Anti-genotoxic and anti-mutagenic activity of Escherichia coli Nissle 1917 as assessed by in vitro tests.

Anti-genotoxic or anti-mutagenic activity has been described for a number of Gram-positive probiotic bacterial species. Here we present evidence that Gram-negative Escherichia coli Nissle 1917 (EcN) also displays anti-genotoxic/anti-mutagenic activity, as assessed in vitro by the Comet Assay and the Ames Test, respectively. This activity was demonstrated by use of the mutagens 4-nitroquinoline-1-oxide (NQO), hydrogen peroxide (H2O2) and benzo(a) pyrene (B[a]P). For both assays and all three test agents the anti-genotoxic/anti-mutagenic activity of EcN was shown to be concentration dependent. By the use of extracts of bacteria that were inactivated by various procedures (heat treatment, ultrasound sonication or ultraviolet light irradiation), mechanistic explanations could be put forward. The proposed mechanisms were enforced by treating the bacterial material with proteinase K prior to testing. The mutagen H2O2 is most likely inactivated by enzymic activity, with catalase a likely candidate, while several explanations can be put forward for inactivation of B[a]P. NQO is most likely inactivated by metabolising enzymes, since the formation of the metabolite 4-aminoquinoline could be demonstrated. In conclusion, the in vitro results presented here make a strong case for antimutagenic properties of EcN.

Interference of Bifidobacterium choerinum or Escherichia coli Nissle 1917 with Salmonella Typhimurium in gnotobiotic piglets correlates with cytokine patterns in blood and intestine.

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.

Cell-free supernatants of Escherichia coli Nissle 1917 modulate human colonic motility: evidence from an in vitro organ bath study.

Abstract Clinical studies have shown that probiotics influence gastrointestinal motility, e.g. Escherichia coli Nissle 1917 (EcN) (Mutaflor) proved to be at least as efficacious as lactulose and more potent than placebo in constipated patients. As the underlying mechanisms are not clarified, the effects of EcN culture supernatants on human colonic motility were assessed in vitro. Human colonic circular smooth muscle strips (n = 94, 17 patients) were isometrically examined in an organ bath and exposed to different concentrations of EcN supernatants. Contractility responses were recorded under (i) native conditions, (ii) electrical field stimulation (EFS), (iii) non-adrenergic non-cholinergic conditions, and (iv) enteric nerve blockade by tetrodotoxin (TTX). As concentrations of acetic acid were increased in EcN supernatants, contractility responses to acetic acid were additionally tested. EcN supernatants significantly increased the maximal tension forces both at low and high concentrations. Neither blockade of both adrenergic and cholinergic nerves nor application of TTX abolished these effects. EFS-induced contractility responses were not altered after exposure to EcN supernatants. Acetic acid elicited effects comparable to EcN supernatants only under TTX conditions. EcN supernatants modulate in vitro contractility of the human colon. As neither partial nor TTX blockade of enteric nerves abolished these effects, EcN supernatants appear to enhance colonic contractility by direct stimulation of smooth muscle cells. Active metabolites may include other substances than acetic acid, as acetic acid only partially resembled the effects elicited by EcN supernatants. The data provide a rationale for therapeutical application of probiotics in gastrointestinal motility disorders.

Escherichia coli strain Nissle 1917 ameliorates experimental colitis via toll-like receptor 2- and toll-like receptor 4-dependent pathways.

