Tumor-specific colonization, tissue distribution, and gene induction by probiotic Escherichia coli Nissle 1917 in live mice

Systemic administration of microorganisms into tumor-bearing mice revealed preferential accumulation in tumors in comparison to clearance in organs such as spleen and liver. Here we compared the efficiency of tumor-specific colonization of pathogenic Salmonella typhimurium strains 14028 and SL1344 to the enteroinvasive Escherichia coli 4608-58 strain and to the attenuated Salmonella flexneri 2a SC602 strain, as well as to the uropathogenic E. coli CFT073, the non-pathogenic E. coli Top10, and the probiotic E. coli Nissle 1917 strain. All strains colonized and replicated in tumors efficiently each resulting in more than 1 x 10(8) colony-forming units per gram tumor tissue. Colonization of spleen and liver were significantly lower when E. coli strains were used in comparison to S. typhimurium and the non-pathogenic strains did not colonize those organs at all. Further investigation of E. coli Nissle 1917 showed that no drastic differences in colonization and amplification were seen when immunocompetent and immunocompromised animals were used, and we were able to show that E. coli Nissle 1917 replicates at the border of live and necrotic tumor tissue. We also demonstrated exogenously applied L-arabinose-dependent gene activation in colonized tumors in live mice. These findings will prepare the way for bacterium-mediated controlled protein delivery to solid tumors.     

Development of strain-specific PCR reactions for the detection of the probiotic Escherichia coli strain Nissle 1917 in fecal samples

PCR was used to establish a specific detection system for the non-pathogenic Escherichia coli strain Nissle 1917 (DSM6601), which is used as a probiotic drug against intestinal disorders and diseases. Five PCR assays have been developed which are based on the chromosomally encoded major fimbrial subunit genes fimA (type 1 fimbriae) and focA (F1C fimbriae), and the two small cryptic plasmids pMUT1 and pMUT2. The assays were validated by testing a collection of 354 different pathogenic and non-pathogenic E. coli strains from various origins, including E. coli K-12, fecal and environmental as well as pathogenic extraintestinal and intestinal E. coli strains. The most specific results were obtained with primers based on DNA sequences from plasmid pMUT2. The plasmid-based PCR assays described can be used to detect E. coli strain Nissle 1917 in feces from patients without prior cultivation.     

The probiotic Escherichia coli strain Nissle 1917 induces gammadelta T cell apoptosis via caspase- and FasL-dependent pathways

Human gammadelta T cells play a vital role in the innate and adaptive immune response to microbial antigens by acting as antigen-presenting cells while at the same time being capable of directly activating CD4(+) T cells. Pathogenic microbes or loss of tolerance toward the host's own microflora trigger many diseases including inflammatory bowel diseases. We previously demonstrated that Escherichia coli Nissle 1917 directly interacts with the adaptive immune system by regulating central T cell functions. Here we aimed to investigate whether E. coli Nissle regulates gammadelta T cell function, thereby linking the innate and adaptive immune system. In our study, we demonstrate that, in contrast to the other probiotic strains tested, E. coli Nissle increased activation, cell cycling and expansion of gammadelta, but not alphabeta T cells. In gammadelta T cells, E. coli Nissle reduced tumor necrosis factor-alpha secretion but increased IL-6 and CXCL8 release. However, after activation, only E. coli Nissle induced gammadelta T cell apoptosis, mediated via Toll-like receptor-2 by caspase- and FasLigand-dependent pathways. gammadelta T cells play an important role in the recognition of microbial antigens and the perpetuation of inflammatory processes. The demonstration that E. coli Nissle, but not the other bacteria tested, profoundly regulate gammadelta T cell function contributes to explaining the biological function of this probiotic strain in inflammatory diseases and provides us with a better understanding of the role of gammadelta T cells.     

