不同抗生素对肝螺杆菌的抑制效应及生态菌对根除肝螺杆菌后再感染的预防作用
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摘要
目的：探讨不同抗生素对H.hepaticus的体外抑制效应，并分析单药敏感的抗生素联合应用时对H.hepaticus的抑制效应，筛选对H.hepaticus敏感的抗生素治疗方案，为进一步在H.hepaticus感染实验动物体内进行根除治疗提供实验依据。
方法：分别采用E-test法及Kirby-Bauer纸片琼脂扩散法，观察不同抗生素对H.hepaticus标准菌株ATCC51450及分离株的最低抑菌浓度（MIC）及抑菌环直径，探讨不同抗生素对H.hepaticus的体外抑制效应，并通过棋盘格稀释法观察对H.hepaticus敏感抗生素联合应用时的联合抑菌分数(FIC)，筛选具有明显协同或累加作用的组合方案。
结果： E-test及Kirby-Bauer纸片琼脂扩散法均证实左氧氟沙星、莫西沙星、阿莫西林、氧氟沙星、克拉霉素、替硝唑、甲硝唑和四环素对H.hepaticus标准菌株ATCC51450和分离株均表现出明显的抑制效应，其中左氧氟沙星和莫西沙星的抑菌效应最强；而头孢类、糖肽类、氯霉素及庆大霉素对H.hepaticus未显示出明显的抑菌效应。随后应用“棋盘格法”分别对H.hepaticus敏感的抗生素间的联合药敏实验进行观察，结果表明，对H.hepaticus敏感的抗生素间的二联组合对H.hepaticus均具有协同或累加的抑菌效应，联合FIC指数均在≤0.5或0.5～1之间，其中左氧氟沙星联合阿莫西林或甲硝唑的的抑制效应最强，其次为莫西沙星联合替硝唑，均具有协同的抑菌效应；其他抗生素的二联组合也呈现出不同程度的累加抑菌效应。
结论：
1.青霉素类中的阿莫西林、大环内酯类中的克拉霉素、硝基咪唑类中的甲硝唑和替硝唑、氟喹诺酮类中的左氧氟沙星和莫西沙星以及四环素等药物在体外对H.hepaticus表现出明显的抑制作用，其中左氧氟沙星和莫西沙星的抑菌效应最强，但头孢类、糖肽类、氯霉素及庆大霉素对H.hepaticus并不敏感。
2.对H.hepaticus具有明显抑制作用的抗生素间的二联组合方案对H.hepaticus均表现出协同或累加的抑菌效应。其中左氧氟沙星联合阿莫西林或甲硝唑的抑菌效应最强，其次为莫西沙星联合替硝唑，表现出明显的协同抑菌效应；其他抗生素的二联组合也呈现出不同程度的累加抑菌效应。
目的：由于H.hepaticus与HP属同一螺杆菌属，本研究组在筛选到对HP具有明显抑制效应的嗜酸乳杆菌的基础上，进一步观察其在体外对H.hepaticus标准菌株ATCC51450及分离株的抑制效应，为进一步开展生态菌预防及治疗H.hepaticus感染奠定基础。
方法：首先采用固体培养打孔法观察本研究组预先筛选的对HP具有明显抑制效应的嗜酸乳杆菌(抗HP嗜酸乳杆菌) L4及L6的培养上清液对H.hepaticus标准菌株ATCC51450及分离株生长的抑制效应，随后采用液体培养法将具有抗HP作用的嗜酸乳杆菌与H.hepaticus共同培养，分别于共同培养6、12、24、36、48h通过倍比稀释菌落计数法及尿素酶活性测定法，观察抗HP嗜酸乳杆菌对H.hepaticus生长及尿素酶活性的抑制效应。
结果：本研究组预先筛选的两株对HP具有明显拮抗效应的嗜酸乳杆菌L4及L6菌株的上清液对H.hepaticus标准菌株ATCC51450及分离株的抑菌环直径明显高于乳酸对照组及布氏肉汤组（P <0.01），校正上清液pH值后仍具有明显的抑制效应；在液体培养条件下抗HP嗜酸乳杆菌菌株（L4）与H.hepaticus标准菌株ATCC51450及分离株共同培养后，H.hepaticus的菌落计数及尿素酶活性明显低于普通嗜酸乳杆菌对照组及单纯H.hepaticus培养组（P <0.05），且随着培养时间的延长其菌落计数及尿素酶活性逐渐下降。
结论：
1.本研究组筛选到的对HP具有明显抑制效应的2株嗜酸乳杆菌的上清液在固体培养条件下可明显抑制H.hepaticus标准菌株及临床分离株的生长，且对H.hepaticus的抑制效应与分泌乳酸无关。
2.本研究组筛选到的对HP具有明显抑制效应的嗜酸乳杆菌在液体培养条件下，与H.hepaticus标准菌株及临床分离株共同培养时，不仅可明显抑制H.hepaticus标准菌株及临床分离株的生长，而且还可以明显抑制其毒性因子尿素酶的活性。
目的：在体外观察不同抗生素单药及联合用药对H.hepaticus的抑制效应以及在体外观察对HP具有拮抗作用的嗜酸乳杆菌对H.hepaticus同样具有明显拮抗作用的基础上，进一步观察不同抗生素单药及联合铋剂方案对H.hepaticus感染BALB/c Cr小鼠的根除率、探讨不同方案于动物体内的根除疗效及其与病理组织学间的关系，并观察抗HP嗜酸乳杆菌对根除H.hepaticus后再感染的预防效应，最终为将来在临床上的应用提供研究依据。
方法：135只SPF级雄性BALB/c Cr小鼠灌饲H.hepaticus后8周时，通过粪便H.hepaticus分离培养，明确小鼠H.hepaticus感染状况；随机处死8只粪便H.hepaticus培养阳性小鼠，分别对盲肠、结肠、回肠、空肠及肝脏标本行H.hepaticus分离培养鉴定、病理组织学检查及H.hepaticus特异性16SrRNA基因扩增，观察H.hepaticus在不同组织中的定植状况及与病理组织学的关系；随后将确定为H.hepaticus感染的110只小鼠随机分为阿莫西林单药治疗组、甲硝唑单药治疗组、克拉霉素单药治疗组、左氧氟沙星单药治疗组、四环素单药治疗组、左氧氟沙星+甲硝唑+铋联合治疗组、阿莫西林+克拉霉素+铋联合治疗组、阿莫西林+左氧氟沙星+铋联合治疗组、四环素+甲硝唑+铋联合治疗组、单纯铋剂对照组以及生理盐水对照组，每组10只小鼠，疗程2周。于抗生素治疗结束后4或8周时，通过粪便分离培养观察不同根除方案对小鼠H.hepaticus的根除率；并于抗生素治疗结束后8、16周时，每组随机处死4只小鼠，对盲肠、结肠及肝脏进行病理组织学检查及H.hepaticus特异性16SrRNA基因扩增，明确根除H.hepaticus对小鼠肠道及肝脏组织H.hepaticus定植及病理组织损伤的影响；最后将16只成功根除H.hepaticus的小鼠随机分成2组，与未根除小鼠同笼饲养，分别每日灌饲嗜酸乳杆菌及生理盐水，分别于同笼饲养1、2、3个月时进行粪便H.hepaticus分离培养，明确嗜酸乳杆菌对根除H.hepaticus后再感染的预防效应。