Toll-like receptors (TLRs) are key components of the innate immune system that trigger antimicrobial host defense responses. The aim of the present study was to analyze the effects of probiotic Escherichia coli Nissle strain 1917 in experimental colitis induced in TLR-2 and TLR-4 knockout mice. Colitis was induced in wild-type (wt), TLR-2 knockout, and TLR-4 knockout mice via administration of 5% dextran sodium sulfate (DSS). Mice were treated with either 0.9% NaCl or 10(7) E. coli Nissle 1917 twice daily, followed by the determination of disease activity, mucosal damage, and cytokine secretion. wt and TLR-2 knockout mice exposed to DSS developed acute colitis, whereas TLR-4 knockout mice developed significantly less inflammation. In wt mice, but not TLR-2 or TLR-4 knockout mice, E. coli Nissle 1917 ameliorated colitis and decreased proinflammatory cytokine secretion. In TLR-2 knockout mice a selective reduction of gamma interferon secretion was observed after E. coli Nissle 1917 treatment. In TLR-4 knockout mice, cytokine secretion was almost undetectable and not modulated by E. coli Nissle 1917, indicating that TLR-4 knockout mice do not develop colitis similar to the wt mice. Coculture of E. coli Nissle 1917 and human T cells increased TLR-2 and TLR-4 protein expression in T cells and increased NF-kappaB activity via TLR-2 and TLR-4. In conclusion, our data provide evidence that E. coli Nissle 1917 ameliorates experimental induced colitis in mice via TLR-2- and TLR-4-dependent pathways.

Effect of E. coli Nissle 1917 on post-inflammatory visceral sensory function in a rat model.

OBJECTIVE: Visceral hyperalgesia (VH) plays a key role for the manifestation of functional gastrointestinal (GI) disorders. In a subgroup of patients, the initial manifestation is preceded by GI inflammation. Recent studies have demonstrated an improvement of inflammation and symptoms during treatment with Escherichia coli Nissle 1917 (EcN). AIM: We aimed to characterize the effects of EcN on visceral sensitivity in a rat model of post-inflammatory VH. METHODS: Male Lewis rats underwent colorectal instillation of trinitrobenzenesulphonic acid (TNBS) plus an equal amount of ethanol (test group) or physiological saline solution (control group). After 28, 35 and 42 days, standardized colorectal distensions were performed and the visceromotor reflex (VMR) of abdominal wall muscles was quantified by electromyographic recording. From day 28 onwards, EcN was administered in drinking water. RESULTS: After TNBS, a significant increase of VMR was observed compared with saline controls over all study days. Administration of EcN reduced the TNBS-induced hyperalgesia [EcN: 863+/-125 microV vs placebo: 1258+/-157 microV (P<0.05)] at day 35, while there were no significant alterations at any other study day. CONCLUSION: The EcN administration caused a significant reduction of VH. Whether EcN might play a role in the treatment of post-infectious functional bowel disorders remains to be investigated in further studies.

Specific proliferative and antibody responses of premature infants to intestinal colonization with nonpathogenic probiotic E. coli strain Nissle 1917.

The aim of this study was to analyze the influence of oral administration of E. coli Nissle 1917 on the systemic humoral and cellular immunity in premature infants. Thirty-four premature infants were colonized with E. coli Nissle 1917 in a randomized, placebo-controlled blinded clinical trial. Stool samples of infants were analyzed repeatedly for the presence of the administered strain. The proliferative response to bacterial antigens of E. coli origin was measured in whole blood of 34 colonized infants and 27 noncolonized controls. E. coli colonization induced a significant increase in the proliferation of blood cells cultivated with bacterial components of E. coli Nissle 1917 and another E. coli strain in colonized infants as compared with noncolonized controls. Significantly higher amounts of specific anti-E. coli Nissle 1917 antibodies (Ab) of immunoglobulin (Ig)A isotype and nonspecific polyclonal IgM were found in the blood of colonized infants compared to noncolonized placebo controls. We concluded that the oral application of E. coli Nissle 1917 after birth significantly stimulates specific humoral and cellular responses and simultaneously induces nonspecific natural immunity.

