为探索大肠杆菌Nissle 1917株(EcN)鞭毛展示的应用,本研究以重组质粒p UC18-fli C为模板,反向PCR产物经DpnⅠ、Exnase^(TM)Ⅱ重组环化,将6His标签基因分别插入无质粒大肠杆菌Nissle 1917(EcNcured of its two cryptic plasmids p MUT1和...为探索大肠杆菌Nissle 1917株(EcN)鞭毛展示的应用,本研究以重组质粒p UC18-fli C为模板,反向PCR产物经DpnⅠ、Exnase^(TM)Ⅱ重组环化,将6His标签基因分别插入无质粒大肠杆菌Nissle 1917(EcNcured of its two cryptic plasmids p MUT1和p MUT2;EcNc)fli C高变区774 bp^775 bp和792 bp^811 bp之间,构建嵌合鞭毛基因片段fliC01和fliC02。分别将其克隆于自杀性载体pRE112中,构建重组质粒pRE112-fliC01和pRE112-fliC02,并分别转化至EcNc中。通过一次重组和二次重组将嵌合鞭毛基因整合于EcNc基因组中,构建无抗性筛选的重组菌株EcNc fliC01和EcNc fliC02。对重组菌株进行生长状况、鞭毛形成以及体外细胞黏附试验和小鼠体内定植试验检测,结果表明EcNc fliC01和EcNc fliC02的生长未受影响,表达的嵌合鞭毛能够被H1多抗或His-Tag单克隆抗体识别并组装形成鞭毛丝;重组菌对IPEC-J2细胞的黏附能力及小鼠肠道定植能力与亲本株EcNc相比无显著性差异。本实验结果为进一步研究EcNc鞭毛展示技术应用于活疫苗研制奠定了基础。展开更多
初步研究大肠杆菌Nissle 1917(E.coli Nissle 1917,EcN)鞭毛蛋白(Flagellin,FliC)高变区的功能,为探讨其作为表面展示系统的可行性打下基础。以EcN无质粒克隆(EcN was cured of its 2 cryptic plasmids pMUT1 and pMUT2 resulting in Ec...初步研究大肠杆菌Nissle 1917(E.coli Nissle 1917,EcN)鞭毛蛋白(Flagellin,FliC)高变区的功能,为探讨其作为表面展示系统的可行性打下基础。以EcN无质粒克隆(EcN was cured of its 2 cryptic plasmids pMUT1 and pMUT2 resulting in EcNc)为实验菌株,分别构建2个鞭毛蛋白基因(fl iC)高变区859-888位、829-858位的缺失突变株和回补菌株。分析突变区域对EcNc的生长性能、生化特性、运动性及抗原性等方面的影响。结果显示:EcNc、两个缺失株和回补株的生长特性无明显差异,且三者生化试验结果一致。缺失突变株在半固体培养基上不能运动,而回补株均恢复了原运动性;缺失突变株均不与H1单因子血清发生凝集,而互补菌株均能产生与EcNc一致的凝集反应。表明高变区的这两个区域尽管不影响菌株的生长性能和生化特性,但与EcN的运动性及抗原性密切相关。这为进一步探索EcN鞭毛展示技术的研究提供了一定的数据。展开更多
AIM: To evaluate the effect of oral Escherichia coli (E. coli) Nissle application on the outcome of intestinal-borne dermatoses.METHODS: In a randomized, controlled, non-blinded prospective clinical trial 82 patients ...AIM: To evaluate the effect of oral Escherichia coli (E. coli) Nissle application on the outcome of intestinal-borne dermatoses.METHODS: In a randomized, controlled, non-blinded prospective clinical trial 82 patients with intestinal-borne facial dermatoses characterized by an erythematous papular-pustular rash were screened. At the initiation visit 37 patients entered the experimental arm and 20 patients constituted the control arm. All 57 patients were treated with a vegetarian diet and conventional topical therapy of the dermatoses with ointments containing tetracycline, steroids and retinoids. In the experimental arm patients received a one month therapy with oral E. coli Nissle at a maintenance dose of 2 capsules daily. The experimental group was compared to a non-treatment group only receiving the diet and topical therapy. The primary outcome parameter was improvement of the dermatoses, secondary parameters included life quality and adverse events. In addition the immunological reaction profile (IgA, interleucin-8 and interferon-α) was determined. Furthermore the changes of stool consistency and the microbiota composition over the time of intervention were recorded.RESULTS: Eighty-nine percent of the patients with acne, papular-pustular rosacea and seborrhoic dermatitis responded to E. coli Nissle therapy with significant amelioration or complete recovery in contrast to 56% in the control arm (P < 0.01). Accordingly, in the E. coli Nissle treated patients life quality improved significantly (P < 0.01), and adverse events were not recorded. The clinical improvement was associated with a significant increase of IgA levels to normal values in serum as well as suppression of the proinflammatory cytokine IL-8 (P < 0.01 for both parameters). In the E. coli Nissle treated group a shift towards a protective microbiota with predominance of bifidobacteria and lactobacteria (> 10<sup>7</sup> CFU/g stool) was observed in 79% and 63% of the patients, respectively (P < 0.01), compared to no change in the control group without E. coli Nissle. Moreover, the detection rate of a pathogenic flora dropped from 73% to 14 % of the patients in the experimental arm (P < 0.01) with no significant change in the control arm (accounting 80% before and 70% after the observation period, P > 0.05). Accordingly, stool consistency, color and smell normalized in the E. coli Nissle treated patients.CONCLUSION: E. coli Nissle protects the mucus barrier by overgrowth of a favorable gut microbiota with less immunoreactive potential which finally leads to clinical improvement of intestinal borne dermatoses.展开更多
