Herdecke, Germany
Herdecke, Germany

Time filter

Source Type

News Article | December 7, 2016
Site: www.nature.com

Strains of the probiotic E. coli Nissle 1917 (EcN; Mutaflor, DSM 6601), the mouse commensal E. coli (cEc), the adherent invasive E. coli (AIEC), and the pathogen S. enterica serovar Typhimurium (STm) used in this study are listed in Supplementary Table 3. All plasmids used in this study are listed in Supplementary Table 4. EcN was provided by Ardeypharm GmbH. The commensal E. coli strain was isolated from mice in our vivarium and does not appear to encode for known antibacterial exoproducts. The AIEC strain is a human isolate from a patient with Crohn’s disease (isolate NRG857c O83:H1)26 and was provided by A. Torres. The STm strain background we employed (IR715) is a fully virulent, nalidixic acid-resistant derivative of STm wild-type isolate ATCC 14028 and does not encode for known antibacterial exoproducts. All strains were routinely grown aerobically in Luria–Bertani (LB) broth (10 g l−1 tryptone, 5 g l−1 yeast extract, 10 g l−1 NaCl) or on LB agar plates at 37 °C. When indicated, antibiotics were added to the medium at the following concentrations: 0.03 mg ml−1 chloramphenicol (Cm); 0.1 mg ml−1 carbenicillin (Carb); 0.05 mg ml−1 kanamycin (Kan); 0.01 mg ml−1 tetracycline (Tet). For animal infections or bacterial administration, all strains were grown in LB medium aerobically at 37 °C overnight. For in vitro growth assays, strains were grown in iron-limiting conditions (nutrient broth supplemented with 0.2 mM 2,2′-dipyridyl dissolved in ethanol; Sigma) aerobically at 37 °C overnight. Restriction enzymes and Phusion High Fidelity DNA Polymerase were purchased from New England Biolabs. Oligonucleotides were synthesized by Fisher Scientific and are listed in Supplementary Tables 5 and 6. Mutants in EcN and STm were constructed using the lambda red recombinase system27. In brief, primers (Supplementary Table 5) homologous to sequences flanking the 5′ and 3′ ends of the target regions were designed (H1 and H2 primers, respectively; Supplementary Table 5) and were used to replace the selected genes with a chloramphenicol- (derived from pKD3), kanamycin- (derived from pKD4) or tetracycline-resistance cassette (Supplementary Table 4). Strain names for the mutants are listed in Supplementary Table 3. To confirm integration of the resistance cassette and deletion of the target, mutant strains and wild-type controls were each assayed using three PCR amplifications (5′ end, 3′ end, deleted target) to validate mutations. Primers (Supplementary Table 5) that flank the target sequence were used in conjunction with a common test primer (C1, C2, K1, K2 or primers for the tetRA cassette) to test for both new-junction fragments. The mchDEF region was amplified from EcN genomic DNA using primers listed in Supplementary Table 5. A region of 4,148 bp was amplified to ensure all regulatory elements were included. The PCR fragment was cloned into plasmid pCR-XL-TOPO using Zero Blunt TOPO PCR Cloning Kit (Invitrogen) according to the manufacturer’s protocol, subcloned into the multiple cloning site of low-copy plasmid pWSK29, and confirmed by Sanger sequencing (Eton Bioscience). The mchDEF mcmI and mchDEF mchI constructs were amplified from EcN genomic DNA using primers listed in Supplementary Table 5. PCR fragments of 4,321 bp (mchDEFmcmI), 4,000 bp (mchDEF) and 400 bp (mchI) were directly assembled with plasmid pWKS30 using the Gibson assembly method. The role of EcN microcins against cEc, AIEC and STm was tested by an in vitro competitive growth assay in iron-rich and iron-limiting conditions. Strains were grown in nutrient broth supplemented with 0.2 mM 2,2′-dipyridyl (Sigma) aerobically at 37 °C overnight. Approximately 5 × 103 CFU ml−1 from an overnight culture were inoculated into 0.1 ml of tissue-culture medium (DMEM/F12 with 10% fetal bovine serum (FBS); Invitrogen), as previously described23, 24. When indicated, medium was prepared with 1 μM ferric iron citrate (Sigma). Wild-type EcN or EcN mutants were inoculated in competition with wild-type STm, STm mutants, cEc, or AIEC. CFUs of each strain were enumerated by plating serial dilutions at 0, 5, 8, 11 and (for some assays) 16 h after inoculation. The Institutional Animal Care and Use Committee at the University of California, Irvine approved all mouse experiments. Eight-to-ten-week-old male and female C57BL/6 Slc11a1+ (also known as Nramp1) mice were bred and housed in a vivarium. For some experiments, mice were purchased from Jackson laboratory and, upon arrival, allowed to acclimate to the new environment for at least one week before the start of an experiment. All mice were housed under specific pathogen-free conditions. Mice were fed an irradiated 2920X Teklad diet (Envigo). For one experimental procedure, mice were fed an irradiated iron-rich diet (1% diet FeSO 2020, Teklad TD 160234; Envigo). Culturing faeces on MacConkey agar indicated that culturable enterobacteria were absent from our mice. Five to ten mice were used per experimental group, as indicated by the number of symbols in each figure panel. Group sizes were based