The probiotic Escherichia coli Nissle 1917 (EcN) is widely used to maintain remission in ulcerative colitis. This is thought to be mediated by various immunomodulatory and barrier-stabilizing

Loading metrics Open Access Peer-reviewed Research Article Francine C. Paim, Ayako Miyazaki, Stephanie N. Langel, David D. Fischer, Juliet Chepngeno, Steven D. Goodman, Gireesh Rajashekara, Linda J. Saif , Anastasia Nickolaevna Vlasova Escherichia coli Nissle 1917 administered as a dextranomar microsphere biofilm enhances immune responses against human rotavirus in a neonatal malnourished pig model colonized with human infant fecal microbiota Husheem Michael, Francine C. Paim, Ayako Miyazaki, Stephanie N. Langel, David D. Fischer, Juliet Chepngeno, Steven D. Goodman, Gireesh Rajashekara, Linda J. Saif, Anastasia Nickolaevna Vlasova x Published: February 16, 2021 Figures AbstractHuman rotavirus (HRV) is a leading cause of diarrhea in children. It causes significant morbidity and mortality, especially in low- and middle-income countries (LMICs), where HRV vaccine efficacy is low. The probiotic Escherichia coli Nissle (EcN) 1917 has been widely used in the treatment of enteric diseases in humans. However, repeated doses of EcN are required to achieve maximum beneficial effects. Administration of EcN on a microsphere biofilm could increase probiotic stability and persistence, thus maximizing health benefits without repeated administrations. Our aim was to investigate immune enhancement by the probiotic EcN adhered to a dextranomar microsphere biofilm (EcN biofilm) in a neonatal, malnourished piglet model transplanted with human infant fecal microbiota (HIFM) and infected with rotavirus. To create malnourishment, pigs were fed a reduced amount of bovine milk. Decreased HRV fecal shedding and protection from diarrhea were evident in the EcN biofilm treated piglets compared with EcN suspension and control groups. Moreover, EcN biofilm treatment enhanced natural killer cell activity in blood mononuclear cells (MNCs). Increased frequencies of activated plasmacytoid dendritic cells (pDC) in systemic and intestinal tissues and activated conventional dendritic cells (cDC) in blood and duodenum were also observed in EcN biofilm as compared with EcN suspension treated pigs. Furthermore, EcN biofilm treated pigs had increased frequencies of systemic activated and resting/memory antibody forming B cells and IgA+ B cells in the systemic tissues. Similarly, the mean numbers of systemic and intestinal HRV-specific IgA antibody secreting cells (ASCs), as well as HRV-specific IgA antibody titers in serum and small intestinal contents, were increased in the EcN biofilm treated group. In summary EcN biofilm enhanced innate and B cell immune responses after HRV infection and ameliorated diarrhea following HRV challenge in a malnourished, HIFM pig model. Citation: Michael H, Paim FC, Miyazaki A, Langel SN, Fischer DD, Chepngeno J, et al. (2021) Escherichia coli Nissle 1917 administered as a dextranomar microsphere biofilm enhances immune responses against human rotavirus in a neonatal malnourished pig model colonized with human infant fecal microbiota. PLoS ONE 16(2): e0246193. Nicholas J. Mantis, New York State Department of Health, UNITED STATESReceived: October 18, 2020; Accepted: January 14, 2021; Published: February 16, 2021Copyright: © 2021 Michael et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are Availability: All relevant data are within the manuscript and its Supporting Information filesFunding: This work was supported by the Bill and Melinda Gates Foundation (OPP 1117467), the NIAID, NIH (R01 A1099451), federal and state funds appropriated to the Ohio Agricultural Research and Development Center, The Ohio State University and from the NIH Office of Dietary Supplements (ODS) supplemental grant interests: The authors have declared that no competing interests exist. IntroductionHuman rotavirus (HRV) is a leading cause of diarrhea in children. It causes significant morbidity and mortality, especially in developing countries [1]. Malnutrition is a major contributor of high mortality due to viral gastroenteritis, including HRV, in countries with low socioeconomic status [2–4]. A number of studies have shown that malnutrition triggers immune dysfunction, including altered innate and adaptive immune responses, impairment of epithelial cell barrier function and/or dysfunction of intestinal epithelial cells [5–10]. Probiotics are increasingly used to enhance oral vaccine responses and to treat enteric infections [11] and ulcerative colitis in children [12]. The probiotic Escherichia coli Nissle (EcN) 1917 has been widely used in the treatment of ulcerative colitis in humans [13]. EcN lacks virulence factors and possesses unique health-promoting properties [14]. The long term persistence of EcN in humans suggests adaption to a host with an established gut microbiome [15]. Our research group has shown that EcN protected gnotobiotic (Gn) piglets against HRV infection and decreased the severity of diarrhea by modulating innate and adaptive immunity, and protecting the intestinal epithelium [16–18]. Oral administration of probiotics is associated with a number of challenges, such as low pH of gastric acid and bile salts in the stomach, effector functions of the host immune system, and competition with commensal and pathogenic bacteria [19]. These factors adversely influence adherence and persistence of probiotics within the host and thus reduce the beneficial effects [20]. Probiotics must survive in gastric acids to reach the small intestine and colonize the host to confer beneficial effects of preventing or moderating gastrointestinal diseases [21]. Encapsulation of lyophilized probiotics have resulted in enhanced bacterial viability [22, 23]. Navarro and his colleagues (2017) have formulated a new synbiotic formulation that employed porous semi-permeable, biocompatible and biodegradable microspheres (dextranomer microspheres) containing readily diffusible prebiotic cargo [24]. Adherence of the probiotic bacteria to the microsphere has a two-fold effect; it facilitates the more formidable biofilm state of probiotics as well as a creates a directed means to provide a high concentration gradient of prebiotics via diffusion of the microsphere cargo. However, currently there are no strategies for improved EcN probiotic efficacy and stability within the malnourished host. Previously we have established a deficient HIFM-transplanted neonatal pig model that recapitulates major aspects of malnutrition seen in children in impoverished countries [5, 6]. The purpose of this study was to investigate a novel probiotic delivery method to prolong the persistence of probiotics in the gut and to enhance their beneficial effects. We hypothesized that oral administration of EcN attached to the surface of biocompatible dextranomar microspheres in a biofilm state will protect against harsh conditions of the stomach and improve gut stability, thus enhancing their beneficial effects with a single administration compared with the repetitive administration of probiotics in the suspension form, which results in transient and often inconsistent outcomes. In addition, administration of probiotics in their suspension state has modest impact on the host’s microbiome [25]. High doses and repeated administration of probiotics are needed to achieve potential health benefits; however, in impoverished countries this poses challenges due to lack of product availability, the limited health care system, and resources [26–28]. Whether the use of the biofilm microsphere can overcome this remains to be established. The multifactorial pathobiology of malnutrition is associated with a vicious cycle of intestinal dysbiosis, epithelial breaches, altered metabolism, impaired immunity, intestinal inflammation, and malabsorption [29, 30]. Malnutrition increases the risk of diarrheal diseases caused by some, but not all, entero-pathogens. Malnutrition can result in impaired immune defenses that compromise gut integrity, and dybiosis that can influence defense against intestinal pathogens in the malnourished host [31]. This in turn limits the ability of probiotics to repair the intestinal epithelium and establish healthy microbiota. These concerns necessitate further research to enhance the stability and persistence of probiotics in malnourished hosts. Probiotics are generally considered safe, however there are some associated risks. These risks are increased if there are chronic medical conditions that weaken the immune system or if there are gut barrier breeches. Possible risks can include: developing an infection, developing resistance to antibiotics, and developing harmful byproducts from the probiotic supplement. Also, in malnourished hosts due to increased intestinal motility, probiotics can be eliminated from the gut faster limiting their beneficial effects [32, 33]. Furthermore, we aimed to investigate whether a single dose of EcN biofilm microspheres enhances immune responses after HRV infection in a malnourished Gn pig model. Previous