BACKGROUND Hepatic encephalopathy(HE)can be considered a result of dysregulated gutliver-brain axis function,where cognitive impairment can be reversed or prevented by the beneficial effects induced by"gut-centri...BACKGROUND Hepatic encephalopathy(HE)can be considered a result of dysregulated gutliver-brain axis function,where cognitive impairment can be reversed or prevented by the beneficial effects induced by"gut-centric"therapies,such as the administration of nonabsorbable disaccharides,nonabsorbable antibiotics,probiotics and prebiotics.AIM To assess the short-term efficacy and safety of the probiotic Escherichia coli Nissle(EcN)1917 strain compared to lactulose and rifaximin in patients with minimal/mild HE.METHODS From January 2017 to March 2020,a total of 45 patients with HE were enrolled in this prospective,single-centre,open-label,randomized study.Participants were randomly assigned at a ratio of 1:1:1 to one of the treatment groups:The EcN group(n=15),lactulose group(n=15)or rifaximin group(n=15)for a 1 mo intervention period.The main primary outcomes of the study were changes in serum ammonia and Stroop test score.The secondary outcomes were markers of a chronic systemic inflammatory response(ІL-6,ІL-8,and IFN-γ)and bacteriology of the stool flora evaluated by specialized nonculture techniques after a 1 mo intervention period.RESULTS Patients who were given rifaximin or EcN showed a more significant reduction in serum ammonia and normalization of Bifidobacteria and Lactobacilli abundance compared to the lactulose group.However,the most pronounced restoration of the symbiotic microflora was associated with EcN administration and characterized by the absence of E.coli with altered properties and pathogenic enterobacteria in patient faeces.In the primary outcome analysis,improvements in the Stroop test parameters in all intervention groups were observed.Moreover,EcN-treated patients performed 15%faster on the Stroop test than the lactulose group patients(P=0.017).Both EcN and rifaximin produced similar significant reductions in the proinflammatory cytokines INF-γ,IL-6 and IL-8.EcN was more efficient than lactulose in reducing proinflammatory cytokine levels.CONCLUSION The use of the probiotic EcN strain was safe and quite efficient for HE treatment.The probiotic reduced the ammonia content and the level of serum proinflammatory cytokines,normalized the gut microbiota composition and improved the cognitive function of patients with HE.The application of the EcN strain was more effective than lactulose treatment.展开更多
AIM： To evaluate bacteriocinogeny in short-term highdose indomethacin administration with or without probiotic Escherichia coli Nissle 1917 （EcN） in experimental pigs.METHODS： Twenty-four pigs entered the study： ...AIM： To evaluate bacteriocinogeny in short-term highdose indomethacin administration with or without probiotic Escherichia coli Nissle 1917 （EcN） in experimental pigs.METHODS： Twenty-four pigs entered the study： Group A （controls）, Group B （probiotics alone）, Group C （indomethacin alone） and Group D （probiotics and indomethacin）. EcN （3.5 × 10^10 bacteria/d for 14 d） and/or indomethacin （15 mg/kg per day for 10 d） were administrated orally. Anal smears before and smears from the small and large intestine were taken from all animals. Bacteriocin production was determined with 6 different indicator strains; all strains were