结果：SPF级雄性BALB/c Cr小鼠灌饲H.hepaticus8周时粪便H.hepaticus培养的阳性率高达88.9%；粪便培养阳性小鼠盲肠组织中均可检测到H.hepaticus定植，结肠、回肠及肝脏组织中也可检测到H.hepaticus菌体，并且小鼠盲肠、结肠及肝脏组织可出现明显的炎症反应。对H.hepaticus感染小鼠进行不同抗生素方案治疗，结果表明单一抗生素治疗组H.hepaticus根除率仅为10～20%，而两种抗生素联合铋剂三联治疗组H.hepaticus根除率可高达90～100%，明显高于单药治疗组、单纯铋剂治疗对照组及未应用抗生素治疗对照组（p<0.05）；成功根除H.hepaticus的小鼠盲肠、结肠及肝脏的病理组织学评分明显低于未根除H.hepaticus小鼠及未应用抗生素治疗对照组小鼠，并明显减少肝脏组织H.hepaticus特异性16SrRNA的检出率。将成功根除H.hepaticus小鼠与未根除小鼠同笼饲养，灌饲本研究组已经筛选到的对HP具有明显拮抗作用的嗜酸乳杆菌组小鼠，同笼饲养1、2、3个月时H.hepaticus再感染的发生率分别为0%、18.75%和31.25%，而灌饲生理盐水对照组小鼠H.hepaticus再感染率分别为50%、68.75%和81.25%，两组比较差异显著，P＜0.05。
结论：
1. BALB/c Cr小鼠灌饲H.hepaticus8周后可成功建立H.hepaticus感染模型，H.hepaticus主要定植在小鼠盲肠及结肠粘膜组织，肝脏及回肠组织中也可发现H.hepaticus定植，并可诱发小鼠盲肠、结肠及肝脏组织的病理损伤。
2.单一抗生素治疗不能完全根除H.hepaticus，而两种抗生素联合铋剂方案有较高的根除率。根除H.hepaticus后可明显减少H.hepaticus在肠道粘膜的检出率，并可使结肠粘膜的病理组织学评分明显下降并减少肝组织H.hepaticus特异性16SrRNA基因的检出率，使肝脏病理组织学得到明显改善。
3. BALB/c Cr小鼠根除H.hepaticus后，灌饲于体外证实对H.hepaticus具有明显抑制效应的嗜酸乳杆菌，可有效预防根除H.hepaticus再感染的发生。
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.
方法：分别采用E-test法及Kirby-Bauer纸片琼脂扩散法，观察不同抗生素对H.hepaticus标准菌株ATCC51450及分离株的最低抑菌浓度（MIC）及抑菌环直径，探讨不同抗生素对H.hepaticus的体外抑制效应，并通过棋盘格稀释法观察对H.hepaticus敏感抗生素联合应用时的联合抑菌分数(FIC)，筛选具有明显协同或累加作用的组合方案。
结果： E-test及Kirby-Bauer纸片琼脂扩散法均证实左氧氟沙星、莫西沙星、阿莫西林、氧氟沙星、克拉霉素、替硝唑、甲硝唑和四环素对H.hepaticus标准菌株ATCC51450和分离株均表现出明显的抑制效应，其中左氧氟沙星和莫西沙星的抑菌效应最强；而头孢类、糖肽类、氯霉素及庆大霉素对H.hepaticus未显示出明显的抑菌效应。随后应用“棋盘格法”分别对H.hepaticus敏感的抗生素间的联合药敏实验进行观察，结果表明，对H.hepaticus敏感的抗生素间的二联组合对H.hepaticus均具有协同或累加的抑菌效应，联合FIC指数均在≤0.5或0.5～1之间，其中左氧氟沙星联合阿莫西林或甲硝唑的的抑制效应最强，其次为莫西沙星联合替硝唑，均具有协同的抑菌效应；其他抗生素的二联组合也呈现出不同程度的累加抑菌效应。
结论：
1.青霉素类中的阿莫西林、大环内酯类中的克拉霉素、硝基咪唑类中的甲硝唑和替硝唑、氟喹诺酮类中的左氧氟沙星和莫西沙星以及四环素等药物在体外对H.hepaticus表现出明显的抑制作用，其中左氧氟沙星和莫西沙星的抑菌效应最强，但头孢类、糖肽类、氯霉素及庆大霉素对H.hepaticus并不敏感。
2.对H.hepaticus具有明显抑制作用的抗生素间的二联组合方案对H.hepaticus均表现出协同或累加的抑菌效应。其中左氧氟沙星联合阿莫西林或甲硝唑的抑菌效应最强，其次为莫西沙星联合替硝唑，表现出明显的协同抑菌效应；其他抗生素的二联组合也呈现出不同程度的累加抑菌效应。
目的：由于H.hepaticus与HP属同一螺杆菌属，本研究组在筛选到对HP具有明显抑制效应的嗜酸乳杆菌的基础上，进一步观察其在体外对H.hepaticus标准菌株ATCC51450及分离株的抑制效应，为进一步开展生态菌预防及治疗H.hepaticus感染奠定基础。
方法：首先采用固体培养打孔法观察本研究组预先筛选的对HP具有明显抑制效应的嗜酸乳杆菌(抗HP嗜酸乳杆菌) L4及L6的培养上清液对H.hepaticus标准菌株ATCC51450及分离株生长的抑制效应，随后采用液体培养法将具有抗HP作用的嗜酸乳杆菌与H.hepaticus共同培养，分别于共同培养6、12、24、36、48h通过倍比稀释菌落计数法及尿素酶活性测定法，观察抗HP嗜酸乳杆菌对H.hepaticus生长及尿素酶活性的抑制效应。
结果：本研究组预先筛选的两株对HP具有明显拮抗效应的嗜酸乳杆菌L4及L6菌株的上清液对H.hepaticus标准菌株ATCC51450及分离株的抑菌环直径明显高于乳酸对照组及布氏肉汤组（P <0.01），校正上清液pH值后仍具有明显的抑制效应；在液体培养条件下抗HP嗜酸乳杆菌菌株（L4）与H.hepaticus标准菌株ATCC51450及分离株共同培养后，H.hepaticus的菌落计数及尿素酶活性明显低于普通嗜酸乳杆菌对照组及单纯H.hepaticus培养组（P <0.05），且随着培养时间的延长其菌落计数及尿素酶活性逐渐下降。