transplantation of Gn pigs with probiotic bacteria demonstrated upregulated innate and adaptive immune responses following HRV infection [16, 17, 34–37]. In this preliminary study, we report increased innate immune and B cell responses after EcN biofilm treatment that were associated with protection against HRV disease and infection in a neonatal malnourished, HIFM pig model. Materials and methods Human Infant Fecal Microbiota (HIFM) The collection and use of HIFM was approved by The Ohio State University Institutional Review Board (IRB). With parental consent, sequential fecal samples were collected from a healthy, two-month-old, exclusively breastfed, vaginally delivered infant. Samples were pooled and diluted to 1:20 (wt/vol) in PBS containing (vol/vol) cysteine and 30% glycerol and stored at -80°C as described previously [5, 6]. Virus HRV (VirHRV) Wa strain passaged 25–26 times in Gn piglets was used to orally inoculate piglets at a dose of 1 × 106 fluorescent focus units (FFU) as described previously [5, 6]. Preparation of biofilm dextrananomer microspheres Anhydrous dextranomer microspheres (Sephadex, GE Healthcare Life Sciences, Pittsburgh, PA) were used. Anhydrous microspheres were hydrated in growth medium at 50 mg per ml and autoclaved for 20 min. Autoclaved microspheres were removed from solution on a vacuum filter apparatus and collected via sterile loop into a filter-sterilized 1M solution of sucrose. The microsphere mixture was vortexed and incubated for 24 hours at room temperature (RT). Sugar was removed from solution on a vacuum filter apparatus and collected via sterile loop. The microspheres were then added to EcN [1 × 109 colony-forming unit (CFU) per ml], pelleted, washed, and re-suspended in sterile saline. EcN was allowed to incubate with the microspheres for 1h at RT to facilitate binding and stored in -80°C in 30% glycerol. Prior to use, microspheres were thawed, mixed 1:1 with Natrel and administered orally. For EcN administered as a suspension, 1 × 109 CFU per ml was pelleted and re-suspended in sterile saline in preparation for oral inoculation. Animal experiments The animal experiments were approved by the Institutional Animal Care and Use Committee at The Ohio State University (OSU). Piglets were derived from near-term sows (purchased from OSU specific pathogen-free swine herd) by hysterectomy and maintained in sterile isolators as described previously [38]. For preliminary investigations, neonatal pigs were randomly assigned to three groups: 1) EcN biofilm (n = 3); 2) EcN suspension (n = 4); and 3) control pigs (n = 3). Pigs were fed a deficient diet of 50% ultra-high temperature pasteurized bovine milk diluted with 50% sterile water which contained half of the recommended protein levels ( that met or exceeded the National Research Council Animal Care Committee’s guidelines for calories, fat, protein and carbohydrates in suckling pigs. All pigs were confirmed free from bacterial and fungal contamination prior to HIFM transplantation by aerobic and anaerobic cultures of rectal swabs. Pigs were orally inoculated with 2ml of diluted HIFM stock at 4 days of age (post-HIFM transplantation day, PTD 0). The pigs were colonized orally with EcN biofilm or EcN suspension at PTD 11. Pigs were then challenged with VirHRV [1 × 106 FFU, post challenge day (PCD) 0] at PTD 13 and euthanized at PTD 27/PCD 14. Post-VirHRV challenge, rectal swabs were collected daily to assess HRV shedding. Blood, spleen, duodenum, and ileum were collected to isolate mononuclear cells (MNCs) as described previously (31, 35, 36). Jejunum was collected to isolate intestinal epithelial cells (IECs) using modified protocols [18, 39–41]. Serum and small intestinal contents (SIC) were collected to determine the HRV specific and total antibody responses [6, 17, 34, 42, 43]. Assessment of clinical signs and detection of HRV shedding Rectal swabs were collected daily post-VirHRV challenge. Fecal consistency was scored as follows; 0, normal; 1, pasty; 2, semi-liquid; and 3, liquid, and pigs with fecal score more than 1 were considered as diarrheic. Rectal swabs were suspended in 2 ml of minimum essential medium (MEM) (Life technologies, Waltham, MA, USA), clarified by centrifugation for 800 × g for 10 minutes at 4°C, and stored at -20°C until quantification of infectious HRV by a cell culture immunofluorescence (CCIF) assay as previously described [44]. Isolation of mononuclear cells (MNCs) Systemic (blood and spleen) and intestinal (duodenum and ileum) tissues were collected to isolate MNCs as described