High resolution crystal structures of T4 phage beta-glucosyltransferase: induced fit and effect of substrate and metal binding.

beta-Glucosyltransferase (BGT) is a DNA-modifying enzyme encoded by bacteriophage T4 that transfers glucose from uridine diphosphoglucose to 5-hydroxymethyl cytosine bases of phage T4 DNA. We report six X-ray structures of the substrate-free and the UDP-bound enzyme. Four also contain metal ions which activate the enzyme, including Mg(2+) in forms 1 and 2 and Mn(2+) or Ca(2+). The substrate-free BGT structure differs by a domain movement from one previously determined in another space group. Further domain movements are seen in the complex with UDP and the four UDP-metal complexes. Mg(2+), Mn(2+) and Ca(2+) bind near the beta-phosphate of the nucleotide, but they occupy slightly different positions and have different ligands depending on the metal and the crystal form. Whilst the metal site observed in these complexes with the product UDP is not compatible with a role in activating glucose transfer, it approximates the position of the positive charge in the oxocarbonium ion thought to form on the glucose moiety of the substrate during catalysis.

T4 phage beta-glucosyltransferase: substrate binding and proposed catalytic mechanism.

beta-Glucosyltransferase (BGT) is a DNA-modifying enzyme encoded by bacteriophage T4 which catalyses the transfer of glucose (Glc) from uridine diphosphoglucose (UDP-Glc) to 5-hydroxymethylcytosine (5-HMC) in double-stranded DNA. The glucosylation of T4 phage DNA is part of a phage DNA protection system aimed at host nucleases. We previously reported the first three-dimensional structure of BGT determined from crystals grown in ammonium sulphate containing UDP-Glc. In this previous structure, we did not observe electron density for the Glc moiety of UDP-Glc nor for two large surface loop regions (residues 68-76 and 109-122). Here we report two further BGT co-crystal structures, in the presence of UDP product (form I) and donor substrate UDP-Glc (form II), respectively. Form I crystals are grown in ammonium sulphate and the structure has been determined to 1.88 A resolution (R -factor 19.1 %). Form II crystals are grown in polyethyleneglycol 4000 and the structure has been solved to 2.3 A resolution (R -factor 19.8 %). The form I structure is isomorphous to our previous BGT UDP-Glc structure. The form II structure, however, has allowed us to model the two missing surface loop regions and thus provides the first complete structural description of BGT. In this low-salt crystal form, we see no electron density for the Glc moiety from UDP-Glc similar to previous observations. Biochemical data however, shows that BGT can cleave UDP-Glc in the absence of DNA acceptor, which probably accounts for the absence of Glc in our UDP-Glc substrate structures. The complete BGT structure now provides a basis for detailed modelling of a BGT HMC-DNA ternary complex. By using the structural similarity between the catalytic core of glycogen phosphorylase (GP) and BGT, we have modelled the position of the Glc moiety in UDP-Glc. From these two models, we propose a catalytic mechanism for BGT and identify residues involved in both DNA binding and in stabilizing a "flipped-out" 5-HMC nucleotide.

Probiotics and E. coli infections in man.

After oral administration of live oral vaccines COLINFANT and MUTAFLOR prepared from non-enteropathogenic E. coli strains, both strains colonized effectively the intestine in full-term and preterm infants and remained for many weeks showing, that they were capable to establish themselves as a resident strain in the infant's gut. The presence of E. coli stimulated significantly antibody production in gut, saliva and serum of colonized infants. An early induction of secretory IgA production is important particularly in formula-fed infants, where it partly replaces the lacking immunoglobulin supplied with mother milk. In full-term and premature infants the early presence of non-pathogenic E. coli strains in the intestine decreased significantly the presence of pathogenic bacterial strains in the intestine but also other mucosal surfaces of the body. The COLINFANT strain decreased the number of nosocomial infections, mortality rate in connection with infection, and the need for antibiotics. Both strains replaced successfully pathogenic strains in carriers after treatment with antibiotics.

Effect of preventive administration of a nonpathogenic Escherichia coli strain on the colonization of the intestine with microbial pathogens in newborn infants.

