Strain Nissle Research Articles

The Carbon Storage Regulator (Csr) System Exerts a Nutrient-Specific Control over Central Metabolism in Escherichia coli Strain Nissle 1917

The role of the post-transcriptional carbon storage regulator (Csr) system in nutrient utilization and in the control of the central metabolism in E. coli reference commensal strain Nissle 1917 was investigated. Analysis of the growth capabilities of mutants altered for various components of the Csr system (csrA51, csrB, csrC and csrD mutations) showed that only the protein CsrA - the key component of the system - exerts a marked role in carbon nutrition. Attenuation of CsrA activity in the csrA51 mutant affects the growth efficiency on a broad range of physiologically relevant carbon sources, including compounds utilized by the Entner-Doudoroff (ED) pathway. Detailed investigations of the metabolomes and fluxomes of mutants and wild-type cells grown on carbon sources representative of glycolysis and of the ED pathway (glucose and gluconate, respectively), revealed significant re-adjusting of central carbon metabolism for both compounds in the csrA51 mutant. However, the metabolic re-adjusting observed on gluconate was strikingly different from that observed on glucose, indicating a nutrient-specific control of metabolism by the Csr system.

Tu1971 Probiotic Escherichia coli Strain Nissle 1917 Binds to Gluconat of Mucin2/ Human Mucus via Its Flagellum

Probiotic E. coli strain Nissle 1917 (EcN), serotype O6:K5:H1, is licensed as a drug for treatment of various diseases of the digestive tract in several countries. Some components of this strain have been identified to be important for its probiotic properties. These include two microcins, the K5 capsule and it's flagellum. The later is not only an organelle for motility but also the inducer of human beta-defensin 2 (hBD2) expression. In contrast to the monotrichously flagellated EcN-WT a peritrichous variant of EcN (EcNATHF) was an even more efficient hBD2 inducer. During screening of EcN mutants for reduced adherence to cryosections of human colon biopsies we found the unflagellated EcN ΔfliC mutant to exhibit strongly reduced adherence. Adherence was restored in the complemented EcN ΔfliC strain and EcNATHF showed even stronger adherence than EcN-WT. However, the number of bacteria adhering to Caco-2 or T24 cells was highest for EcN ΔfliC, followed by EcN-WT and the complemented EcNΔfliC and lowest for EcNATHF. Presence or absence of type 1, F1C or curli pili neither affected adherence of EcN to cryosections nor to Caco-2 or T24 cells. The obvious difference between the cryosections and the two cell lines employed was the presence of mucus in the cryosections. These findings indicated binding of EcN to human mucus via its flagellum. This hypothesis could be verified by (i) demonstrating flagellum-dependent adherence of EcN to the mucin producing human gut epithelial cell line LS174T, porcine mucin2 and isolated human mucus, (ii) a dose dependent reduction of EcN adherence to cryosections, mucin2 and human mucus after preincubation of EcN with mucin2 or isolated human mucus, (iii) binding of isolated EcN flagella to mucin2or human mucus-coated wells. In order to identify the mucus component to which EcN's flagellum binds, EcN-flagella were preincubated with carbohydrates known to be constituents of mucus. Only gluconate reduced binding of the pretreated flagella significantly in a dose dependent manner. Similarly, gluconate highly significantly inhibited adherence of EcNWT and EcNATHF to mucin2 as well as to the cryosections. Finally, attempts were made to identify the FliC domain, responsible for binding to mucin2/human mucus. Domain D3 of FliC is the most surface exposed one. A D3 deletion mutant of EcN was no longer motile but expressed a flagellum. Isolated flagella of this mutant bound about tenfold more efficient to mucin2/human mucus compared to WT-flagella. Obviously, domain D3 is not necessary for binding. Binding of the D3-less flagella to mucin2/human mucus was also inhibitable by gluconate. Probably, domain D2 of FliC is mediating binding of FliC to mucus, because this domain should be more accessible in the D3-less flagella. Construction of a D2und D2/D3-less FliC expressing EcN mutant is in progress.

β-Carotene Biosynthesis in Probiotic Bacteria.