结论：
1.本研究组筛选到的对HP具有明显抑制效应的2株嗜酸乳杆菌的上清液在固体培养条件下可明显抑制H.hepaticus标准菌株及临床分离株的生长，且对H.hepaticus的抑制效应与分泌乳酸无关。
2.本研究组筛选到的对HP具有明显抑制效应的嗜酸乳杆菌在液体培养条件下，与H.hepaticus标准菌株及临床分离株共同培养时，不仅可明显抑制H.hepaticus标准菌株及临床分离株的生长，而且还可以明显抑制其毒性因子尿素酶的活性。
目的：在体外观察不同抗生素单药及联合用药对H.hepaticus的抑制效应以及在体外观察对HP具有拮抗作用的嗜酸乳杆菌对H.hepaticus同样具有明显拮抗作用的基础上，进一步观察不同抗生素单药及联合铋剂方案对H.hepaticus感染BALB/c Cr小鼠的根除率、探讨不同方案于动物体内的根除疗效及其与病理组织学间的关系，并观察抗HP嗜酸乳杆菌对根除H.hepaticus后再感染的预防效应，最终为将来在临床上的应用提供研究依据。
方法：135只SPF级雄性BALB/c Cr小鼠灌饲H.hepaticus后8周时，通过粪便H.hepaticus分离培养，明确小鼠H.hepaticus感染状况；随机处死8只粪便H.hepaticus培养阳性小鼠，分别对盲肠、结肠、回肠、空肠及肝脏标本行H.hepaticus分离培养鉴定、病理组织学检查及H.hepaticus特异性16SrRNA基因扩增，观察H.hepaticus在不同组织中的定植状况及与病理组织学的关系；随后将确定为H.hepaticus感染的110只小鼠随机分为阿莫西林单药治疗组、甲硝唑单药治疗组、克拉霉素单药治疗组、左氧氟沙星单药治疗组、四环素单药治疗组、左氧氟沙星+甲硝唑+铋联合治疗组、阿莫西林+克拉霉素+铋联合治疗组、阿莫西林+左氧氟沙星+铋联合治疗组、四环素+甲硝唑+铋联合治疗组、单纯铋剂对照组以及生理盐水对照组，每组10只小鼠，疗程2周。于抗生素治疗结束后4或8周时，通过粪便分离培养观察不同根除方案对小鼠H.hepaticus的根除率；并于抗生素治疗结束后8、16周时，每组随机处死4只小鼠，对盲肠、结肠及肝脏进行病理组织学检查及H.hepaticus特异性16SrRNA基因扩增，明确根除H.hepaticus对小鼠肠道及肝脏组织H.hepaticus定植及病理组织损伤的影响；最后将16只成功根除H.hepaticus的小鼠随机分成2组，与未根除小鼠同笼饲养，分别每日灌饲嗜酸乳杆菌及生理盐水，分别于同笼饲养1、2、3个月时进行粪便H.hepaticus分离培养，明确嗜酸乳杆菌对根除H.hepaticus后再感染的预防效应。
结果：SPF级雄性BALB/c Cr小鼠灌饲H.hepaticus8周时粪便H.hepaticus培养的阳性率高达88.9%；粪便培养阳性小鼠盲肠组织中均可检测到H.hepaticus定植，结肠、回肠及肝脏组织中也可检测到H.hepaticus菌体，并且小鼠盲肠、结肠及肝脏组织可出现明显的炎症反应。对H.hepaticus感染小鼠进行不同抗生素方案治疗，结果表明单一抗生素治疗组H.hepaticus根除率仅为10～20%，而两种抗生素联合铋剂三联治疗组H.hepaticus根除率可高达90～100%，明显高于单药治疗组、单纯铋剂治疗对照组及未应用抗生素治疗对照组（p<0.05）；成功根除H.hepaticus的小鼠盲肠、结肠及肝脏的病理组织学评分明显低于未根除H.hepaticus小鼠及未应用抗生素治疗对照组小鼠，并明显减少肝脏组织H.hepaticus特异性16SrRNA的检出率。将成功根除H.hepaticus小鼠与未根除小鼠同笼饲养，灌饲本研究组已经筛选到的对HP具有明显拮抗作用的嗜酸乳杆菌组小鼠，同笼饲养1、2、3个月时H.hepaticus再感染的发生率分别为0%、18.75%和31.25%，而灌饲生理盐水对照组小鼠H.hepaticus再感染率分别为50%、68.75%和81.25%，两组比较差异显著，P＜0.05。
结论：
1. BALB/c Cr小鼠灌饲H.hepaticus8周后可成功建立H.hepaticus感染模型，H.hepaticus主要定植在小鼠盲肠及结肠粘膜组织，肝脏及回肠组织中也可发现H.hepaticus定植，并可诱发小鼠盲肠、结肠及肝脏组织的病理损伤。
2.单一抗生素治疗不能完全根除H.hepaticus，而两种抗生素联合铋剂方案有较高的根除率。根除H.hepaticus后可明显减少H.hepaticus在肠道粘膜的检出率，并可使结肠粘膜的病理组织学评分明显下降并减少肝组织H.hepaticus特异性16SrRNA基因的检出率，使肝脏病理组织学得到明显改善。
3. BALB/c Cr小鼠根除H.hepaticus后，灌饲于体外证实对H.hepaticus具有明显抑制效应的嗜酸乳杆菌，可有效预防根除H.hepaticus再感染的发生。
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.
引文
[1] Vandenplas Y. Helicobacter pylori infection. World J. Gastroenterol,2000;6:20-31.
[2] Sakai T, Ognra Y, Narita J, et al. Simultaneous early adenocarcinoma andmucosa-associated lymphoid tissue（MALT）lymphoma of the stomachassociated with Helicobacter pylori infection. Gastric Cancer,2003;8:191-196.