on previous studies that detected a tenfold difference with statistical significance. For one experiment, ten-week-old Swiss Webster germ-free mice were used. Swiss Webster germ-free mice were maintained in sterile isolators and were fed a double-irradiated diet (Purina 5066, Charles River Rodent 18% Vac Pac). For this experimental procedure, three mice per experimental group were used (see figure panels). Mice were randomly grouped in cages of a maximum five animals per cage. Similar numbers of male and female mice were used in each experimental group. No blinding was performed, with the exception of histopathology. Drinking water was replaced with either filter-sterilized water (mock treatment) or with a filter-sterilized solution of 4% (w/v) dextran sulphate sodium (DSS; relative molecular mass 36,000–50,000; MP Biomedicals) in water as indicated. For DSS-treated mice, 3 days before the end of the experiment, drinking water was switched for 24 h to filter-sterilized water. Drinking water was then replaced with either filter-sterilized water (mock treatment) or with a filter-sterilized solution of 2% (w/v) DSS. At 4 days after the start of DSS treatment, animals were orally inoculated with a 1:1 ratio of 5 × 108 CFU of wild-type EcN and mutants in competition with cEc or AIEC strains (resuspended in 0.1 ml LB broth). Faecal material was collected each day after bacterial administration, resuspended in sterile PBS and the bacterial load was determined by plating serial tenfold dilutions on selective agar plates. Animals were euthanized 5 days after inoculation. Caecal tissue was collected and then flash-frozen in liquid nitrogen and stored at −80 °C for later isolation of mRNA and protein. The caecal tip was fixed in 10% formalin for histopathology. Faecal material and caecal content were collected in sterile PBS and the bacterial load for the E. coli strains was determined by plating serial tenfold dilutions on selective agar plates. At some time points, a mouse did not produce faecal material, hence an n value is displayed for a given time point that is less than the experiment’s indicated n value for that group. To differentiate EcN from other E. coli strains in biological samples, the strains were differentially marked with the low-copy number pACYomega (Cm) or pHP45omega (Carb) (Supplementary Table 4), and markers were swapped in some experiments. Plasmids were stably maintained throughout the infection and similar colonies were counted in other differential media such as on MacConkey agar (not shown). When noted, the ratio of two strains in the faeces and caecal content was calculated by dividing the output ratio (EcN CFU/cEc or AIEC CFU) by the input ratio (EcN CFU/cEc or AIEC CFU). The competitive index was calculated for isogenic strains by dividing the output ratio (EcN wild-type CFU:mutant CFU) by the input ratio (EcN wild-type CFU:mutant CFU). C57BL/6 Nramp1+ mice were treated with streptomycin (100 μl of a 200 mg ml−1 solution in sterile water) one day before infection. The following day, mice were orally inoculated with 1 × 109 CFU of STm (resuspended in 0.1 ml LB broth) or with a 1:1 ratio of 5 × 108 CFU each of STm and wild-type EcN, or EcN mchDEF strain for competitive colonization experiments. For therapeutic administration of EcN after STm infection, C57BL/6 Nramp1+ mice were treated with streptomycin (100 μl of a 200 mg ml−1 solution in sterile water) one day before STm infection. The following day, mice were orally inoculated with 1 × 109 CFU of STm. At days 2 and 5 after STm infection, mice were either mock-treated or treated with 1 × 109 CFU of wild-type EcN or EcN mchDEF. Faecal material was collected each day after bacterial administration and resuspended in sterile PBS. Bacterial load for STm and EcN strains was then determined by plating serial tenfold dilutions on selective agar plates. At some time points, a mouse did not produce faecal material, hence an n value is displayed for a given time point that is less than the experiment’s indicated n value for that group. At day 7 following infection, mice were euthanized and a portion of the caecum was flash-frozen in liquid nitrogen then stored at −80 °C for later isolation of mRNA and protein. The caecal tip was fixed in 10% formalin for histopathology. Bacteria in the faecal content were counted by plating serial tenfold dilutions on LB agar plates containing the appropriate antibiotics. To selectively identify STm from EcN strain in the faecal content, strains were differentially marked with the low-copy number pACYomega (Cm) or pHP45omega (Carb) plasmids. Plasmids were stably maintained throughout the infection and similar colonies were counted in other differential media such as MacConkey agar (not shown). When noted, the ratio of two strains was calculated by dividing the output ratio (EcN CFU/STm CFU) by the input ratio (EcN CFU/STm CFU). Faecal samples were collected before DSS treatment (day −4), at day 4 after DSS treatment (day 0; before inoculation of bacteria) and at day 5 after administration of commensal E. coli in competition with wild-type EcN or EcN mchDEF. Faeces were snap-frozen in liquid