Susceptibility to deadly diarrheal diseases is partly due to widespread pediatric vitamin A deficiency. To increase vitamin A coverage in malnourished children, we propose to engineer a probiotic bacterium that will produce β-carotene in the intestine, which will be metabolized to vitamin A. Such a therapy has the potential to broadly stimulate mucosal immunity and simultaneously reduce the incidence and duration of diarrheal disease. To that end, a β-carotene-producing variant of the probiotic Escherichia coli strain Nissle 1917 (EcN-BETA) was generated. Notably, the strain produces β-carotene under anaerobic conditions, reflective of the gut environment. EcN-BETA also retains β-carotene production capability after lyophilization, suggesting that it may be amenable to dry formulation. Moreover, EcN-BETA activates murine dendritic cells in vitro, suggesting that the presence of β-carotene may not diminish the immunostimulatory capacity of EcN. Finally, we present a framework through which further improvements may enable approaches such as the one described in this report to yield innovative life-saving therapies for the developing world.

Antagonistic effects of probiotic Escherichia coli Nissle 1917 on EHEC strains of serotype O104:H4 and O157:H7

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 2h of incubation. Thereafter, EHEC counts declined. At 24h, the numbers of viable EDL933 was at or slightly below the numbers at the time of inoculation. The amount of Stx2 after 24h 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.

More than a marine propeller – the flagellum of the probiotic Escherichia coli strain Nissle 1917 is the major adhesin mediating binding to human mucus

The flagellum of the probiotic Escherichia coli strain Nissle 1917 (EcN) is not just responsible for motility, but also for EcN's ability to induce the production of human β-defensin 2. Here, we report a third function of this EcN organell. In this study we investigated the role of the EcN flagellum in adhesion to different host tissues by ex vivo and in vitro studies. Ex vivo studies with cryosections of human gut biopsies revealed that the flagellum of EcN is most likely important for efficient adhesion to the human intestinal tract. These results and in vitro studies with different epithelial cells indicated that the presence of mucus is important for efficient mediation of adhesion by the flagellum of EcN. We observed direct interaction between isolated flagella from EcN wild type and porcine mucin 2 as well as human mucus. However, we could not observe any interaction of the flagella with murine mucus. For the first time, we identified the mucus component gluconate as one receptor for the binding of flagella from EcN and were able to exclude the flagellin domain D3 as a responsible interaction partner. We propose that the flagellum of EcN is its major adhesin in vivo, which enables this probiotic strain to compete efficiently for binding sites on host tissue with several bacterial pathogens.

Construction of recombinant E. coli Nissle 1917 (EcN) strains for the expression and secretion of defensins

The probiotic Escherichia coli strain Nissle 1917 (EcN) is one of the few probiotics licensed as a medication in several countries. Best documented is its effectiveness in keeping patients suffering from ulcerative colitis (UC) in remission. This might be due to its ability to induce the production of human β-defensin 2 (HBD2) in a flagellin-dependent way in intestinal epithelial cells. In contrast to ulcerative colitis, for Crohn's disease (CD) convincing evidence is lacking that EcN might be clinically effective, most likely due to the genetically based inability of sufficient defensin production in CD patients.As a first step in the development of an alternative approach for the treatment of CD patients, EcN strains were constructed which were able to produce human α-defensin 5 (HD5) or β-defensin 2 (HBD2). For that purpose, codon-optimized defensin genes encoding either the proform with the signal sequence of human α-defensin 5 (HD5) or the gene encoding HBD2 with or without the signal sequence were cloned in an expression vector plasmid under the control of the T7 promoter. Synthesis of the encoded defensins was shown by Western blots after induction of expression and lysis of the recombinant EcN strains. Recombinant mature HBD2 with an N-terminal His-tag could be purified by Ni-column chromatography and showed antimicrobial activity against E. coli, Salmonella enterica serovar Typhimurium and Listeria monocytogenes. In a second approach, that part of the HBD2 gene which encodes mature HBD2 was fused with the yebF gene. The resulting fusion protein YebFMHBD2 was secreted from the encoding EcN mutant strain after induction of expression. Presence of YebFMHBD2 in the medium was not the result of leakage from the bacterial cells, as demonstrated in the spent culture supernatant by Western blots specific for β-galactosidase and maltose-binding protein. The dialyzed and concentrated culture supernatant inhibited the growth of E. coli, S. enterica serovar Typhimurium and L. monocytogenes in radial diffusion assays as well as in liquid culture. This demonstrates EcN to be a suitable probiotic E. coli strain for the production of certain defensins.