NF-kappaB- and AP-1-mediated induction of human beta defensin-2 in intestinal epithelial cells by Escherichia coli Nissle 1917: a novel effect of a probiotic bacterium

Little is known about the defensive mechanisms induced in epithelial cells by pathogenic versus probiotic bacteria. The aim of our study was to compare probiotic bacterial strains such as Escherichia coli Nissle 1917 with nonprobiotic, pathogenic and nonpathogenic bacteria with respect to innate defense mechanisms in the intestinal mucosal cell. Here we report that E. coli strain Nissle 1917 and a variety of other probiotic bacteria, including lactobacilli--in contrast to more than 40 different E. coli strains tested--strongly induce the expression of the antimicrobial peptide human beta-defensin-2 (hBD-2) in Caco-2 intestinal epithelial cells in a time- and dose-dependent manner. Induction of hBD-2 through E. coli Nissle 1917 was further confirmed by activation of the hBD-2 promoter and detection of the hBD-2 peptide in the culture supernatants of E. coli Nissle 1917-treated Caco-2 cells. Luciferase gene reporter analyses and site-directed mutagenesis experiments demonstrated that functional binding sites for NF-kappaB and AP-1 in the hBD-2 promoter are required for induction of hBD-2 through E. coli Nissle 1917. Treatment with the NF-kappaB inhibitor Helenalin, as well as with SP600125, a selective inhibitor of c-Jun N-terminal kinase, blocked hBD-2 induction by E. coli Nissle 1917 in Caco-2 cells. SB 202190, a specific p38 mitogen-activated protein kinase inhibitor, and PD 98059, a selective inhibitor of extracellular signal-regulated kinase 1/2, were ineffective. This report demonstrates that probiotic bacteria may stimulate the intestinal innate defense through the upregulation of inducible antimicrobial peptides such as hBD-2. The induction of hBD-2 may contribute to an enhanced mucosal barrier to the luminal bacteria.     

Expressing cytotoxic compounds in Escherichia coli Nissle 1917 for tumor-targeting therapy

Abnormal blood vessels and hypoxic and necrotic regions are common features of solid tumors and related to the malignant phenotype and therapy resistance. Certain obligate or facultative anaerobic bacteria exhibit inherent ability to colonize and proliferate within solid tumors in vivo. Escherichia coli Nissle 1917 (EcN), a non-pathogenic probiotic in European markets, has been known to proliferate selectively in the interface between the viable and necrotic regions of solid tumors. The objective of this study was to establish a tumor-targeting therapy system using the genetically engineered EcN for targeted delivery of cytotoxic compounds, including colibactin, glidobactin and luminmide. Biosynthetic gene clusters of these cytotoxic compounds were introduced into EcN and the corresponding compounds were detected in the resultant recombinant EcN strains. The recombinant EcN showed significant cytotoxic activity in vitro and in vivo as well, and significantly suppressed the tumor growth. Together, this study confirmed efficient tumor-targeting colonization of EcN and demonstrated its potentiality in the tumor-specific delivery of cytotoxic compounds as a new tumor-targeting therapy system.     

Sensitivity to Escherichia coli Nissle 1917 in mice is dependent on environment and genetic background

Escherichia coli Nissle 1917 (EcN) is a well-characterized probiotic bacterium. Although genomic comparisons of EcN with the uropathogenic E. coli strain CFT073 revealed high degrees of similarity, EcN is generally considered a non-pathogenic organism. However, as recent evidence suggests that EcN is capable of inducing inflammatory responses in host intestinal epithelial cells, we aimed to investigate potential pathogenic properties of EcN in an in vivo model using various germ-free (GF) mouse strains. With the exception of C3H/HeJZtm mice, which carry a defective toll-like receptor (TLR)4-allele, no lesions were obvious in mice of different strains orally inoculated with EcN for 1 week, although organ cultures (blood, lung, mesenteric lymph node, pancreas, spleen, liver and kidney) tested positive to various degrees. C3H/HeJZtm mice inoculated with EcN became clinically ill and the majority died or had to be euthanized. Organs of all gnotobiotic C3H/HeJZtm mice were positive for EcN by culture; major histological findings were moderate to severe pyogranulomatous serositis, typhlitis and pancreatitis. Histological findings were corroborated by highly elevated tumour necrosis factor (TNF) serum levels. Lesions were not detected in specified pathogen free maintained C3H/HeJZtm mice, GF C3H/HeJ mice lacking the interleukin-10 gene, or GF C3H/HeJZtm mice that were inoculated with E. coli K12 strain MG1655 as a control. In addition, mild histological lesions were detected in Ztm:NMRI mice 3 months after oral inoculation with EcN. This study shows that EcN is capable of displaying a virulent phenotype in GF C3H/HeJZtm mice. Whether this phenotype is linked to the bacterium's probiotic nature should be the focus of further studies.     