[3]王江滨，亓文骞,李冰，秦少游，等幽门螺杆菌感染与胃肠外疾病.中华消化杂志,2011;5(31):358–360.
[4] Kowalski M. Helicobacter pylori(H. pylori)infection in coronary arterydisease：influence of H. pylori eradication on coronary artery lumen afterpercuteneous transluminal coronary angioplasty. The detection of H. pylorispecific DNA in human coronary atherosclerotic plaque. J PhysiolPharmacol,2001;52(Supple1):3-31.
[5] Suzuk T, Matsushima M, Masui A, et al. Effect of Helicobacter pylorieradication in patients with chronic：diopathic thrombocyto purpura-arandomized controlled trial. Am J Gastroenterrol,2005;100(6):1265-1270.
[6] Roussos A, Tsimpoukas F, Anastasakou E, et al. Helicobacter pyloriseroprevalence in patients with chronic bronchitis. J Gastroenterol,2002;37(5):332-335.
[7] Fox JG, Dewhirst FE, Tully JG, et al. Helicobacter hepaticus Sp. Now, amicroaerophilic bacterium isolated from lives and interstinal mucosalscrapings from mice[J]. Clinical Microbiology,1994,32(5):1238-1245.
[8] Ananieva O，Nilsson I，Vorobjova T，Uibo R，Wadstr m T. Immuneresponses to bile-tolerant helicobacter species in patients with chronic liverdiseases, a randomized population group, and healthy blood donors. ClinDiagn Lab Immunol.2002，9(6)：1160~1164.
[9]季尚玮，王颂，王江滨，等，我国不同品系实验室小鼠肝螺杆菌感染状况调查.中华消化杂志.2010，30（9）：597-601
[10]Blacky,et al.In vitro activity of fosfomycin alone and in combination withamoxicillin, clarithromycin and metronidazole against Helicobacter pyloricompared with combined clarithromycin and metronidazole. Eur J ClinMicrobiol Infect Dis (2005)24:276–279
[11]Yoko Yanagawa,Yoshimasa Yamamoto,et,al. A Combination Effect ofEpigallocatechin Gallate, a Major Compound of Green Tea Catechins,with Antibiotics on Helicobacter pylori Growth In Vitro. CurrentMicrobiology Vol.47(2003), pp.244–249
[12]Lin E, Stanek R J, Mufson M A. Lack of synergy of erythromycincombined with penicillin or cefotaxime againstStreptococcus pneumoniaein vitro[J].Antimicrob Agents Chemother,2003,47(3):1151
[13]Makoto Sasaki，Tsutomu Mizoshita, Takashi Mizushima, et,al. Effect ofplaunotol in combination with clarithromycin against clarithromycin-resistant Helicobacter pylori in vitro and in vivo. Journal of AntimicrobialChemotherapy (2007)60,1060–1063
[14]Rocha M，Avenaud P，Ménard A，Le Bail B，Balabaud C，Bioulac-SageP，de Magalh es Queiroz DM，Mégraud F. Association of Helicobacterspecies with hepatitis C cirrhosis with or without hepatocellular carcinoma.Gut．2005，54：396~401
[15]Nilsson I et al.Serum antibodies to Helicobacter hepaticus and Helicobacterpylori in patients with chronic liver disease. Gut,2000;46(3):410-414
[16]Ward JM, Fox JG, Anver MR, et a1. Chronic active hepatitis andassociated livertum orsinmice caused by a persistent bacterial itfection witha novel Helicobacter speces[J]. Natl CancerInst,1994,86(16):1222-1227
[17]Shames B, Fox JG, Dewhirst F, et al. Identification of widespreadHelicobacter hepaticus infection in feces in commercial mouse colonies byculture and PCR assay. J. Clin. Microbiol.,1995,33:2968–2972.
[18] Russell RJ,et al.Use of antibiotics to prevent hepatitis and typhlitis in malescid mice spontaneously infected with Helicobacter hepaticus.Lab AnimSci,1995,45(4):373-378
[19]Foltz CJ.Fox JG,Yan L,Shames B.Evaluation of antibiotic therapies foreradication of Helicobacter hepaticus. Antimicrob Agents Chemother1995;39:1292-4.
[20]Rogers AB. How gut microbes promote extraintestinal cancers. GutMicrobes2:1,52-57; January/February2011.
[1] Sakai T, Ognra Y, Narita J, et al. Simμltaneous early adenocarcinoma andmucosa-associated lymphoid tissue(MALT)lymphoma of the stomachassociated with Helicobacter pylori infection[J]. Gastric Cancer,2003,8:191-196.
[2] Herrera AG.Helicobacter pylori and food products:a public healthproblem[J].Methods Mol Biol,2004,268:297-301.
[3] Eidt S,Stolte M.The significance of Helicobacter pylori in relation to gastriccancer and lymphoma[J].Eur J Gastroenterol Hepatol,1995,7(4):318-321.
[4]Vandenplas Y. Helicobacter pylori infection. World J. Gastroenterol,2000,6:20-31.
[5] Parsonnet J.Bacterial infection as a cause of cancer[J].Environ Health Prespect,1995,103(l8):263-268.
[6] Forman D.Helicobacter pylori and gastric cancer[J].Scand J Gastr-oenterolSuppl,1996,215:48-51.
[7]王江滨，亓文骞,李冰，秦少游，等幽门螺杆菌感染与胃肠外疾病.中华消化杂志,2011;5(31):358–360.
[8] Kabir AM, Aiba Y, Takagi A, et al. Prevention of Helicobacter pyloriinfection by lactobacilli in a gnotobiotic murine model. Gut.1997;41(1):49-55.
[9] Lesbros-Pantoflickova D,Corthesy-Theulaz. Helicobacter pylori andProbiotics. J Nutr.2007;137(3):812S-818S.
[10] Lorca GL, Wadstrom T, Valdez GF, et al. Lactobacillusacidophilusautolysins inhibit Helicobacter pylori in vitro. Curr Microbiol.2001;42:39–44.
[11]王锦鸿，王江滨，李岩，等，抗HP嗜酸乳杆菌的筛选及其相关生物学特征的研究，中国免疫学杂志，2007；23，300-305.
[12] Fox JG, Dewhirst FE, Tully JG, Paster BJ, Yan L, Taylor NS, Collins MJ,Jr Gorelick PL, Ward JM. Helicobacter hepaticus sp. nov., amicroaerophilic bacterium isolated from livers and intestinal mucosalscrapings from mice. J Clin Microbiol1994,32(5):1238-45.