Experimental Administration of the Probiotic Escherichia coli Strain Nissle 1917 Results in Decreased Diversity of E. coli Strains in Pigs

The strain Escherichia coli Nissle 1917 (EcN) is widely used as an efficient probiotic in therapy and prevention of human infectious diseases, especially of the intestinal system. Concurrently, small adult pigs are being used as experimental omnivore models to study human gastrointestinal functions. EcN bacteria were applied to 6 adult healthy female pigs in a 2-week trial. 6 Control animals remained untreated. Altogether, 164 and 149 bacterial strains were isolated from smear samples taken from gastrointestinal mucosa in the experimental and control group, respectively. Each individual E. coli strain was then tested for the presence of 29 bacteriocin-encoding determinants as well as for DNA markers of A, B1, B2 and D phylogenetic groups. A profound reduction of E. coli genetic variance (from 32 variants to 13 ones, P=0.0006) was found in the experimental group, accompanied by a lower incidence of bacteriocin producers in the experimental group when compared to control (21.3 and 34.9%, respectively; P=0.007) and by changes in the incidence of individual bacteriocin types. The experimental administration of EcN strain was not sufficient for stable colonization of porcine gut, but induced significant changes in the enterobacterial microbiota.

E. coli Nissle 1917 Affects Salmonella Adhesion to Porcine Intestinal Epithelial Cells

BackgroundThe probiotic Escherichia coli strain Nissle 1917 (EcN) has been shown to interfere in a human in vitro model with the invasion of several bacterial pathogens into epithelial cells, but the underlying molecular mechanisms are not known.Methodology/Principal FindingsIn this study, we investigated the inhibitory effects of EcN on Salmonella Typhimurium invasion of porcine intestinal epithelial cells, focusing on EcN effects on the various stages of Salmonella infection including intracellular and extracellular Salmonella growth rates, virulence gene regulation, and adhesion. We show that EcN affects the initial Salmonella invasion steps by modulating Salmonella virulence gene regulation and Salmonella SiiE-mediated adhesion, but not extra- and intracellular Salmonella growth. However, the inhibitory activity of EcN against Salmonella invasion always correlated with EcN adhesion capacities. EcN mutants defective in the expression of F1C fimbriae and flagellae were less adherent and less inhibitory toward Salmonella invasion. Another E. coli strain expressing F1C fimbriae was also adherent to IPEC-J2 cells, and was similarly inhibitory against Salmonella invasion like EcN.ConclusionsWe propose that EcN affects Salmonella adhesion through secretory components. This mechanism appears to be common to many E. coli strains, with strong adherence being a prerequisite for an effective reduction of SiiE-mediated Salmonella adhesion.

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.

Testing probiotic strain Escherichia coli Nissle 1917 (Mutaflor) for its ability to reduce carriage of multidrug-resistant E. coli by elderly residents in long-term care facilities

A high carriage rate of multidrug-resistant Escherichia coli (MDREC) was observed in elderly residents in long-term care facilities. A double-blinded, placebo-controlled trial was carried out to determine whether the probiotic product E. coli strain Nissle 1917 (Mutaflor) would compete with MDREC in the bowel and thereby reduce the prevalence of the multiresistant bacteria in faeces and urine. Sixty-nine patients excreting norfloxacin-resistant E. coli were randomized to probiotic or placebo groups and administered capsules twice daily. The daily dose of probiotic was 5×10(9)-5×10(10) bacteria. Faecal and urine samples were cultured at baseline and during and after the treatment period. A reduction in baseline carriage was not influenced by probiotic administration. The probiotic strain was detected in faecal specimens collected during the treatment period of only two out of 12 probiotic group subjects that were tested. Genotyping of norfloxacin-resistant E. coli isolates showed that 32 strains were prevalent among the patients. Thus, E. coli Nissle 1917 does not have the capacity to compete effectively with MDREC in the bowel of elderly patients.

Modulation of natural immunity in the gut by Escherichia coli strain Nissle 1917

The beneficial effect of probiotic Escherichia coli strain Nissle 1917 (EcN) suggests the gut epithelium plays a basic role in immune interactions with bacteria. Contrary to other commensal strains of Escherichia coli, EcN profoundly modulates the gut barrier to elevate its resistance to microbial pathogens. The present review documents the properties of EcN that have led to the protection of gnotobiotic pigs against lethal enteric infections. This effect could be important in light of the growing number of acquired deficiencies that paralyze gut immunity in humans.