In a randomized, double-blind study, 27 healthy newborn infants were colonized with the nonpathogenic Escherichia coli strain Nissle 1917 (E. coli DSM 6601, Mutaflor) during the first 5 days of life by daily oral inoculation of 1 ml of a suspension with 10(8) living cells. A second group of 27 newborns, used as controls, received a placebo suspension (1 ml of phosphate-buffered saline) instead. Stool samples were taken on days 1, 2, 3, 5, and 21, and 6 months after birth. All samples were examined for the presence of the nonpathogenic E. coli strain and of pathogenic and potentially pathogenic microorganisms. The administered E: coli strain was detected in the stools of the colonized newborns from day 2 and remained present throughout the study in more than 90% of these infants. Colonization with true and potential bacterial pathogens was significantly reduced in infants receiving E. coli strain Nissle 1917 compared to the placebo group--both with respect to numbers of pathogens and to the spectrum of species.

[The development of an aerobic intestinal microflora in newborn infants]. / Die Entwicklung der aeroben Darmflora bei Neugeborenen.

The microbial colonization of the intestinal tract was studied in 60 newborn babies from three hospitals, with special regard to the typical hospital "problem" strains. Escherichia coli was the predominant initial colonizing species. Opportunistic pathogens were repeatedly detected. Some 30% of E. coli strains showed beta-hemolysis. Distinct differences between the three clinics were noted with respect to the detection frequency of the species isolated, hemolytic bacteria, the predominating E. coli strains, the occurrence of Candida albicans, and the time course of colonization of the gut by E coli. The microbial environment of the respective neonatal unit has a great influence on the initial intestinal colonization by aerobic microorganisms.

Regulation of steroidogenesis in rat adrenal gland: identification of the bifunctional, hormone-sensitive cholesterol esterase--triacylglycerol lipase enzyme protein and its discrimination from hormone-insensitive lipases.

The activities of hormone-sensitive cholesterol esterase and hormone-sensitive triacylglycerol lipase from rat adrenal glands were enhanced about 2-fold by means of ether stress and showed parallel elution profiles on a Sepharose CL-6B column. Both enzymatic activities were inhibited to a similar extent by DFP after separation from hormone-insensitive lipase on heparin-Sepharose. Fractions from the gel filtration column containing the two hormone-sensitive enzymes showed incorporation of tritium-labelled DFP into only one polypeptide of Mr 84 000. From these results we conclude that both hormone-sensitive activities reside on one polypeptide of Mr 84 000, thus providing further support to the concept that the different hormone-sensitive acylester hydrolase activities in steroid-secreting tissues as well as in adipose tissue are performed by the same bifunctional enzyme. In addition to the hormone-sensitive enzyme, rat adrenals contained high amounts of neutral triacylglycerol lipase activity which was not affected by stress. The latter enzyme was resistant to high salt concentrations, was less susceptible to inhibition by DFP, but could be inhibited completely by the addition of antibodies raised against rat liver lipase, thus most probably representing the adrenal liver lipase-like triacylglycerol lipase.

Purification and properties of the fatty acid synthetase complex from the marine dinoflagellate, Crypthecodinium cohnii.

De novo biosynthesis of fatty acids in the heterotrophic marine dinoflagellate, Crypthecodinium cohnii, has been studied in vitro. Fatty acid synthetase was located in the cytosol and its activity was dependent on acetyl-CoA, malonyl-CoA, NADPH2 and NADH2. The enzyme was purified 100-fold using ion-exchange chromatography on DEAE-Sephadex A-25, adsorption to hydroxyapatite and gel filtration on Sepharose 4B columns. Very active endogenous proteases were separated from the fatty acid synthetase at the first step of purification. The purified enzyme had a molecular weight of about 400000, as judged from gel filtration, sucrose density gradient centrifugation and polyacrylamide gel electrophoresis under non-denaturing conditions. Polyacrylamide gel electrophoresis under denaturing conditions in the presence of SDS and urea revealed one major protein band of Mr 180000, suggesting that the enzyme is composed of two multifunctional subunits of apparently identical molecular weight. Reaction products of the C. cohnii fatty acid synthetase are free fatty acids due to the presence of a thioesterase activity in the purified enzyme complex. The main product is palmitate. Docosahexaenoic acid (C22:6, n-3), the major fatty acid component of C. cohnii lipids, is not directly synthesized by the enzyme.