Escherichia coli Nissle 1917 distinctively modulates T-cell cycling and expansion via toll-like receptor 2 signaling

Although the probiotic Escherichia coli strain Nissle 1917 has been proven to be efficacious for the treatment of inflammatory bowel diseases, the underlying mechanisms of action still remain elusive. The aim of the present study was to analyze the effects of E. coli Nissle 1917 on cell cycling and apoptosis of peripheral blood and lamina propria T cells (PBT and LPT, respectively). Anti-CD3-stimulated PBT and LPT were treated with E. coli Nissle 1917-conditioned medium (E. coli Nissle 1917-CM) or heat-inactivated E. coli Nissle 1917. Cyclin B1, DNA content, and caspase 3 expression were measured by flow cytometry to assess cell cycle kinetics and apoptosis. Protein levels of several cell cycle and apoptosis modulators were determined by immunoblotting, and cytokine profiles were determined by cytometric bead array. E. coli Nissle 1917-CM inhibits cell cycling and expansion of peripheral blood but not mucosal T cells. Bacterial lipoproteins mimicked the effect of E. coli Nissle 1917-CM; in contrast, heat-inactivated E. coli Nissle 1917, lipopolysaccharide, or CpG DNA did not alter PBT cell cycling. E. coli Nissle 1917-CM decreased cyclin D2, B1, and retinoblastoma protein expression, contributing to the reduction of T-cell proliferation. E. coli Nissle 1917 significantly inhibited the expression of interleukin-2 (IL-2), tumor necrosis factor alpha, and gamma interferon but increased IL-10 production in PBT. Using Toll-like receptor 2 (TLR-2) knockout mice, we further demonstrate that the inhibition of PBT proliferation by E. coli Nissle 1917-CM is TLR-2 dependent. The differential reaction of circulating and tissue-bound T cells towards E. coli Nissle 1917 may explain the beneficial effect of E. coli Nissle 1917 in intestinal inflammation. E. coli Nissle 1917 may downregulate the expansion of newly recruited T cells into the mucosa and limit intestinal inflammation, while already activated tissue-bound T cells may eliminate deleterious antigens in order to maintain immunological homeostasis.     

Tumor-targeted delivery of TAT-Apoptin fusion gene using Escherichia coli Nissle 1917 to colorectal cancer

In view of the high incidence and mortality of the colorectal cancer, the limited efficacy and serious adverse effect of the conventional treatment, a novel alternative treatment needs to be developed. Recent studies have demonstrated that the targeted therapy as an alternative treatment showed a promising prospect. We hypothesized that construct a recombination non-pathogenic Escherichia coli Nissle 1917 (EcN), inserting a fusion gene TAT-Apoptin into this probiotic vector, as a targeted therapy strategy for patients of colorectal cancer. Compared with conventional treatments for tumors, the recombination EcN containing TAT-Apoptin fusion gene is capable of tumor-specific colonization, secretary expression and efficient intracellular delivery and therefore able to reduce the incidence of side effect and promote the efficiency of treatment.     

The K5 Capsule of Escherichia coli Strain Nissle 1917 Is Important in Mediating Interactions with Intestinal Epithelial Cells and Chemokine Induction