Genome dynamics and its impact on evolution of Escherichia coli

The Escherichia coli genome consists of a conserved part, the so-called core genome, which encodes essential cellular functions and of a flexible, strain-specific part. Genes that belong to the flexible genome code for factors involved in bacterial fitness and adaptation to different environments. Adaptation includes increase in fitness and colonization capacity. Pathogenic as well as non-pathogenic bacteria carry mobile and accessory genetic elements such as plasmids, bacteriophages, genomic islands and others, which code for functions required for proper adaptation. Escherichia coli is a very good example to study the interdependency of genome architecture and lifestyle of bacteria. Thus, these species include pathogenic variants as well as commensal bacteria adapted to different host organisms. In Escherichia coli, various genetic elements encode for pathogenicity factors as well as factors, which increase the fitness of non-pathogenic bacteria. The processes of genome dynamics, such as gene transfer, genome reduction, rearrangements as well as point mutations contribute to the adaptation of the bacteria into particular environments. Using Escherichia coli model organisms, such as uropathogenic strain 536 or commensal strain Nissle 1917, we studied mechanisms of genome dynamics and discuss these processes in the light of the evolution of microbes.

Safety of ProbioticEscherichia coliStrain Nissle 1917 Depends on Intestinal Microbiota and Adaptive Immunity of the Host

Probiotics are viable microorganisms that are increasingly used for treatment of a variety of diseases. Occasionally, however, probiotics may have adverse clinical effects, including septicemia. Here we examined the role of the intestinal microbiota and the adaptive immune system in preventing translocation of probiotics (e.g., Escherichia coli Nissle). We challenged C57BL/6J mice raised under germfree conditions (GF-raised C57BL/6J mice) and Rag1(-/-) mice raised under germfree conditions (GF-raised Rag1(-/-) mice) and under specific-pathogen-free conditions (SPF-raised Rag1(-/-) mice) with probiotic E. coli strain Nissle 1917, strain Nissle 1917 mutants, the commensal strain E. coli mpk, or Bacteroides vulgatus mpk. Additionally, we reconstituted Rag1(-/-) mice with CD4(+) T cells. E. coli translocation and dissemination and the mortality of mice were assessed. In GF-raised Rag1(-/-) mice, but not in SPF-raised Rag1(-/-) mice or GF-raised C57BL/6J mice, oral challenge with E. coli strain Nissle 1917, but not oral challenge with E. coli mpk, resulted in translocation and dissemination. The mortality rate was significantly higher for E. coli strain Nissle 1917-challenged GF-raised Rag1(-/-) mice (100%; P < 0.001) than for E. coli strain Nissle 1917-challenged SPF-raised Rag1(-/-) mice (0%) and GF-raised C57BL/6J mice (0%). Translocation of and mortality due to strain E. coli Nissle 1917 in GF-raised Rag1(-/-) mice were prevented when mice were reconstituted with T cells prior to strain E. coli Nissle 1917 challenge, but not when mice were reconstituted with T cells after E. coli strain Nissle 1917 challenge. Cocolonization experiments revealed that E. coli mpk could not prevent translocation of strain E. coli Nissle 1917. Moreover, we demonstrated that neither lipopolysaccharide structure nor flagella play a role in E. coli strain Nissle 1917 translocation and dissemination. Our results suggest that if both the microbiota and adaptive immunity are defective, translocation across the intestinal epithelium and dissemination of the probiotic E. coli strain Nissle 1917 may occur and have potentially severe adverse effects. Future work should define the possibly related molecular factors that promote probiotic functions, fitness, and facultative pathogenicity.

Probiotic Escherichia coli strain Nissle 1917 outcompetes intestinalpathogens during biofilm formation

Many bacterial infections are associated with biofilm formation. Bacterial biofilms can develop on essentially all kinds of surfaces, producing chronic and often intractable infections. Escherichia coli is an important pathogen causing a wide range of gastrointestinal infections. E. coli strain Nissle 1917 has been used for many decades as a probiotic against a variety of intestinal disorders and is probably the best field-tested E. coli strain in the world. Here we have investigated the biofilm-forming capacity of Nissle 1917. We found that the strain was a good biofilm former. Not only was it significantly better at biofilm formation than enteropathogenic, enterotoxigenic and enterohaemorrhagic E. coli strains, it was also able to outcompete such strains during biofilm formation. The results support the notion of bacterial prophylaxis employing Nissle 1917 and may partially explain why the strain has a beneficial effect on many intestinal disorders.