[13]季尚玮，王颂，王江滨，等，我国不同品系实验室小鼠肝螺杆菌感染状况调查.中华消化杂志.2010，30（9）：597-601
[14] Handa Y, Saitoh, Kavaguchi M, et al. Association of Helocobacter pyloriand diffuse type gastric cancer. J Gastroenterol.1996;31:29
[15] Kuipirs EJ, Uyterlinde SM, Pinz AS, et al. Long-term consequence ofHelicobacter pylori gastritis. Lancet.1995;345:1525-1528.
[16] Horstman M, Erttmann R, Winkler K, et al. Relapse of MALT lymphomaassociated with Helicobacter pylori after antibiotic treatment. Lancet.1994;343:1098
[17] Kumar D, Dhar A, Dattagupta S, et al. Pre and post eradicationgastricinflammation in Helicobacter pylori-associated duodenal ulcer.Indian J Gastroenterol.2002;21(1):7-10.
[18] Graham DY, Lew GM, Klein PD, et al. Effect of treatment of Helicobacterpylori infection on the long-term recurrence of gastric or duodenal ulcer.Ann Zntern Med.1992;116:705-708
[19] Konstantinov SR, Awati AA, Williams BA, Miller BG, Jones P, Stokes CR,Akkermans AD, Smidt H, de Vos WM. Post-natal development of theporcine microbiota composition and activities. Environ Microbiol2006,8(7):1191-9.
[20] Timmerman H M.Together strong: probiotics to promote health in peopleand animals [J]. Tijdschr Diergeneeskd,2006;131（19）:697-700.
[21] Midolo PD, Lambert JR, Hull R, Luo F, Grayson ML. In vitro inhibitionof Helicobacter pylori NCTC11637by organic acids and lactic acidbacteria. J Appl Bacteriol1995,79(4):475-9.
[22] Bernet MF, Brassart D, Neeser JR, Servin AL. Lactobacillus acidophilusLA1binds to cultured human intestinal cell lines and inhibits cellattachment and cell invasion by enterovirulent bacteria. Gut1994,35(4):483-9
[23] Coconnier MH, Lievin V, Hemery E, Servin AL. Antagonistic activityagainst Helicobacter infection in vitro and in vivo by the humanLactobacillus acidophilus strain LB. Appl Environ Microbiol1998,64(11):4573-80.
[24] Yeung PS, Sanders ME, Kitts CL, Cano R, Tong PS. Species-specificidentification of commercial probiotic strains. J Dairy Sci2002,85(5):1039-51.
[25] Fox JG, Yan L, Shames B, Campbell J, Murphy JC, Li X. Persistenthepatitis and enterocolitis in germfree mice infected with Helicobacterhepaticus. Infect Immun1996,64(9):3673-81.
[1] Sakai T, Ognra Y, Narita J, et al. Simμltaneous early adenocarcinoma andmucosa-associated lymphoid tissue(MALT)lymphoma of the stomachassociated with Helicobacter pylori infection[J]. Gastric Cancer,2003,8:191-196.
[2] Herrera AG.Helicobacter pylori and food products:a public healthproblem[J].Methods Mol Biol,2004,268:297-301.
[3] Eidt S,Stolte M.The significance of Helicobacter pylori in relation to gastriccancer and lymphoma[J].Eur J Gastroenterol Hepatol,1995,7(4):318-321.
[4]Vandenplas Y. Helicobacter pylori infection. World J. Gastroenterol,2000,6:20-31.
[5] Parsonnet J.Bacterial infection as a cause of cancer[J].Environ Health Prespect,1995,103(l8):263-268.
[6] Forman D.Helicobacter pylori and gastric cancer[J].Scand J Gastr-oenterolSuppl,1996,215:48-51.
[7]王锦鸿，王江滨，李岩，等，抗HP嗜酸乳杆菌的筛选及其相关生物学特征的研究，中国免疫学杂志，2007；23，300-305.
[8]Ward JM, Fox JG, Anver MR, et a1. Chronic active hepatitis and associatedlivertum orsinmice caused by a persistent bacterial infection with a novelHelicobacter speces[J]. Natl CancerInst,1994,86(16):1222-1227.
[9] LI XT,Fox JG,Mark T,et al. SCID/NCr Mice Naturally Infected withHelicobacter hepaticus Develop Progressive Hepatitis,ProliferativeTyphlitis,and Colitis. Infection and Immuity, Nov.1998,66:5477-5484.
[10]Matthew H. Myles, Robert S. Livingston, Beth A. Livingston, Jennifer M.Criley,and Craig L. Franklin*.Analysis of Gene Expression in Ceca ofHelicobacter hepaticus-Infected A/JCr Mice before and after Developmentof Typhlitis. Infection and Immunity.2003,71(7):3885–3893.
[11]Kowalski M.Helicobacter pylori（H.pylori）infection in coronary arterdisease：influence of H.pylori eradication on coronary artery lumen aftepercuteneous transluminal coronary angioplasty.The detection of H.pylospecific DNA in human coronary atherosclerotic plaque.J PhysioPharmacol.2001,52（Supple1）:3-31.
[12]Suzuk T， Matsushima M,Masui A,et al.Effect of Helicobacter pyloeradication in patients with chronic：diopathic thrombocyto purpura－randomized controlled trial.Am J Gastroenterrol.2005,100（6）:1265～1270.
[13]Roussos A,Tsimpoukas F,Anastasakou E,et al.Helicobacter pyloriseroprevalence in patients with chronic bronchitis.J Gastroenterol.2002,37（5）:332-335.
[14]王静，季尚玮，李岩，王江滨等.幽门螺杆菌感染慢性阻塞性肺病模型的建立及特征分析.中华消化杂志.2011,31(11):757-760.
[15]Whary MT,Fox JG.Natural and experimental Helicobacter infections.Comp.Med,2004,54:128-158.
[16]Erdman SE, Poutahidis T, Tomczak, et al. CD4+CD25+regulatory Tlymphocytes inhibit microbially induced colon cancer in Rag2-deficientmice. Am. J. Pathol.,2003,162:691–702.
[17]Maurer KJ, Ihrig MM, Rogers AB, et al. Identification of cholelithogenicenterohepatic Helicobacter species and their role in murine cholesterolgallstone formation. Gastroenterology,2005,128:1023–1033.
[18]Franklin CL, Riley LK, Livingston RS, et al. Enterohepatic lesions in SCIDmice infected with Helicobacter bilis. Lab. Anim. Sci.,1998,48:334–339.