polymerase chain reaction tested for the presence of 29 individual bacteriocinencoding determinants. RESULTS： The general microbiota profile was rather uniform in all animals but there was a broad diversity in coliform bacteria （parallel genotypes A, B1, B2 and D found）. In total, 637 bacterial strains were tested, mostly Escherichia coli （E. coli. There was a higher incidence of non-E：, coli strains among samples taken from the jejunum and ileum compared to that of the colon and rectum indicating predominance of E. coil strains in the large intestine. Bacteriocinogeny was found in 24/77 （31%） before and in 155/560 （28%） isolated bacteria at the end of the study. Altogether, 13 individual bacteriocin types （out of 29 tested） were identified among investigated strains. Incidence of four E. coli genotypes was equally distributed in all groups of E. coil strains, with majority of genotype A （ranging from 81% to 88%）. The following types of bacteriocins were most commonly revealed： colicins Ia/Ib （44%）, microcin V （18%）, colicin E1 （16%） and microcin H47 （6%）. There was a difference in bacteriocinogeny between control group A （52/149, 35%） and groups with treatment at the end of the study： B： 31/122 （25%, P = 0.120）; C： 43/155 （28%, P = 0.222）; D： 29/134 （22%, P = 0.020）. There was a significantly lower prevalence of colicin Ib, microcins H47 and V （probiotics group, P 〈 0.001）, colicin E1 and microcin H47 （indomethacin group, P 〈 0.001） and microcins H47 and V （probiotics and indomethacin group, P = 0.025） compared to controis. Escherichia fergusonil （E. fergusoniO was identi-fled in 6 animals （6/11 isolates from the rectum）. One strain was non-colicinogenic, while all other strains of E. fergusonii solely produced colicin El. All animals started and remained methanogenic despite the fact that EcN is a substantial hydrogen producer. There was an increase in breath methane （after the treatment） in 5/6 pigs from the indomethacin group （C）. CONCLUSION： EcN did not exert long-term liveabilib/ in the porcine intestine, All experimental pigs remained methanogenic, Indomethacin and EcN administered together might produce the worst impact on bacteriocinogeny.展开更多
文摘初步研究大肠杆菌Nissle 1917(E.coli Nissle 1917,EcN)鞭毛蛋白(Flagellin,FliC)高变区的功能,为探讨其作为表面展示系统的可行性打下基础。以EcN无质粒克隆(EcN was cured of its 2 cryptic plasmids pMUT1 and pMUT2 resulting in EcNc)为实验菌株,分别构建2个鞭毛蛋白基因(fl iC)高变区859-888位、829-858位的缺失突变株和回补菌株。分析突变区域对EcNc的生长性能、生化特性、运动性及抗原性等方面的影响。结果显示:EcNc、两个缺失株和回补株的生长特性无明显差异,且三者生化试验结果一致。缺失突变株在半固体培养基上不能运动,而回补株均恢复了原运动性;缺失突变株均不与H1单因子血清发生凝集,而互补菌株均能产生与EcNc一致的凝集反应。表明高变区的这两个区域尽管不影响菌株的生长性能和生化特性,但与EcN的运动性及抗原性密切相关。这为进一步探索EcN鞭毛展示技术的研究提供了一定的数据。
文摘AIM: To evaluate the effect of oral Escherichia coli (E. coli) Nissle application on the outcome of intestinal-borne dermatoses.METHODS: In a randomized, controlled, non-blinded prospective clinical trial 82 patients with intestinal-borne facial dermatoses characterized by an erythematous papular-pustular rash were screened. At the initiation visit 37 patients entered the experimental arm and 20 patients constituted the control arm. All 57 patients were treated with a vegetarian diet and conventional topical therapy of the dermatoses with ointments containing tetracycline, steroids and retinoids. In the experimental arm patients received a one month therapy with oral E. coli Nissle at a maintenance dose of 2 capsules daily. The experimental group was compared to a non-treatment group only receiving the diet and topical therapy. The primary outcome parameter was improvement of the dermatoses, secondary parameters included life quality and adverse events. In addition the immunological reaction profile (IgA, interleucin-8 and interferon-α) was determined. Furthermore the changes of stool consistency and the microbiota composition over the time of