Escherichia coli strain Nissle 1917 has been widely used as a probiotic for the treatment of inflammatory bowel disorders and shown to have immunomodulatory effects. Nissle 1917 expresses a K5 capsule, the expression of which often is associated with extraintestinal and urinary tract isolates of E. coli. In this paper, we investigate the role of the K5 capsule in mediating interactions between Nissle 1917 and intestinal epithelial cells. We show that the loss of capsule significantly reduced the level of monocyte chemoattractant protein 1 (MCP-1), RANTES, macrophage inflammatory protein 2α (MIP-2α), MIP-2β, interleukin-8, and gamma interferon-inducible protein 10 induction by Nissle 1917 in both Caco-2 cells and MCP-1 induction in ex vivo mouse small intestine. The complementation of the capsule-minus mutation confirmed that the effects on chemokine induction were capsule specific. The addition of purified K5, but not K1, capsular polysaccharide to the capsule-minus Nissle 1917 at least in part restored chemokine induction to wild-type levels. The purified K5 capsular polysaccharide alone was unable to stimulate chemokine production, indicating that the K5 polysaccharide was acting to mediate interactions between Nissle 1917 and intestinal epithelial cells. The induction of chemokine by Nissle 1917 was generated predominantly by interaction with the basolateral surface of Caco-2 cells, suggesting that Nissle 1917 will be most effective in inducing chemokine expression where the epithelial barrier is disrupted.A probiotic has been defined as “live microorganisms which when administered in adequate amounts confer a health benefit on the host” (20). These benefits include the balancing and restoration of the intestinal microflora, repair of intestinal barrier functions (54), expression of bacteriocins (36), immunomodulatory effects (18, 43, 47, 53), and antagonizing epithelial colonization and invasion by pathogens (2). Escherichia coli strain Nissle 1917 was isolated from the feces of a soldier who did not develop diarrhea during a severe outbreak of shigellosis (38). Despite exhibiting a serotype (O6:K5:H1) that is characteristic of E. coli strains associated with urinary tract infections, Nissle 1917 apparently is nonpathogenic (25, 53) and has been used widely in preventing infectious diarrheal diseases (7, 14, 27, 37, 52, 53), the treatment of inflammatory bowel diseases such as ulcerative colitis and Crohn''s disease (7, 23, 32, 33), and to prevent the colonization of the digestive tract of neonates by pathogens (35). Recently, there has been a growing interest in investigating the immunomodulatory effect of Nissle 1917. Previous studies showed that colonization by Nissle 1917 may lead to an alteration of the hosts'' cytokine repertoire (13, 49), increased immunoglobulin A secretion (14), lymphocyte or macrophage activation (13), the modulation of CD4⁺ clonal expansion (47), the stimulation of antimicrobial peptide production by intestinal epithelial cells (39, 52, 54), and alterations of the pro- and anti-inflammatory balance of local cytokines (49). Recently it has been shown that Nissle 1917 activates γδT cells, stimulating CXCL8 and interleukin-6 (IL-6) release but inhibiting tumor necrosis factor alpha (TNF-α) secretion (26). Following activation, Nissle 1917 induced apoptosis in activated γδT cells, indicating a key role for Nissle 1917 in interacting with the subset of T cells that operate at the interface between the adaptive and innate immune responses (26). Nissle 1917 also has been shown to express a direct anti-inflammatory activity on epithelial cells by blocking TNF-α-induced IL-8 secretion through a NF-κB-independent mechanism (28). Although the immunomodulatory effects of Nissle 1917 are well documented, the contribution of individual microbial components in mediating such effects is less well understood. So far, only a role for flagellin in mediating the induction of human β-defensin expression by Nissle 1917 has been established (44). Nissle 1917 expresses a K5 capsule on its cell surface, and a number of roles for polysaccharide capsules in the virulence of E. coli have been proposed, including resistance to phagocytosis and complement-mediated killing and the increased colonization of the host (42). In contrast, in the case of other encapsulated pathogens, it has been shown that the expression of a polysaccharide capsule can affect the induction of chemokines following attachment to host cells (6, 17, 22, 24, 40, 41, 45, 50). The aim of the present study was to investigate the role of the K5 capsule in mediating the immunomodulatory activity of Nissle 1917.     

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.     