nitrogen, then DNA was later extracted using the QIAamp DNA Stool Kit (Qiagen) according to the manufacturer’s instructions with modifications as previously described18. Because DSS interferes with PCR, we used the following procedure (developed by W. Zhu and S. Winter) to eliminate DSS from samples. Samples were eluted in 200 μl of ultra-pure water and combined with 80 mg of KCl followed by vortexing for 2 min. The mixture was then incubated on ice for 30 min and centrifuged at 16,800g at 4 °C for 30 min. The supernatant was then transferred to a new tube and 1/10 volume of 3 M NaAc (pH 5.2) was added. We then added 2.5 ml of pure ethanol to the mixture, vortexed and centrifuged at 16,800g at 4 °C for 30 min. The supernatant was removed and 1 ml of 70% ethanol was added to the pellet, vortexed and centrifuged at 16,800g for 5 min at room temperature. This wash step was repeated 3–4 times. The pellet was then air-dried at 55 °C and resuspended in 30 μl of ultra-pure water. DNA extracted from faecal samples was amplified by a two-step PCR enrichment of 16S rDNA (V4 region) with primers 515F and 806R modified by addition of barcodes for multiplexing, then sequenced on an Illumina MiSeq system (UC Davis HMSB Facility). Sequences were processed and analysed by employing the QIIME pipeline v1.9.1 (ref. 28) with default settings, except as noted. In brief, paired-end sequences were joined, quality-filtered, reverse-complemented and chimaera-filtered (usearch61 option; RDP gold database); operational taxonomic units (OTUs) were picked (usearch61 option; enable_rev_strand_match True) at 97% similarity; taxonomy was assigned (confidence 0.8) with the RDP classifier. Greengenes database v13_8 was used in the open-reference OTU picking workflow. A bacterial RNA mini-kit (Bio-Rad Aurum Total) was used to extract RNA from bacterial cultures. EcN strains were grown in nutrient broth supplemented with 0.2 mM 2,2′-dipyridyl aerobically at 37 °C overnight. Approximately 104 CFU ml−1 from an overnight culture were inoculated into 5 ml of tissue culture medium (DMEM/F12 plus 10% FBS, Invitrogen), as previously described23, 24. When indicated, iron citrate was added to the medium at a final concentration of 1 μM. At 7 h post-inoculation, 2 × 109 CFU were used to extract RNA. An additional DNase treatment (Ambion) was done before the generation of cDNA with reverse transcription reagents (Roche). For analysis of gene expression by quantitative real-time PCR, total RNA was extracted from caecal tissue with TRI Reagent (Molecular Research Center). RNA from DSS-treated mice was further purified using the Dynabeads mRNA DIRECT Purification Kit (Life Technologies) according to manufacturer recommendations. Reverse-transcription reagents (Roche) were employed to generate cDNA from all RNA samples. Real-time PCR was performed using SYBR Green (Roche) and the Roche Lightcycler 480 Instrument II system (Roche). Data were analysed using the comparative method. Target gene transcription of each tissue sample was normalized to the respective levels of Actb mRNA (β-actin). For qPCR analysis of bacterial transcripts, transcription of mcmA and mchB was normalized to bacterial gapA mRNA levels. Data represent at least three independent experiments. DNA contamination was less than 1% for all bacterial amplicons, as determined by separate mock reactions lacking reverse transcriptase. All primers used are listed in Supplementary Table 6. Tissue samples were fixed in 10% formalin, processed according to standard procedures for paraffin embedding, sectioned at 5 mm, and stained with haematoxylin and eosin. The pathology score of caecal samples was determined by blinded examination of caecal sections from a board-certified pathologist using previously published methods18. Each section was evaluated for the presence of neutrophils, mononuclear infiltrate, submucosal oedema, surface erosions, inflammatory exudates, and cryptitis. Inflammatory changes were scored from 0 to 4 according to the following scale: 0, none; 1, low; 2, moderate; 3, high; 4, extreme. The inflammation score was calculated by adding up all scores obtained for each parameter and interpreted as follows: 0–2, within normal limit; 3–5, mild; 6–8, moderate; ≥8, severe. The experiments were not randomized. No statistical methods were used to predetermine sample size. Prism 6 and 7 software (GraphPad) was used for statistical analysis. Bacterial growth curves were analysed by unpaired Student’s t-test. To compare bacterial CFUs in faeces and caecal content, mRNA expression, and eubacterial taxa present at greater than 0.5% relative abundance in at least one sample, we applied a non-parametric Mann–Whitney–Wilcoxon test. We chose this test because it can be applied to data with normal or unknown distribution. P-values for all statistical comparisons presented in the main text, figures and Extended Data are available in Supplementary Table 7. The data that support the findings of this study are available from the corresponding author upon request. Faecal microbiota 16S rRNA gene sequencing data were deposited in the European Nucleotide Archive under accession PRJEB15700.