intervention were recorded.RESULTS: Eighty-nine percent of the patients with acne, papular-pustular rosacea and seborrhoic dermatitis responded to E. coli Nissle therapy with significant amelioration or complete recovery in contrast to 56% in the control arm (P < 0.01). Accordingly, in the E. coli Nissle treated patients life quality improved significantly (P < 0.01), and adverse events were not recorded. The clinical improvement was associated with a significant increase of IgA levels to normal values in serum as well as suppression of the proinflammatory cytokine IL-8 (P < 0.01 for both parameters). In the E. coli Nissle treated group a shift towards a protective microbiota with predominance of bifidobacteria and lactobacteria (> 10<sup>7</sup> CFU/g stool) was observed in 79% and 63% of the patients, respectively (P < 0.01), compared to no change in the control group without E. coli Nissle. Moreover, the detection rate of a pathogenic flora dropped from 73% to 14 % of the patients in the experimental arm (P < 0.01) with no significant change in the control arm (accounting 80% before and 70% after the observation period, P > 0.05). Accordingly, stool consistency, color and smell normalized in the E. coli Nissle treated patients.CONCLUSION: E. coli Nissle protects the mucus barrier by overgrowth of a favorable gut microbiota with less immunoreactive potential which finally leads to clinical improvement of intestinal borne dermatoses.
文摘BACKGROUND Hepatic encephalopathy(HE)can be considered a result of dysregulated gutliver-brain axis function,where cognitive impairment can be reversed or prevented by the beneficial effects induced by"gut-centric"therapies,such as the administration of nonabsorbable disaccharides,nonabsorbable antibiotics,probiotics and prebiotics.AIM To assess the short-term efficacy and safety of the probiotic Escherichia coli Nissle(EcN)1917 strain compared to lactulose and rifaximin in patients with minimal/mild HE.METHODS From January 2017 to March 2020,a total of 45 patients with HE were enrolled in this prospective,single-centre,open-label,randomized study.Participants were randomly assigned at a ratio of 1:1:1 to one of the treatment groups:The EcN group(n=15),lactulose group(n=15)or rifaximin group(n=15)for a 1 mo intervention period.The main primary outcomes of the study were changes in serum ammonia and Stroop test score.The secondary outcomes were markers of a chronic systemic inflammatory response(ІL-6,ІL-8,and IFN-γ)and bacteriology of the stool flora evaluated by specialized nonculture techniques after a 1 mo intervention period.RESULTS Patients who were given rifaximin or EcN showed a more significant reduction in serum ammonia and normalization of Bifidobacteria and Lactobacilli abundance compared to the lactulose group.However,the most pronounced restoration of the symbiotic microflora was associated with EcN administration and characterized by the absence of E.coli with altered properties and pathogenic enterobacteria in patient faeces.In the primary outcome analysis,improvements in the Stroop test parameters in all intervention groups were observed.Moreover,EcN-treated patients performed 15%faster on the Stroop test than the lactulose group patients(P=0.017).Both EcN and rifaximin produced similar significant reductions in the proinflammatory cytokines INF-γ,IL-6 and IL-8.EcN was more efficient than lactulose in reducing proinflammatory cytokine levels.CONCLUSION The use of the probiotic EcN strain was safe and quite efficient for HE treatment.The probiotic reduced the ammonia content and the level of serum proinflammatory cytokines,normalized the gut microbiota composition and improved the cognitive function of patients with HE.The application of the EcN strain was more effective than lactulose treatment.