Decisive role of lipopolysaccharide in activating nitric oxide and cytokine production by the probiotic Escherichia coli strain Nissle 1917

Effects of Gram-negative probiotic E. coli strain Nissle 1917 (EcN) on the production of nitric oxide (NO) and cytokines were determined in cultures of resident peritoneal cells of rats. The cells (2 x 10(6)/mL) were cultured for 24 h in the presence of live EcN suspension (EcN-Susp), bacteria-free supernatant of this suspension (Sup-EcN), and LPS of EcN origin (LPS-EcN). The biosynthesis of NO was substantially enhanced using live bacteria counts as low as 10(3)/mL applied in the form of EcN-Susp. The same NO-enhancing effect was produced by the correspondingly diluted Sup-EcN. It was found that Sup-EcN contained relatively high amounts of LPS. Administration of the LPS-EcN mimicked the high NO-augmenting activities of both Sup-EcN and EcN-Susp. However, the activity of LPS-EcN was significantly less pronounced than were the activities of Sup-EcN and EcN-Susp containing identical amounts of LPS. The NO-stimulatory effects of the EcN preparations were completely inhibited by polymyxin B. All LPS-EcN and correspondingly diluted Sup-EcN and EcN-Susp stimulated the secretion of cytokines TNF-alpha, IL-1beta, IL-6, IL-10 and VEGF. Also these effects were abrogated by polymyxin B. In contrast to the effects on NO production, the cytokine-stimulatory effects were significantly less pronounced after the exposure of the cells to Sup-EcN and EcN-Susp than to the identical amounts of LPS-EcN. It may be concluded that the in vitro stimulatory effects of EcN on NO and cytokine production are mediated by LPS. It is suggested that the immunostimulatory activity of LPS is modulated by EcN-derived factor(s), the nature of which remains to be identified.

Sever Sepsis After Probiotic Treatment With Escherichia coli NISSLE 1917

To the Editors: We report a case of an eutrophic preterm infant with a gestational age of 28 weeks and a birth weight of 935 g. The early postnatal phase was uncomplicated. On postnatal day 13, the infant developed gastroenteritis due to a rotavirus and adenovirus coinfection. A blood culture had no growth. To facilitate the physiologic intestinal bacterial flora on day 15 of life, we initiated a probiotic treatment with Escherichia coli NISSLE 1917 in the recommended dosage for this age group (1 mL once a day), with an immediate positive effect on the oral feeding. On postnatal day 25, a dramatic deterioration occurred with the infant showing a sepsis syndrome with fever, apnea attacks and hypotension. Blood cultures yielded E. coli, which was showing pulsed field gelelectrophoresis to be E. coli Nissle 1917 with clonal identity to the strain used therapeutically (Fig. 1). After intensified therapy with vasopressors, antibiotic treatment active against the isolated strain (meropenem in combination with vancomycine because of the central venous catheter), and intravenous immunoglobulins the patient stabilized and at 10 weeks of age was discharged in good clinical condition.FIGURE 1.: Result of the pulsed field gel electrophoresis. Lane 1: E. coli strain NISSLE 1917. Lane 2: E. coli strain isolated of the blood culture.Colonization with potentially pathogenic Gram negative bacteria is a risk factor for the development of necrotizing enterocolitis. Therefore, probiotics as live microbial supplements in preterms are used with the objective to support the intestinal colonization as well as to prevent infections or side effects of antibiotic drugs.1 It is unknown whether probiotic treatment increases or decreases the risk of invasive infection in preterm infants weighing less than 1500 g. The safety of this therapy is uncertain. Because of the immaturity of the gastrointestinal tract and of the local and systemic immune system in preterm infants the risk of systemic infections is increased. In a small study with 27 healthy newborns, E. coli strain NISSLE 1917 colonized the intestines within a few days and was well tolerated.2 Despite the fact that many studies have shown that E. coli NISSLE can replace pathogenic bacteria in the intestinal flora by means of intensive adherence to epithelial cells, analysis of the genomic structure has revealed a potential pathogenicity.3 Sepsis associated with probiotic supplementation in preterm infants with a very low birth weight (<1500 g) has not been previously reported. The immaturity of the immune system as well as the gastrointestinal tract combined with an increased permeability of the intestinal wall related to infection with rotavirus and adenovirus resulting in translocation of bacteria from the mucous membranes to the blood stream. After the neonatal period the probiotic prophylaxis is associated with an increased risk of sepsis in patients with severe acute pancreatitis and AIDS.4,5 In infancy, only a few case reports exist describing sepsis or meningitis complicated by Lactobacillus GG and Saccharomyces cerevisiae independent of probiotic supplementation.6,7 All cases of sepsis related to probiotics have occurred in patients with underlying immune compromise or chronic disease. Many case reports of probiotic sepsis describe patients with preexisting intestinal pathology, a common indication for probiotic use, but also associated with an increased risk of probiotic translocation from the intestinal mucosa. The presence of a central venous catheter is also a frequent finding and is considered as a possible source of sepsis. Studies with athymic neonatal mice have shown that the colonization with probiotics can lead to increased mortality.8 This finding suggests that the immaturity of the immune system in preterm infants put them at particularly high risk for probiotic-associated sepsis. The present case report should prompt a discussion of pros and cons of probiotic use. Additional systematical investigations and well organized clinical studies are needed to clarify risk factors for the development of septic complications after a treatment with probiotics. Katrin Guenther, MD Eberhardt Straube, MD Wolfgang Pfister, MD Albrecht Guenther, MD Axel Huebler, MD Department of Pediatrics Friedrich-Schiller University Jena, Germany