Kruis W.,University of Cologne | Chrubasik S.,Albert Ludwigs University of Freiburg | Boehm S.,University of Cologne | Stange C.,Ardeypharm GmbH | Schulze J.,Ardeypharm GmbH
International Journal of Colorectal Disease | Year: 2012

Purpose To study the therapeutic effects of probiotic Escherichia coli Nissle 1917 (EcN) in irritable bowel syndrome (IBS) and identify subgroups benefiting most. Background Some trials investigating therapeutic effects in irritable bowel syndrome have shown benefits in IBS subgroups only. Probiotic treatment seems to be promising. Methods Patients with irritable bowel syndrome (120; Rome II) were recruited to a prospective double-blind study and randomized to either EcN (n=60) or placebo (n= 60) given for 12 weeks. Objectives were to describe efficacy and safety of EcN in different groups of irritable bowel syndrome. Outcome was assessed by 'Integrative Medicine Patient Satisfaction Scale'. Results Altogether, the responder rate was higher in the EcN than in the placebo group. However, only after 10 and 11 weeks, the differences were significant (Δ 20.0% points [95% CI 2.6; 37.4], p=0.01 and Δ 18.3% points [95% CI 1.0; 35.7], p=0.02, respectively). The best response was observed in the subgroup of patients with gastroenteritis or antibiotics prior to irritable bowel syndrome onset (Δ 45.7% points, p=0.029). No significant differences were observed in any other subgroup. Both treatment groups showed similar adverse events and tolerance. Conclusions Probiotic EcN shows effects in irritable bowel syndrome, especially in patients with altered enteric microflora, e.g. after gastroenterocolitis or administration of antibiotics. © 2011 CARS.