Safety of Probiotic Escherichia coli Strain 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.The human gastrointestinal tract contains a complex symbiotic microbiota that is estimated to comprise more than 40,000 species and in some regions more than 10¹¹ organisms (14) and that helps maintain immune homeostasis in the gut-associated lymphoid tissues (20, 29), optimize nutritional uptake (13), and support development of the gut (40). The thick mucus layer that overlies the entire intestinal epithelium and an effective immune system keep this enormous bacterial load strictly sequestered on the luminal side of the gut, preventing penetration across the epithelial barrier (20).The importance of the cross talk between the microbiota, intestinal epithelial cells, and the innate and adaptive portions of the immune system is indicated by a variety of intestinal pathological conditions, including Crohn''s disease, ulcerative colitis, pouchitis, irritable bowel syndrome, and necrotizing enterocolitis (NEC). Immature or genetically compromised immunity (25) results in exaggerated intestinal inflammation (26) or disruption or altered composition of the intestinal mucosa, which in turn disturbs the homeostasis between the human host and its intestinal symbionts. Pathological events change the relative balance between beneficial and aggressive enteric symbionts, turn beneficial bacteria into pathogens (36), or select for novel opportunistic pathogens (2, 28). A qualitatively and quantitatively changed gastrointestinal microbiota, often described as small bowel bacterial overgrowth (SIBO) (14, 33) or dysbiosis (26), may contribute substantially to local chronic inflammation in a vicious cycle and provoke bacterial translocation that leads to fatal sepsis.This concept provides the rationale for selective therapeutic manipulation of the abnormal microbiota by probiotics for the intestinal diseases that have been described; probiotics are defined as viable microorganisms with beneficial physiological or therapeutic activities (36). Various in vitro and animal studies with probiotics, including Escherichia coli strain Nissle 1917, have demonstrated the capacity of probiotics to reduce intestinal inflammation (29, 42), to strengthen the intestinal barrier against pathogens (15, 46), to increase the host innate immune functions (37), or to prevent adherent and invasive E. coli strains from adhering to and invading human intestinal epithelial cells (9). Indeed, limited clinical trials using E. coli strain Nissle 1917 or other microorganisms have suggested that this therapeutic strategy is efficacious in patients with chronic idiopathic inflammatory bowel diseases (IBD) (23, 34, 35, 36), irritable bowel syndrome (32), and NEC (25).However, all probiotic bacterial species are not equally beneficial, and each species may have individual mechanisms of action due to specific metabolic activities and cellular structures (10). Some case reports even seem to indicate that probiotics, including E. coli strain Nissle 1917, might promote sepsis (6, 24), and severe adverse effects of probiotics have been observed in patients suffering from acute pancreatitis (3, 5).Host characteristics, specifically characteristics of the existing microbiota and the intestinal immune status, are often not considered when probiotics are used as therapeutic agents, particularly in genetically or therapeutically immunocompromised patients, including very-low-birth-weight preterm infants (25) and severely ill IBD patients receiving anti-inflammatory therapy (11).The fact that living bacteria that may turn out to be opportunistic pathogens are therapeutically administered to patients with impaired immune functions and an altered intestinal microbiota raises two important basic questions. First, how important is a functional adaptive immune system for preventing adverse clinical effects of probiotics, such as translocation, bacteremia, and death? Lymphocytes of the gut-associated lymphoid tissue play an essential role in controlling proliferation and differentiation of intestinal epithelial cells (22) and in the maintenance of gut integrity (12). Second, how important is the microbiota in this context? Symbionts have been shown to activate epithelial innate immune signaling pathways that are needed to fight microbial pathogens (20).To answer the questions mentioned above, we employed mice having a targeted disruption of recombinase-activating gene 1 (Rag1^−/−), in which T- and B-lymphocyte development is arrested at the CD4⁻ CD8⁻ double-negative thymocyte or B220⁺ CD43⁺ pro-B-cell stage (30). To study the role of the physiological microbiota, these mice were raised under germfree (GF) or specific-pathogen-free (SPF) conditions and were challenged with E. coli strain Nissle 1917. Here we present evidence indicating that when there is a profound deficiency in the adaptive immune system in the presence of both functional innate immunity and the intestinal microbiota, translocation and dissemination of E. coli strain Nissle 1917 do not occur; however, in the absence of the intestinal microbiota, substantial translocation of E. coli strain Nissle 1917 causes high rates of mortality in Rag1^−/− mice. We also show that adoptive transfer of naïve CD4⁺ T cells to Rag1^−/− mice raised under GF conditions (GF-raised Rag1^−/− mice) after E. coli strain Nissle 1917 challenge increases the mortality rate significantly. Our data might explain why immunocompromised patients that have an immature or disrupted intestinal microbiota and are treated with probiotics have an enhanced risk for severe side effects due to bacterial translocation. Furthermore, we describe a T-cell-mediated pathogenic mechanism that is involved in a fatal outcome for immunocompromised mice fed the probiotic E. coli strain Nissle 1917.     