In vivo replication of T4 and T7 bacteriophages in germ-free mice colonized with Escherichia coli

The gut transit of T4 phages was studied in axenic mice mono-colonized with the non-pathogenic Escherichia coli strain K-12. Thirty minutes, 1 and 2 h after phage feeding, T4 phage had reached the jejunum, ileum and cecum, respectively. Phage was found in the lumen and was also associated with the mucosa. One day later no phage was detected in the feces. Compared to germ-free control animals, oral T4 phage led to a 300-fold higher fecal phage titer in mice mono-colonized with E. coli strain WG-5. The in vivo T4 phage replication was transient and reached peak fecal titers about 8 h after oral phage application followed by a rapid titer decrease over two days. Similar data were obtained in mice colonized with E. coli strain Nissle. In contrast, orally applied T7 phage experienced a massive and sustained in vivo replication in mice mono-colonized with E. coli strain WG-5 irrespective whether phage or E. coli host was applied first. T7 phage replication occurred mainly in the large intestine. High titers of T7 phage and high E. coli cell counts coexisted in the feces. The observation of only 20% T7 phage-resistant fecal E. coli colonies suggests a refuge model where phage-sensitive E. coli cells are physically or physiologically protected from phage infection in the gut. The difference between T7 and T4 with respect to gut replication might partly reflect their distinct in vitro capacity to replicate on slowly growing cells.

Characterization of cellulose production in Escherichia coli Nissle 1917 and its biological consequences

Bacterial species of the Enterobacteriaceae family produce cellulose and curli fimbriae as extracellular matrix components, and their synthesis is positively regulated by the transcriptional activator CsgD. In this group of bacteria, cellulose biosynthesis is commonly regulated by CsgD via the GGDEF domain protein AdrA, a diguanylate cyclase that produces cyclic-diguanylic acid (c-di-GMP), an allosteric activator of cellulose synthase. In the probiotic Escherichia coli strain Nissle 1917 and its recent clonal isolates, CsgD activates the production of curli fimbriae at 28 degrees C, but neither CsgD nor AdrA is required for the c-di-GMP-dependent biosynthesis of cellulose at 28 degrees C and 37 degrees C. In these strains, the GGDEF domain protein YedQ, a diguanylate cyclase that activates cellulose biosynthesis in certain E. coli strains, is not required for cellulose biosynthesis and it has in fact evolved into a novel protein. Cellulose production in Nissle 1917 is required for adhesion of bacteria to the gastrointestinal epithelial cell line HT-29, to the mouse epithelium in vivo, and for enhanced cytokine production. The role of cellulose in this strain is in contrast to the role of cellulose in the commensal strain E. coli TOB1. Consequently, the role of cellulose in bacterial-host interaction is dependent on the E. coli strain background.