[19]Maggio-Price L, Shows D, Waggie K, et al. Helicobacter bilis infectionaccelerates and H.hepaticus infection delays the development of colitis inmultiple drug resistance-deficient (mdr1a-/-) mice. Am. J. Pathol.,2002,160:739–751.
[20]Myles MH, Livingston RS, Franklin CL. Pathogenicity of Helicobacterrodentium in A/JCr and SCID mice. Comp. Med.,2004,54(5):549–557.
[21]Fox JG, Dewhirst FE, Tully JG, et al. Helicobacter hepaticus Sp. Now,amicroaerophilic bacterium isolated from lives and interstinal mucosalscrapings from mice[J]. Clinical Microbiology,1994,32(5):1238-45.
[22]季尚玮，王颂，王江滨，等，我国不同品系实验室小鼠肝螺杆菌感染状况调查.中华消化杂志.2010，30（9）：597-601
[23]Blacky,et al.In vitro activity of fosfomycin alone and in combination withamoxicillin, clarithromycin and metronidazole against Helicobacter pyloricompared with combined clarithromycin and metronidazole. Eur J ClinMicrobiol Infect Dis (2005)24:276–279
[24]Yoko Yanagawa,Yoshimasa Yamamoto,et,al. A Combination Effect ofEpigallocatechin Gallate, a Major Compound of Green Tea Catechins,with Antibiotics on Helicobacter pylori Growth In Vitro. CurrentMicrobiology Vol.47(2003), pp.244–249
[25]Lin E, Stanek R J, Mufson M A. Lack of synergy of erythromycincombined with penicillin or cefotaxime againstStreptococcus pneumoniaein vitro[J].Antimicrob Agents Chemother,2003,47(3):1151
[26]Makoto Sasaki，Tsutomu Mizoshita, Takashi Mizushima, et,al. Effect ofplaunotol in combination with clarithromycin against clarithromycin-resistant Helicobacter pylori in vitro and in vivo. Journal of AntimicrobialChemotherapy (2007)60,1060–1063
[27] Nancy ST, Xu SL, Nambiar P, et al. Enterohepatic Helicobacter Speciesare Prevalent in Mice from Commercial and Academic Institutions in Asia,Europe, and North America. Journal of clinical microbiology,2007,45(7):2166–2172.
[28] Shames B, Fox JG, Dewhirst F, et al. Identification of widespreadHelicobacter hepaticus infection in feces in commercial mouse coloniesby culture and PCR assay. J. Clin. Microbiol.,1995,33:2968–2972.
[29] Ananieva O，Nilsson I，Vorobjova T，Uibo R，Wadstr m T. Immuneresponses to bile-tolerant helicobacter species in patients with chronicliver diseases, a randomized population group, and healthy blood donors.Clin Diagn Lab Immunol.2002，9(6)：1160~1164.
[30] Russell RJ,et al.Use of antibiotics to prevent hepatitis and typhlitis in malescid mice spontaneously infected with Helicobacter hepaticus.Lab AnimSci,1995,45(4):373-378
[31]李红艳、季尚玮、王颂、王江滨等，不同品系小鼠肝螺杆菌感染实验模型的建立及病理特征分析中华消化杂志2011，11.743-738.
[2] Sakai T, Ognra Y, Narita J, et al. Simultaneous early adenocarcinoma andmucosa-associated lymphoid tissue（MALT）lymphoma of the stomachassociated with Helicobacter pylori infection. Gastric Cancer,2003;8:191-196.
[3]王江滨，亓文骞,李冰，秦少游，等幽门螺杆菌感染与胃肠外疾病.中华消化杂志,2011;5(31):358–360.
[4] Kowalski M. Helicobacter pylori(H. pylori)infection in coronary arterydisease：influence of H. pylori eradication on coronary artery lumen afterpercuteneous transluminal coronary angioplasty. The detection of H. pylorispecific DNA in human coronary atherosclerotic plaque. J PhysiolPharmacol,2001;52(Supple1):3-31.
[5] Suzuk T, Matsushima M, Masui A, et al. Effect of Helicobacter pylorieradication in patients with chronic：diopathic thrombocyto purpura-arandomized controlled trial. Am J Gastroenterrol,2005;100(6):1265-1270.
[6] Roussos A, Tsimpoukas F, Anastasakou E, et al. Helicobacter pyloriseroprevalence in patients with chronic bronchitis. J Gastroenterol,2002;37(5):332-335.
[7] Fox JG, Dewhirst FE, Tully JG, et al. Helicobacter hepaticus Sp. Now, amicroaerophilic bacterium isolated from lives and interstinal mucosalscrapings from mice[J]. Clinical Microbiology,1994,32(5):1238-1245.
[8] Ananieva O，Nilsson I，Vorobjova T，Uibo R，Wadstr m T. Immuneresponses to bile-tolerant helicobacter species in patients with chronic liverdiseases, a randomized population group, and healthy blood donors. ClinDiagn Lab Immunol.2002，9(6)：1160~1164.
[9]季尚玮，王颂，王江滨，等，我国不同品系实验室小鼠肝螺杆菌感染状况调查.中华消化杂志.2010，30（9）：597-601
[10]Blacky,et al.In vitro activity of fosfomycin alone and in combination withamoxicillin, clarithromycin and metronidazole against Helicobacter pyloricompared with combined clarithromycin and metronidazole. Eur J ClinMicrobiol Infect Dis (2005)24:276–279
[11]Yoko Yanagawa,Yoshimasa Yamamoto,et,al. A Combination Effect ofEpigallocatechin Gallate, a Major Compound of Green Tea Catechins,with Antibiotics on Helicobacter pylori Growth In Vitro. CurrentMicrobiology Vol.47(2003), pp.244–249
[12]Lin E, Stanek R J, Mufson M A. Lack of synergy of erythromycincombined with penicillin or cefotaxime againstStreptococcus pneumoniaein vitro[J].Antimicrob Agents Chemother,2003,47(3):1151
[13]Makoto Sasaki，Tsutomu Mizoshita, Takashi Mizushima, et,al. Effect ofplaunotol in combination with clarithromycin against clarithromycin-resistant Helicobacter pylori in vitro and in vivo. Journal of AntimicrobialChemotherapy (2007)60,1060–1063
[14]Rocha M，Avenaud P，Ménard A，Le Bail B，Balabaud C，Bioulac-SageP，de Magalh es Queiroz DM，Mégraud F. Association of Helicobacterspecies with hepatitis C cirrhosis with or without hepatocellular carcinoma.Gut．2005，54：396~401
[15]Nilsson I et al.Serum antibodies to Helicobacter hepaticus and Helicobacterpylori in patients with chronic liver disease. Gut,2000;46(3):410-414
[16]Ward JM, Fox JG, Anver MR, et a1. Chronic active hepatitis andassociated livertum orsinmice caused by a persistent bacterial itfection witha novel Helicobacter speces[J]. Natl CancerInst,1994,86(16):1222-1227
[17]Shames B, Fox JG, Dewhirst F, et al. Identification of widespreadHelicobacter hepaticus infection in feces in commercial mouse colonies byculture and PCR assay. J. Clin. Microbiol.,1995,33:2968–2972.