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.     

Probiotic Escherichia coli Nissle 1917 activates DC and prevents house dust mite allergy through a TLR4‐dependent pathway

Experimental animal and human studies have demonstrated that probiotic strains have beneficial effects on allergy. Here we report that the probiotic Escherichia coli Nissle 1917 strain (EcN) is able to activate DC, as shown by important cytokine synthesis together with up‐regulation of membrane expression of CD40, CD80 and CD86. This EcN‐induced DC activation was strictly dependent on the TLR4 signaling pathway and was also associated with stimulation of NF‐κB and MAPK. We next investigated the prophylactic potential of i.n. co‐administration of EcN with a recombinant form of Der p 1 (ProDer p 1) in a murine model of mite allergy. I.n. vaccinations with EcN plus ProDer p 1 prevented the subsequent allergic response following Der p 1 sensitization and airway challenge with aerosolized mite extracts through the induction of an allergen‐specific IgG2a response, the prevention of specific IgE production and a strong reduction of IL‐5 secretion by allergen‐restimulated splenocytes. EcN alone or in combination with ProDer p 1 inhibited the development of airway eosinophilia and neutrophilia. This in vivo protective effect of EcN was, in part, mediated by TLR4 signaling. Our results suggest that EcN represents an efficient adjuvant to prevent allergic responses.     

Enhanced wound healing by recombinant Escherichia coli Nissle 1917 via human epidermal growth factor receptor in human intestinal epithelial cells: therapeutic implication using recombinant probiotics

The gastrointestinal mucosa has a remarkable ability to repair damage with the support of epidermal growth factor (EGF), which stimulates epithelial migration and proliferative reepithelialization. For the treatment of mucosal injuries, it is important to develop efficient methods for the localized delivery of mucoactive biotherapeutics. The basic idea in the present study came from the assumption that an intestinal probiotic vehicle can carry and deliver key recombinant medicinal proteins to the injured epithelial target in patients with intestinal ulcerative diseases, including inflammatory bowel disease. The study was focused on the use of the safe probiotic E. coli Nissle 1917, which was constructed to secrete human EGF in conjunction with the lipase ABC transporter recognition domain (LARD). Using the in vitro physically wounded monolayer model, ABC transporter-mediated EGF secretion by probiotic E. coli Nissle 1917 was demonstrated to enhance the wound-healing migration of human enterocytes. Moreover, the epithelial wound closure was dependent on EGF receptor-linked activation, which exclusively involved the subsequent signaling pathway of the mitogen-activated protein kinase kinase (MEK) extracellular-related kinases 1 and 2 (ERK1/2). In particular, the migrating frontier of the wounded edge displayed the strongest EGF receptor-linked signaling activation in the presence of the recombinant probiotic. The present study provides a basis for the clinical application of human recombinant biotherapeutics via an efficient, safe probiotic vehicle.     

Escherichia coli EDL933 Requires Gluconeogenic Nutrients To Successfully Colonize the Intestines of Streptomycin-Treated Mice Precolonized with E. coli Nissle 1917

Escherichia coli MG1655, a K-12 strain, uses glycolytic nutrients exclusively to colonize the intestines of streptomycin-treated mice when it is the only E. coli strain present or when it is confronted with E. coli EDL933, an O157:H7 strain. In contrast, E. coli EDL933 uses glycolytic nutrients exclusively when it is the only E. coli strain in the intestine but switches in part to gluconeogenic nutrients when it colonizes mice precolonized with E. coli MG1655 (R. L. Miranda et al., Infect Immun 72:1666–1676, 2004, http://dx.doi.org/10.1128/IAI.72.3.1666-1676.2004). Recently, J. W. Njoroge et al. (mBio 3:e00280-12, 2012, http://dx.doi.org/10.1128/mBio.00280-12) reported that E. coli 86-24, an O157:H7 strain, activates the expression of virulence genes under gluconeogenic conditions, suggesting that colonization of the intestine with a probiotic E. coli strain that outcompetes O157:H7 strains for gluconeogenic nutrients could render them nonpathogenic. Here we report that E. coli Nissle 1917, a probiotic strain, uses both glycolytic and gluconeogenic nutrients to colonize the mouse intestine between 1 and 5 days postfeeding, appears to stop using gluconeogenic nutrients thereafter in a large, long-term colonization niche, but continues to use them in a smaller niche to compete with invading E. coli EDL933. Evidence is also presented suggesting that invading E. coli EDL933 uses both glycolytic and gluconeogenic nutrients and needs the ability to perform gluconeogenesis in order to colonize mice precolonized with E. coli Nissle 1917. The data presented here therefore rule out the possibility that E. coli Nissle 1917 can starve the O157:H7 E. coli strain EDL933 of gluconeogenic nutrients, even though E. coli Nissle 1917 uses such nutrients to compete with E. coli EDL933 in the mouse intestine.     