Differential crosstalk between epithelial cells, dendritic cells and bacteria in a co-culture model

Intestinal epithelial cells (IECs) provide a primary physical barrier against commensal and pathogenic bacteria, but the influence of IECs in the regulation of the associated mucosal immune system remains largely unknown. The network of dendritic cells (DCs) in the vicinity of IECs is known to play a crucial role in the regulation of gut homeostasis. We investigated the cross-talk between murine IECs (m-IC cl2 cell line), bone marrow derived DCs and different bacteria using an in vitro Transwell ® co-culture model. IECs responded poorly to different Gram-positive lactic acid bacteria (LAB) and to a Staphylococcus aureus strain . In contrast two Escherichia coli strains, including the probiotic strain Nissle 1917, strongly activated IECs, as evidenced by the induction of different chemokines. While a differential maturation of DCs is observed upon direct stimulation with the various bacteria, DC maturation across the epithelial barrier was only observed upon challenge of the apical surface of the IECs with both E. coli strains and LPS. These results suggested that the capacity of bacteria to induce pro-inflammatory signals through the epithelial barrier is not linked to their pathogenic or commensal status, but seem to be dependent on the presence of specific surface factors. As already reported, we confirmed that m-IC cl2 cells are highly susceptible to LPS. It is highly possible, at least in this model, that free LPS is the “specific factor” key to activate IEC or BMDC. Moreover, IECs are broadly unresponsive to Gram-positive bacterial components, notably TLR-2 ligands, in contrast to Gram-negative bacterial components. These results suggest that the gut epithelium will sense the commensal bacteria in a different way, and seems to be unresponsive to Gram positive bacteria in particular to LAB.

The non-pathogenicEscherichia colistrain Nissle 1917 – features of a versatile probiotic

Probiotics are non-pathogenic living microorganisms which, when administered in adequate amounts, confer a health benefi t on the host. Probiotic microorganisms most often are constituents of special foodstuffs, such as fermented milk products or food supplements. They are also employed as feed additives in livestock breeding and as active components of medical remedies in human and veterinary medicine. In the food industry, probiotics traditionally are lactic acid bacteria (LAB), especially lactobacilli and lactococci, although bifi dobacteria and strepcococci (enterococci) are also used. In livestock breeding and medicine, besides lactic acid bacteria other non-pathogenic microorganisms with health-promoting characteristics, e.g. certain strains of yeast (‘ Saccharomyces boulardii ’) and Escherichia coli ( E. coli Nissle 1917), are also applied. This review focuses on the probiotic E. coli strain Nissle 1917 (EcN), its origin and medical history, microbiology, genetics, biological activities, safety, and toxicological aspects. Furthermore, clinical trials performed with this special E. coli strain will be summarized. The EcN strain was detected and isolated by Alfred Nissle in 1917 because of its antagonistic activity against some pathogenic enterobacteria. With respect to its metabolic capacities, EcN is a typical E. coli strain. It is a non-pathogenic member of the Escherichia coli family, because it does does not carry pathogenic adhesion factors and does not produce any enterotoxins or cytotoxins, it is not invasive, not uropathogenic, and is rapidly killed by non-specifi c defense factors of blood serum. Besides this, EcN has some special characteristics that distinguish it from other pathogenic and non-pathogenic E. coli strains and are thought to be important for its probiotic activities. Thus, EcN carries so-called genomic islands (GEIs), integrated into its chromosome. On these GEIs, gene clusters coding for several fi tness factors are present, e.g. genes for the production of microcins which inhibit the growth of other enterobacteria. Another strain-specifi c feature of EcN is a special lipopolysaccharide (LPS) in its outer cell membrane, being responsible for the fact that EcN exhibits immunomodulating properties without showing immunotoxic effects. The immunomodulating properties mainly focus on anti-infl ammatory activities that are important for the clinical effi cacy in remission maintenance of ulcerative colitis. Also of importance is the bacterial–epithelial crosstalk between EcN and the intestinal epithelial cells, leading to a strengthening of the epithelial barrier and the curing of ‘leaky gut’ phenomena. The restoration of a disturbed gut barrier by EcN is thought to be due to the stimulation of epithelial defensin production as well as to a ‘sealing effect’ on the tight junctions of the enterocytes. EcN has also been shown to induce the development of the gut immune system in animal models and human newborns. In addition, it has been found that products of EcN metabolism, probably acetic acid, promote colonic motility that might be helpful for therapeutic application in chronically constipated patients. Randomized controlled clinical trials (RCTs) have shown EcN to be therapeutically effective in rather diverse indications, such as ulcerative colitis, chronic constipation, and acute and protracted diarrhea. Key words: EcN, medical history, microbiology, genetics, biological activities, safety, toxicological aspects, clinical trials