[18] Russell RJ,et al.Use of antibiotics to prevent hepatitis and typhlitis in malescid mice spontaneously infected with Helicobacter hepaticus.Lab AnimSci,1995,45(4):373-378
[19]Foltz CJ.Fox JG,Yan L,Shames B.Evaluation of antibiotic therapies foreradication of Helicobacter hepaticus. Antimicrob Agents Chemother1995;39:1292-4.
[20]Rogers AB. How gut microbes promote extraintestinal cancers. GutMicrobes2:1,52-57; January/February2011.
[1] Sakai T, Ognra Y, Narita J, et al. Simμltaneous early adenocarcinoma andmucosa-associated lymphoid tissue(MALT)lymphoma of the stomachassociated with Helicobacter pylori infection[J]. Gastric Cancer,2003,8:191-196.
[2] Herrera AG.Helicobacter pylori and food products:a public healthproblem[J].Methods Mol Biol,2004,268:297-301.
[3] Eidt S,Stolte M.The significance of Helicobacter pylori in relation to gastriccancer and lymphoma[J].Eur J Gastroenterol Hepatol,1995,7(4):318-321.
[4]Vandenplas Y. Helicobacter pylori infection. World J. Gastroenterol,2000,6:20-31.
[5] Parsonnet J.Bacterial infection as a cause of cancer[J].Environ Health Prespect,1995,103(l8):263-268.
[6] Forman D.Helicobacter pylori and gastric cancer[J].Scand J Gastr-oenterolSuppl,1996,215:48-51.
[7]王江滨，亓文骞,李冰，秦少游，等幽门螺杆菌感染与胃肠外疾病.中华消化杂志,2011;5(31):358–360.
[8] Kabir AM, Aiba Y, Takagi A, et al. Prevention of Helicobacter pyloriinfection by lactobacilli in a gnotobiotic murine model. Gut.1997;41(1):49-55.
[9] Lesbros-Pantoflickova D,Corthesy-Theulaz. Helicobacter pylori andProbiotics. J Nutr.2007;137(3):812S-818S.
[10] Lorca GL, Wadstrom T, Valdez GF, et al. Lactobacillusacidophilusautolysins inhibit Helicobacter pylori in vitro. Curr Microbiol.2001;42:39–44.
[11]王锦鸿，王江滨，李岩，等，抗HP嗜酸乳杆菌的筛选及其相关生物学特征的研究，中国免疫学杂志，2007；23，300-305.
[12] Fox JG, Dewhirst FE, Tully JG, Paster BJ, Yan L, Taylor NS, Collins MJ,Jr Gorelick PL, Ward JM. Helicobacter hepaticus sp. nov., amicroaerophilic bacterium isolated from livers and intestinal mucosalscrapings from mice. J Clin Microbiol1994,32(5):1238-45.
[13]季尚玮，王颂，王江滨，等，我国不同品系实验室小鼠肝螺杆菌感染状况调查.中华消化杂志.2010，30（9）：597-601
[14] Handa Y, Saitoh, Kavaguchi M, et al. Association of Helocobacter pyloriand diffuse type gastric cancer. J Gastroenterol.1996;31:29
[15] Kuipirs EJ, Uyterlinde SM, Pinz AS, et al. Long-term consequence ofHelicobacter pylori gastritis. Lancet.1995;345:1525-1528.
[16] Horstman M, Erttmann R, Winkler K, et al. Relapse of MALT lymphomaassociated with Helicobacter pylori after antibiotic treatment. Lancet.1994;343:1098
[17] Kumar D, Dhar A, Dattagupta S, et al. Pre and post eradicationgastricinflammation in Helicobacter pylori-associated duodenal ulcer.Indian J Gastroenterol.2002;21(1):7-10.
[18] Graham DY, Lew GM, Klein PD, et al. Effect of treatment of Helicobacterpylori infection on the long-term recurrence of gastric or duodenal ulcer.Ann Zntern Med.1992;116:705-708
[19] Konstantinov SR, Awati AA, Williams BA, Miller BG, Jones P, Stokes CR,Akkermans AD, Smidt H, de Vos WM. Post-natal development of theporcine microbiota composition and activities. Environ Microbiol2006,8(7):1191-9.
[20] Timmerman H M.Together strong: probiotics to promote health in peopleand animals [J]. Tijdschr Diergeneeskd,2006;131（19）:697-700.
[21] Midolo PD, Lambert JR, Hull R, Luo F, Grayson ML. In vitro inhibitionof Helicobacter pylori NCTC11637by organic acids and lactic acidbacteria. J Appl Bacteriol1995,79(4):475-9.
[22] Bernet MF, Brassart D, Neeser JR, Servin AL. Lactobacillus acidophilusLA1binds to cultured human intestinal cell lines and inhibits cellattachment and cell invasion by enterovirulent bacteria. Gut1994,35(4):483-9
[23] Coconnier MH, Lievin V, Hemery E, Servin AL. Antagonistic activityagainst Helicobacter infection in vitro and in vivo by the humanLactobacillus acidophilus strain LB. Appl Environ Microbiol1998,64(11):4573-80.
[24] Yeung PS, Sanders ME, Kitts CL, Cano R, Tong PS. Species-specificidentification of commercial probiotic strains. J Dairy Sci2002,85(5):1039-51.
[25] Fox JG, Yan L, Shames B, Campbell J, Murphy JC, Li X. Persistenthepatitis and enterocolitis in germfree mice infected with Helicobacterhepaticus. Infect Immun1996,64(9):3673-81.
[1] Sakai T, Ognra Y, Narita J, et al. Simμltaneous early adenocarcinoma andmucosa-associated lymphoid tissue(MALT)lymphoma of the stomachassociated with Helicobacter pylori infection[J]. Gastric Cancer,2003,8:191-196.
[2] Herrera AG.Helicobacter pylori and food products:a public healthproblem[J].Methods Mol Biol,2004,268:297-301.