Joeres-Nguyen-Xuan T.H.,Evangelisches Krankenhaus Kalk | Boehm S.K.,Evangelisches Krankenhaus Kalk | Joeres L.,Ardeypharm GmbH | Schulze J.,Ardeypharm GmbH | Kruis W.,Evangelisches Krankenhaus Kalk
Inflammatory Bowel Diseases | Year: 2010

Background: Mesalamine and the probiotic E. coli Nissle 1917 (EcN) are both effective agents for the treatment of ulcerative colitis. A combined therapy may have more than additive efficacy. However, mesalamine may have antimicrobial effects on EcN. Materials and Methods: In this prospective, randomized, double-blind, placebo-controlled study, 48 healthy volunteers took EcN in a run-in phase for 17 days (5-50 × 109 viable bacteria od). If stool samples became positive for EcN, volunteers received combination treatment with EcN plus either mesalamine (1500 mg twice a day) or placebo for 1 week. Fecal samples were further tested for EcN in 2- to 3-day intervals until a maximum of 48 weeks after treatment. Patient diaries, blood, and urine were checked to assess safety, compliance, and tolerance. Results: During run-in, viable EcN were detected in 45 of the 48 volunteers (94%); 2 volunteers were positive before taking EcN. From days 1 to 7 of combination treatment (n = 40), the number of EcN-positive volunteers varied between 70% and 80% in the mesalamine group and between 85% and 95% in the placebo group. Differences between the groups were not significant (normal approximation: day 3, P > 0.15; day 5, P > 0.25; day 7, P > 0.076). At treatment discontinuation, 16 of 20 volunteers in the mesalamine group and 15 of 20 volunteers in the placebo group were EcN positive, whereas this figure dropped continuously up to week 12 after discontinuation (mesalamine, 7 of 20; placebo, 4 of 20). No differences between the groups were seen with regard to tolerance and safety. Conclusions: The combination of EcN and mesalamine has no significant effect on the survival of EcN in healthy volunteers. Copyright © 2009 Crohn's & Colitis Foundation of America, Inc.


Rund S.A.,University of Würzburg | Rohde H.,University of Hamburg | Sonnenborn U.,Ardeypharm GmbH | Oelschlaeger T.A.,University of Würzburg
International Journal of Medical Microbiology | Year: 2013