基金Supported by Research project MZO 00179906 from the Ministry of Health of the Czech RepublicSupported by institutional support from the Czech Republic(MSM0021622415)Supported by research grants GAR 305/080535 and NS9665-4/2008(Ministry of Health of the Czech Republic)
文摘AIM： To evaluate bacteriocinogeny in short-term highdose indomethacin administration with or without probiotic Escherichia coli Nissle 1917 （EcN） in experimental pigs.METHODS： Twenty-four pigs entered the study： Group A （controls）, Group B （probiotics alone）, Group C （indomethacin alone） and Group D （probiotics and indomethacin）. EcN （3.5 × 10^10 bacteria/d for 14 d） and/or indomethacin （15 mg/kg per day for 10 d） were administrated orally. Anal smears before and smears from the small and large intestine were taken from all animals. Bacteriocin production was determined with 6 different indicator strains; all strains were polymerase chain reaction tested for the presence of 29 individual bacteriocinencoding determinants. RESULTS： The general microbiota profile was rather uniform in all animals but there was a broad diversity in coliform bacteria （parallel genotypes A, B1, B2 and D found）. In total, 637 bacterial strains were tested, mostly Escherichia coli （E. coli. There was a higher incidence of non-E：, coli strains among samples taken from the jejunum and ileum compared to that of the colon and rectum indicating predominance of E. coil strains in the large intestine. Bacteriocinogeny was found in 24/77 （31%） before and in 155/560 （28%） isolated bacteria at the end of the study. Altogether, 13 individual bacteriocin types （out of 29 tested） were identified among investigated strains. Incidence of four E. coli genotypes was equally distributed in all groups of E. coil strains, with majority of genotype A （ranging from 81% to 88%）. The following types of bacteriocins were most commonly revealed： colicins Ia/Ib （44%）, microcin V （18%）, colicin E1 （16%） and microcin H47 （6%）. There was a difference in bacteriocinogeny between control group A （52/149, 35%） and groups with treatment at the end of the study： B： 31/122 （25%, P = 0.120）; C： 43/155 （28%, P = 0.222）; D： 29/134 （22%, P = 0.020）. There was a significantly lower prevalence of colicin Ib, microcins H47 and V （probiotics group, P 〈 0.001）, colicin E1 and microcin H47 （indomethacin group, P 〈 0.001） and microcins H47 and V （probiotics and indomethacin group, P = 0.025） compared to controis. Escherichia fergusonil （E. fergusoniO was identi-fled in 6 animals （6/11 isolates from the rectum）. One strain was non-colicinogenic, while all other strains of E. fergusonii solely produced colicin El. All animals started and remained methanogenic despite the fact that EcN is a substantial hydrogen producer. There was an increase in breath methane （after the treatment） in 5/6 pigs from the indomethacin group （C）. CONCLUSION： EcN did not exert long-term liveabilib/ in the porcine intestine, All experimental pigs remained methanogenic, Indomethacin and EcN administered together might produce the worst impact on bacteriocinogeny.