[3] Eidt S,Stolte M.The significance of Helicobacter pylori in relation to gastriccancer and lymphoma[J].Eur J Gastroenterol Hepatol,1995,7(4):318-321.
[4]Vandenplas Y. Helicobacter pylori infection. World J. Gastroenterol,2000,6:20-31.
[5] Parsonnet J.Bacterial infection as a cause of cancer[J].Environ Health Prespect,1995,103(l8):263-268.
[6] Forman D.Helicobacter pylori and gastric cancer[J].Scand J Gastr-oenterolSuppl,1996,215:48-51.
[7]王锦鸿，王江滨，李岩，等，抗HP嗜酸乳杆菌的筛选及其相关生物学特征的研究，中国免疫学杂志，2007；23，300-305.
[8]Ward JM, Fox JG, Anver MR, et a1. Chronic active hepatitis and associatedlivertum orsinmice caused by a persistent bacterial infection with a novelHelicobacter speces[J]. Natl CancerInst,1994,86(16):1222-1227.
[9] LI XT,Fox JG,Mark T,et al. SCID/NCr Mice Naturally Infected withHelicobacter hepaticus Develop Progressive Hepatitis,ProliferativeTyphlitis,and Colitis. Infection and Immuity, Nov.1998,66:5477-5484.
[10]Matthew H. Myles, Robert S. Livingston, Beth A. Livingston, Jennifer M.Criley,and Craig L. Franklin*.Analysis of Gene Expression in Ceca ofHelicobacter hepaticus-Infected A/JCr Mice before and after Developmentof Typhlitis. Infection and Immunity.2003,71(7):3885–3893.
[11]Kowalski M.Helicobacter pylori（H.pylori）infection in coronary arterdisease：influence of H.pylori eradication on coronary artery lumen aftepercuteneous transluminal coronary angioplasty.The detection of H.pylospecific DNA in human coronary atherosclerotic plaque.J PhysioPharmacol.2001,52（Supple1）:3-31.
[12]Suzuk T， Matsushima M,Masui A,et al.Effect of Helicobacter pyloeradication in patients with chronic：diopathic thrombocyto purpura－randomized controlled trial.Am J Gastroenterrol.2005,100（6）:1265～1270.
[13]Roussos A,Tsimpoukas F,Anastasakou E,et al.Helicobacter pyloriseroprevalence in patients with chronic bronchitis.J Gastroenterol.2002,37（5）:332-335.
[14]王静，季尚玮，李岩，王江滨等.幽门螺杆菌感染慢性阻塞性肺病模型的建立及特征分析.中华消化杂志.2011,31(11):757-760.
[15]Whary MT,Fox JG.Natural and experimental Helicobacter infections.Comp.Med,2004,54:128-158.
[16]Erdman SE, Poutahidis T, Tomczak, et al. CD4+CD25+regulatory Tlymphocytes inhibit microbially induced colon cancer in Rag2-deficientmice. Am. J. Pathol.,2003,162:691–702.
[17]Maurer KJ, Ihrig MM, Rogers AB, et al. Identification of cholelithogenicenterohepatic Helicobacter species and their role in murine cholesterolgallstone formation. Gastroenterology,2005,128:1023–1033.
[18]Franklin CL, Riley LK, Livingston RS, et al. Enterohepatic lesions in SCIDmice infected with Helicobacter bilis. Lab. Anim. Sci.,1998,48:334–339.
[19]Maggio-Price L, Shows D, Waggie K, et al. Helicobacter bilis infectionaccelerates and H.hepaticus infection delays the development of colitis inmultiple drug resistance-deficient (mdr1a-/-) mice. Am. J. Pathol.,2002,160:739–751.
[20]Myles MH, Livingston RS, Franklin CL. Pathogenicity of Helicobacterrodentium in A/JCr and SCID mice. Comp. Med.,2004,54(5):549–557.
[21]Fox JG, Dewhirst FE, Tully JG, et al. Helicobacter hepaticus Sp. Now,amicroaerophilic bacterium isolated from lives and interstinal mucosalscrapings from mice[J]. Clinical Microbiology,1994,32(5):1238-45.
[22]季尚玮，王颂，王江滨，等，我国不同品系实验室小鼠肝螺杆菌感染状况调查.中华消化杂志.2010，30（9）：597-601
[23]Blacky,et al.In vitro activity of fosfomycin alone and in combination withamoxicillin, clarithromycin and metronidazole against Helicobacter pyloricompared with combined clarithromycin and metronidazole. Eur J ClinMicrobiol Infect Dis (2005)24:276–279
[24]Yoko Yanagawa,Yoshimasa Yamamoto,et,al. A Combination Effect ofEpigallocatechin Gallate, a Major Compound of Green Tea Catechins,with Antibiotics on Helicobacter pylori Growth In Vitro. CurrentMicrobiology Vol.47(2003), pp.244–249
[25]Lin E, Stanek R J, Mufson M A. Lack of synergy of erythromycincombined with penicillin or cefotaxime againstStreptococcus pneumoniaein vitro[J].Antimicrob Agents Chemother,2003,47(3):1151
[26]Makoto Sasaki，Tsutomu Mizoshita, Takashi Mizushima, et,al. Effect ofplaunotol in combination with clarithromycin against clarithromycin-resistant Helicobacter pylori in vitro and in vivo. Journal of AntimicrobialChemotherapy (2007)60,1060–1063
[27] Nancy ST, Xu SL, Nambiar P, et al. Enterohepatic Helicobacter Speciesare Prevalent in Mice from Commercial and Academic Institutions in Asia,Europe, and North America. Journal of clinical microbiology,2007,45(7):2166–2172.
[28] Shames B, Fox JG, Dewhirst F, et al. Identification of widespreadHelicobacter hepaticus infection in feces in commercial mouse coloniesby culture and PCR assay. J. Clin. Microbiol.,1995,33:2968–2972.
[29] Ananieva O，Nilsson I，Vorobjova T，Uibo R，Wadstr m T. Immuneresponses to bile-tolerant helicobacter species in patients with chronicliver diseases, a randomized population group, and healthy blood donors.Clin Diagn Lab Immunol.2002，9(6)：1160~1164.
[30] Russell RJ,et al.Use of antibiotics to prevent hepatitis and typhlitis in malescid mice spontaneously infected with Helicobacter hepaticus.Lab AnimSci,1995,45(4):373-378
[31]李红艳、季尚玮、王颂、王江滨等，不同品系小鼠肝螺杆菌感染实验模型的建立及病理特征分析中华消化杂志2011，11.743-738.