Lethal Escherichia coli and Salmonella typhimurium endotoxemia is mediated through different pathways

Despite the differences in the molecular structure between lipopolysaccharides (LPS) isolated from Escherichia coli, Klebsiella pneumoniae or Salmonella typhimurium, the potential differences in their biological effects in vivo have not been investigated. In the present study, TNF and LT double knock-out (TNF-/-LT-/-) mice were almost as susceptible as TNF+/+LT+/+ controls to S. typhimurium LPS, but they were significantly more resistant to lethal endotoxemia induced by E. coli or K. pneumoniae LPS. The effect was not due to endotoxin-associated proteins. In the knock-out mice, this difference in lethality was accompanied by decreased interleukin-1 (IL-1) and interferon-gamma (IFN-gamma) production after challenge with E. coli LPS, whereas after S. typhimurium LPS more IL-1 and IFN-gamma were produced. In contrast, more IL-10 was produced after challenge of mice with E. coli LPS than with S. typhimurium LPS. The hypothesis that a combination of pro-inflammatory cytokines is responsible for the mortality after S. typhimurium LPS was suggested by experiments in mice deficient in IL-1beta-converting enzyme (ICE-/- mice). ICE-/-mice, lacking mature IL-1beta and IL-18, but also defective in IFN-gamma and TNF production, were completely protected against both E. coli and S. typhimurium LPS. Experiments in Toll-like receptor (TLR)-4 defective mice suggested that the difference is not due to differential activation of TLR4. In conclusion, TNF and LT play a central role in the lethality due to E. coli LPS, whereas the lethal effects of S. typhimurium LPS are mediated through mechanisms also involving other cytokines such as IFN-gamma, IL-1 and IL-18.     

Environmental factors influence the production of enterobactin, salmochelin, aerobactin, and yersiniabactin in Escherichia coli strain Nissle 1917

The probiotic Escherichia coli strain Nissle 1917 produces four siderophores: the catecholates enterobactin and salmochelin, the hydroxamate aerobactin, and the mixed-type siderophore yersiniabactin. We studied the influence of pH, temperature, and carbon source on the production of these four siderophores. Yersiniabactin and salmochelin were maximally produced under neutral to alkaline conditions (pH 7.0 and 7.6, respectively), whereas aerobactin was maximally produced at a more acidic pH (pH 5.6), which agrees with the slightly higher complex stability of hydroxamates at acidic pH values compared to the catecholates. Under nearly all conditions studied, catecholate siderophore production was higher with glycerol than with glucose as the carbon source. Yersiniabactin production was also higher with glycerol as the carbon source at pH 7.0. At 42 °C, strain Nissle 1917 grew poorly or not at all because of the iron-limiting conditions. In a competition experiment between wild-type strain Nissle 1917 and a mutant of this strain with a deletion in the yersiniabactin operon, the wild-type overgrew the mutant at pH 7.0 and 7.6 and not at pH 5.6. These results agree with yersiniabactin production being of greater advantage at neutral and slightly alkaline pH values. The production of four siderophores may help the probiotic E. coli Nissle 1917 to compete with other E. coli strains in the colon. The probiotic strain Nissle 1917 used in our experiments has many characteristics in common with uropathogenic E. coli and other pathogenic strains which also secrete these siderophores. Uropathogenic E. coli strains may need the multitude of siderophores to adapt to the pH of urine, which varies between pH 4.6 and 8.0.     