The largest EHEC outbreak up to now in Germany occurred in 2011. It was caused by the non-O157:H7 Shiga-toxinogenic enterohemorrhagic E. coli strain O104:H4. This strain encodes in addition to the Shiga toxin 2 (Stx2), responsible for the hemolytic uremic syndrome (HUS), several adhesins such as aggregative adherence fimbriae. Currently, there is no effective prophylaxis and treatment available for EHEC infections in humans. Especially antibiotics are not indicated for treatment as they may induce Stx production, thus worsening the symptoms. Alternative therapeutics are therefore desperately needed. We tested the probiotic Escherichia coli strain Nissle 1917 (EcN) for antagonistic effects on two O104:H4 EHEC isolates from the 2011 outbreak and on the classical O157:H7 EHEC strain EDL933. These tests included effects on adherence, growth, and Stx production in monoculture and co-culture together with EcN. The inoculum of each co-culture contained EcN and the respective EHEC strain either at a ratio of 1:1 or 10:1 (EcN:EHEC). Adhesion of EHEC strains to Caco-2 cells and to the mucin-producing LS-174T cells was reduced significantly in co-culture with EcN at the 1:1 ratio and very dramatically at the 10:1 ratio. This inhibitory effect of EcN on EHEC adherence was most likely not due to occupation of adhesion sites on the epithelial cells, because in monocultures EcN adhered with much lower bacterial numbers than the EHEC strains. Both EHEC strains of serotype O104:H4 showed reduced growth in the presence of EcN (10:1 ratio). EHEC strain EDL933 grew in co-culture with EcN only during the first 2. h of incubation. Thereafter, EHEC counts declined. At 24. h, the numbers of viable EDL933 was at or slightly below the numbers at the time of inoculation. The amount of Stx2 after 24. h co-incubation with EcN (EcN:EHEC ratio 10:1) was for all 3 EHEC strains tested significantly reduced in comparison to EHEC monocultures.Obviously, EcN shows very efficient antagonistic activity on the EHEC strains of serotype O104:H4 and O157:H7 tested here regarding adherence to human gut epithelial cells, bacterial growth, and Stx2 production in vitro. © 2012 Elsevier GmbH.


Matthes H.,Community Hospital Havelhoehe | Wolff C.,Ardeypharm GmbH | Schulze J.,Ardeypharm GmbH
BMC Complementary and Alternative Medicine | Year: 2010

Background: Probiotics are effective in inflammatory bowel diseases. Clinical effectiveness and dose dependency of E. coli Nissle (EcN) enemas were investigated in ulcerative colitis (UC).Methods: In a double-blind study, 90 patients with moderate distal activity in UC were randomly assigned to treatment with either 40, 20, or 10 ml enemas (N = 24, 23, 23) containing 10E8 EcN/ml or placebo (N = 20). The study medication was taken once daily for at least 2 weeks. After 2, 4 and/or 8 weeks the clinical DAI was assessed together with tolerance to treatment. Patients who reached clinical DAI ≤ 2 within that time were regarded as responders.Results: According to ITT analysis the number of responders was not significantly higher in the EcN group than in the placebo group (p = 0.4430, 2-sided). However, the Jonckheere-Terpstra rank correlation for dose-dependent efficacy indicated a significant correlation of per-protocol responder rates (p = 0.0446, 2-sided). Time to remission was shortest with EcN 40 ml, followed by EcN 20 ml. The number of adverse events did not differ notably.Conclusion: In contrast to ITT analysis, efficacy of rectal EcN application was significant in PP and points to EcN as a well tolerated treatment alternative in moderate distal UC.Trial registration: German Clinical Trials Register DRK00000234. © 2010 Matthes et al; licensee BioMed Central Ltd.


Splichalova A.,Academy of Sciences of the Czech Republic | Splichal I.,Academy of Sciences of the Czech Republic | Sonnenborn U.,Ardeypharm GmbH | Rada V.,Czech University of Life Sciences
Journal of Microbiological Methods | Year: 2014

An agar selective enumeration of necrotoxigenic Escherichia coli O55 (NTEC2) and probiotic E. coli Nissle 1917, using modified MacConkey agar, was developed to study bacterial interference between these E. coli strains in a gnotobiotic piglet model. Replacement of lactose with saccharose in the agar enables the direct visual enumeration of red colonies of E. coli O55 and yellow colonies of E. coli Nissle 1917 that are co-cultured in the same Petri dish. A total of 336 colonies (168 for each color) were subjected to strain-specific PCR identification with LNA probes. Sensitivity, specificity, and positive and negative predictive values were 96.43%, 95.83%, 95.86% and 96.41% respectively in E. coli O55, and 98.21%, 97.02%, 97.06% and 98.19% respectively in E. coli Nissle 1917. Color-based enumeration of both E. coli strains in colonic contents and mesenteric lymph nodes homogenates of gnotobiotic piglets demonstrated the applicability of this method for the gnotobiotic piglet model of enteric diseases. © 2014 Elsevier B.V.


