, amoxicillin+metronidazole+bismuth combination therapygroup, amoxicillin+clarithromycin+bismuth combination therapy group,amoxicillin+levofloxacin+bismuth combination therapy group, tetracycline+metronidazole+bismuth combination therapy group, bismuth monotherapycontrol group and physiological saline control group,10mice in each group for2weeks. After finishing antibiotics treatment4and8weeks, the eradicatingrate was observed using fecus H. hepaticus culture, and4mice in each groupwere executed, histology was tested and H.hepaticus specific16SrRNA genewas amplificated to evaluate the influence of eradicating H.hepaticus on mice bowel and liver tissue H.h epaticus engraftment and histopathological damage;at the end,16mice eradicating H.hepaticus successfully were divided into2groups and co-raised with mice eeradicating H.hepaticus unsuccessfully, themice were feed with Lactobacillus acidophilus and physiological saline. After1,2,3months, H.hepaticus were cultured from fecus to the preventive effect ofLactobacillus acidophilus on reinfected H.hepaticus in BALB/cCr mice whichhad eradicated H. hepaticus.Results: At week8, fecal H.hepaticus culture positivity rate of SPF maleBALB/c Cr mice fed H.hepaticus was high to88.9%; in the mice of stoolculture positive,H.hepaticus could be detected colonization in cecal tissues andin ileum, colon and liver tissues, H.hepaticus strains were also be detected andobvious inflammatory was found in cecum, colon and liver tissue. TreatedH.hepaticus mice with different antibiotic programs, the results showed thattreatment with a mono-antibiotic H.hepaticus eradication rate was10~20%,and in the two-antibiotics combined with bismuth groups, H.hepaticuseradication rates were high up to90~100%, significantly higher thanmonotherapy group, bismuth monotherapy group and control group (P <0.05);Co-raise the mice successfully eradicated H.hepaticus and ones unsuccessfullyeradicated H.hepaticus,the results showed the reinfected with H.Hepaticusafter1,2,3months were0%,18.75%and31.25%, and in saline control groupH.hepaticus reinfection rates were50%,68.75%and81.25%, P <0.05.Conclusion:1. By feeding BALB/c Cr mice with H.hepaticus for8weeks,H.hepaticus infection model ould be successfully established. H.hepaticus majorcolonizated in murine cecal and colonic mucosa, and in liver and intestinaltissue H.hepaticus colonization can also be found, which can induce mousecaecum, colon and liver pathological injury.2. Monoantibiotic therapy can not totally eradicate H.hepaticus, and two kinds of antibiotics combined with bismuth program get higher eradicationrates. Eradication of H.hepaticus could significantly reduce the detection rate ofH.hepaticus in the intestinal mucosa, and can make the colonic mucosalhistopathologic scores decrease significantly,reduce the liver tissue H.hepaticusspecific16SrRNA gene detection rates, and the histopathology of liver areimproved obviously.3. After BALB/c Cr mice eradicated H.hepaticus,Lactobacillus acidophilusshowed obvious inhibitory effect on H.hepaticus in vitro and could effectivelyprevent the re-infection of H.hepaticus.