Preventive Effects of Escherichia coli Strain Nissle 1917 on Acute and Chronic Intestinal Inflammation in Two Different Murine Models of Colitis

Escherichia coli strain Nissle 1917 (EcN) is as effective in maintaining remission in ulcerative colitis as is treatment with mesalazine. This study aims to evaluate murine models of acute and chronic intestinal inflammation to study the antiinflammatory effect of EcN in vivo. Acute colitis was induced in mice with 2% dextran-sodium sulfate (DSS) in drinking water. EcN was administered from day −2 to day +7. Chronic colitis was induced by transfer of CD4⁺ CD62L⁺ T lymphocytes from BALB/c mice in SCID mice. EcN was administered three times/week from week 1 to week 8 after cell transfer. Mesenteric lymph node (MLN) cytokine secretion (of gamma interferon [IFN-γ], interleukin 5 [IL-5], IL-6, and IL-10) was measured by enzyme-linked immunosorbent assay. Histologic sections of the colon were analyzed by using a score system ranging from 0 to 4. Intestinal contents and homogenized MLN were cultured, and the number of E. coli-like colonies was determined. EcN was identified by repetitive extragenic palindromic (REP) PCR. EcN administration to DSS-treated mice reduced the secretion of proinflammatory cytokines (IFN-γ, 32,477 ± 6,377 versus 9,734 ± 1,717 [P = 0.004]; IL-6, 231 ± 35 versus 121 ± 17 [P = 0.02]) but had no effect on the mucosal inflammation. In the chronic experimental colitis of the transfer model, EcN ameliorated the intestinal inflammation (histology score, 2.7 ± 0.2 versus 1.9 ± 0.3 [P = 0.02]) and reduced the secretion of proinflammatory cytokines. Translocation of EcN and resident E. coli into MLN was observed in the chronic colitis model but not in healthy controls. Administration of EcN ameliorated acute and chronic experimental colitis by modifying proinflammatory cytokine secretion but had no influence on the acute DSS-induced colitis. In this model, preexisting colitis was necessary for translocation of EcN and resident E. coli into MLN.     

L-fucose stimulates utilization of D-ribose by Escherichia coli MG1655 DeltafucAO and E. coli Nissle 1917 DeltafucAO mutants in the mouse intestine and in M9 minimal medium

Escherichia coli MG1655 uses several sugars for growth in the mouse intestine. To determine the roles of L-fucose and D-ribose, an E. coli MG1655 DeltafucAO mutant and an E. coli MG1655 DeltarbsK mutant were fed separately to mice along with wild-type E. coli MG1655. The E. coli MG1655 DeltafucAO mutant colonized the intestine at a level 2 orders of magnitude lower than that of the wild type, but the E. coli MG1655 DeltarbsK mutant and the wild type colonized at nearly identical levels. Surprisingly, an E. coli MG1655 DeltafucAO DeltarbsK mutant was eliminated from the intestine by either wild-type E. coli MG1655 or E. coli MG1655 DeltafucAO, suggesting that the DeltafucAO mutant switches to ribose in vivo. Indeed, in vitro growth experiments showed that L-fucose stimulated utilization of D-ribose by the E. coli MG1655 DeltafucAO mutant but not by an E. coli MG1655 DeltafucK mutant. Since the DeltafucK mutant cannot convert L-fuculose to L-fuculose-1-phosphate, whereas the DeltafucAO mutant accumulates L-fuculose-1-phosphate, the data suggest that L-fuculose-1-phosphate stimulates growth on ribose both in the intestine and in vitro. An E. coli Nissle 1917 DeltafucAO mutant, derived from a human probiotic commensal strain, acted in a manner identical to that of E. coli MG1655 DeltafucAO in vivo and in vitro. Furthermore, L-fucose at a concentration too low to support growth stimulated the utilization of ribose by the wild-type E. coli strains in vitro. Collectively, the data suggest that L-fuculose-1-phosphate plays a role in the regulation of ribose usage as a carbon source by E. coli MG1655 and E. coli Nissle 1917 in the mouse intestine.