Splichalova A.,Academy of Sciences of the Czech Republic | Trebichavsky I.,Academy of Sciences of the Czech Republic | Rada V.,Czech University of Life Sciences | Vlkova E.,Czech University of Life Sciences | And 2 more authors.
Clinical and Experimental Immunology | Year: 2011

The colonization, translocation and protective effect of two intestinal bacteria - PR4 (pig commensal strain of Bifidobacterium choerinum) or EcN (probiotic Escherichia coli strain Nissle 1917) - against subsequent infection with a virulent LT2 strain of Salmonella enterica serovar Typhimurium were studied in gnotobiotic pigs after oral association. The clinical state of experimental animals correlated with bacterial translocation and levels of inflammatory cytokines [a chemokine, interleukin (IL)-8, a proinflammatory cytokine, tumour necrosis factor (TNF)-α and an anti-inflammatory cytokine, IL-10] in plasma and intestinal lavages. Gnotobiotic pigs orally mono-associated with either PR4 or EcN thrived, and bacteria were not found in their blood. No significant inflammatory cytokine response was observed. Mono-association with Salmonella caused devastating septicaemia characterized by high levels of IL-10 and TNF-α in plasma and TNF-α in the intestine. Di-associated gnotobiotic pigs were given PR4 or EcN for 24 h. Subsequently, they were infected orally with Salmonella and euthanized 24 h later. Pigs associated with bifidobacteria before Salmonella infection suffered from severe systemic infection and mounted similar cytokine responses as pigs infected with Salmonella alone. In contrast, EcN interfered with translocation of Salmonella into mesenteric lymph nodes and systemic circulation. Pigs pre-associated with EcN thrived and their clinical condition correlated with the absence of IL-10 in their plasma and a decrease of TNF-α in plasma and ileum. © 2010 The Authors. Clinical and Experimental Immunology © 2010 British Society for Immunology.


Sabharwal H.,University of Munster | Cichon C.,University of Munster | Olschlager T.A.,University of Würzburg | Sonnenborn U.,Ardeypharm GmbH | Alexander Schmidt M.,University of Munster
Infection and Immunity | Year: 2016

Bacterium-host interactions in the gut proceed via directly contacted epithelial cells, the host's immune system, and a plethora of bacterial factors. Here we characterized and compared exemplary cytokine and microRNA (miRNA) responses of human epithelial and THP-1 cells toward the prototype enteropathogenic Escherichia coli (EPEC) strain E2348/69 (O127:H6) and the probiotic strain Escherichia coli Nissle 1917 (EcN) (O6:K5:H1). Human T84 and THP-1 cells were used as cell culture-based model systems for epithelial and monocytic cells. Polarized T84 monolayers were infected apically or basolaterally. Bacterial challenges from the basolateral side resulted in more pronounced cytokine and miRNA responses than those observed for apical side infections. Interestingly, the probiotic EcN also caused a pronounced transcriptional increase of proinflammatory CXCL1 and interleukin- 8 (IL-8) levels when human T84 epithelial cells were infected from the basolateral side. miR-146a, which is known to regulate adaptor molecules in Toll-like receptor (TLR)/NF-κB signaling, was found to be differentially regulated in THP-1 cells between probiotic and pathogenic bacteria. To assess the roles of flagella and flagellin, we employed several flagellin mutants of EcN. EcN flagellin mutants induced reduced IL-8 as well as CXCL1 responses in T84 cells, suggesting that flagellin is an inducer of this cytokine response. Following infection with an EPEC type 3 secretion system (T3SS) mutant, we observed increased IL-8 and CXCL1 transcription in T84 and THP-1 cells compared to that in wild-type EPEC. This study emphasizes the differential induction of miR-146a by pathogenic and probiotic E. coli strains in epithelial and immune cells as well as a loss of probiotic properties in EcN interacting with cells from the basolateral side. © 2016, American Society for Microbiology.


Hummel S.,University of Munster | Veltman K.,University of Munster | Cichon C.,University of Munster | Sonnenborn U.,Ardeypharm GmbH | Schmidt M.A.,University of Munster
Applied and Environmental Microbiology | Year: 2012

The intestinal ecosystem is balanced by dynamic interactions between resident and incoming microbes, the gastrointestinal barrier, and the mucosal immune system. However, in the context of inflammatory bowel diseases (IBD), where the integrity of the gastrointestinal barrier is compromised, resident microbes contribute to the development and perpetuation of inflammation and disease. Probiotic bacteria have been shown to exert beneficial effects, e.g., enhancing epithelial barrier integrity. However, the mechanisms underlying these beneficial effects are only poorly understood. Here, we comparatively investigated the effects of four probiotic lactobacilli, namely, Lactobacillus acidophilus, L. fermentum, L. gasseri, and L. rhamnosus, in a T84 cell epithelial barrier model. Results of DNA microarray experiments indicating that lactobacilli modulate the regulation of genes encoding in particular adherence junction proteins such as E-cadherin and β-catenin were confirmed by quantitative reverse transcription-PCR (qRT-PCR). Furthermore, we show that epithelial barrier function is modulated by Gram-positive probiotic lactobacilli via their effect on adherence junction protein expression and complex formation. In addition, incubation with lactobacilli differentially influences the phosphorylation of adherence junction proteins and the abundance of protein kinase C (PKC) isoforms such as PKCδ that thereby positively modulates epithelial barrier function. Further insight into the underlying molecular mechanisms triggered by these probiotics might also foster the development of novel strategies for the treatment of gastrointestinal diseases (e.g., IBD). © 2012, American Society for Microbiology.


Veltman K.,University of Munster | Hummel S.,University of Munster | Cichon C.,University of Munster | Sonnenborn U.,Ardeypharm GmbH | Schmidt M.A.,University of Munster
International Journal of Biochemistry and Cell Biology | Year: 2012

In the intestine, dysregulation of miRNA is associated with inflammation, disruption of the gastrointestinal barrier, and the onset of gastrointestinal disorders. This study identifies miRNAs involved in the maintenance of intercellular junctions and barrier integrity. For the functional identification of barrier affecting miRNAs, we took advantage of the barrier-enforcing effects of the probiotic bacterium Escherichia coli Nissle 1917 (EcN) which can be monitored by enhanced transepithelial resistance (TER). miRNA-profiling of T84 monolayers prior and after co-incubation with EcN revealed for the first time differentially regulated miRNAs (miR-203, miR-483-3p, miR-595) targeting tight junction (TJ) proteins. Using real-time PCR, Western blotting and specific miRNA mimics, we showed that these miRNAs are involved in the regulation of barrier function by modulating the expression of regulatory and structural components of tight junctional complexes. Furthermore, specific inhibitors directed at these miRNA abrogated the disturbance of tight junctions induced by enteropathogenic E. coli (EPEC). The half-maximal inhibitory concentration (IC 50) was determined to 340 nM by monitoring inhibitor kinetics. In summary, we conclude that specific miRNAs effect regulatory as well as structural proteins of the junctional complex which in turn are involved in the barrier enhancing effect of EcN. Hence, we suggest that the application of miRNAs might be refined and further developed as a novel supportive strategy for the treatment of gastrointestinal disorders. © 2011 Elsevier Ltd. All rights reserved.

Loading Ardeypharm GmbH collaborators
Loading Ardeypharm GmbH collaborators