previously [36, 45, 46]. The purified MNCs were re-suspended in E-RPMI 1640. The viability of each MNCs preparation was determined by trypan blue exclusion (≥95%). Flow cytometry analysis Freshly isolated MNCs were stained to assess frequencies of conventional dendritic cells (DCs) (cDCs, SWC3a+CD4-CD11R1+) and plasmacytoid DCs (pDCs, SWC3a+CD4+CD11R1-), MHC II and CD103 marker expression on DCs were used in our experiments. Frequencies of IgA+ B lymphocytes were determined by identifying CD79β and IgA expression in MNCs as reported previously [34]. Similarly, frequencies of memory/resting (CD79β+CD2-CD21-) and activated (CD79β+CD2+CD21-) B cells among systemic and intestinal MNCs were determined as described previously [34]. Appropriate isotype matched control antibodies were included. Subsequently, 50,000 events were acquired per sample using BD Accuri C6 flow cytometer (BD Biosciences, San Jose, CA, USA). Data were analyzed using C6 flow sampler software. NK cytotoxicity assay Total blood MNCs and K562 cells were used as effector and target cells, respectively. Effector: target cell ratios of 10:1, 5:1, 1:1 and were used and the assay was done as described previously [47, 48]. HRV-specific and total antibody responses The HRV specific and total antibody titers in serum and SIC were detected by enzyme-linked immunosorbent assay (ELISA) as described previously [6, 17, 34, 42, 43]. To determine the intestinal antibody responses, small intestinal contents (SIC) were collected with protease inhibitors in the medium. HRV-specific Antibody Secreting Cells (ASCs) responses HRV and isotype-specific antibody secretion in MNCs isolated from blood, spleen, duodenum and ileum were analyzed by ELISPOT assay as described previously [17, 34, 42, 43]. Isolation of Intestinal Epithelial Cells (IECs) and extraction of RNA The IECs were isolated from jejunum (mid gut) using a modified protocol adapted from Paim et al. [18, 49]. The viability and numbers of IECs were determined by the trypan blue exclusion method (70–80%). IECs were stored at −80°C in 500 μl of RNAlater tissue collection buffer (Life technologies, Carlsbad, CA, USA) until further analysis. Total RNA from IECs was extracted using Direct-Zol RNA Miniprep (Zymo Research, Irvine, CA, USA) according to the manufacturer’s instructions. The RNA concentrations and purity were measured using NanoDrop 2000c spectrophotometer (Thermo Scientific, Wilmington, DE, USA). Real-time quantitative RT-PCR (qRT-PCR) of CgA, MUC2, PCNA, SOX9 and villin gene mRNA levels in Intestinal Epithelial Cells (IECs) qRT-PCR was performed using equal amounts of total RNA (75 ng) with Power SYBR Green RNA-to-CT 1 step RT-PCR kit (Applied Biosystems, Foster, CA, USA). The primers for enteroendocrine cells chromogramin A (CgA), goblet cells mucin 2 (MUC2), transient amplifying progenitor cells proliferating cell nuclear antigen (PCNA), intestinal epithelial stem cells transcription factor SRY-box9 (SOX9), enterocytes (villin) and β-actin were based on previously published data [18, 39–41]. Relative gene expression of CgA, MUC2, PCNA, SOX9 and villin were normalized to β-actin and expressed as fold change using the 2-ΔΔCt method [50]. Statistical analysis All statistical analyses were performed using GraphPad Prism version 6 (GraphPad software, Inc., La Jolla, CA). Log10 transformed isotype ELISA antibody titers that were analyzed using one-way ANOVA followed by Duncan’s multiple range test. Data represent the mean numbers of HRV specific antibody secreting cells per 5 × 105 mononuclear cells and analyzed using non-parametric t-test (Mann-Whitney). HRV shedding and diarrheal analysis were performed using two way ANOVA followed by Bonferroni posttest. *P values < **P values < and ***P values < Error bars indicate the standard error of mean. Results EcN biofilm treatment reduced fecal HRV shedding and protected malnourished pigs from diarrhea post HRV challenge Analysis revealed that EcN biofilm treated malnourished pigs had shorter and delayed onset of HRV shedding as compared with the EcN suspension and the control group pigs (Table 1). A significant reduction in fecal virus peak titers shed was observed both in EcN biofilm (GMT = FFU/ml) and EcN suspension groups (GMT = FFU/ml), as compared with the control pigs (GMT = FFU/ml). In addition, EcN biofilm and EcN suspension groups had decreased peak shedding titers at PCD 2 as compared with that of control pigs (S1 Fig). EcN biofilm treatment shortened the mean duration of viral shedding to days as compared with and days in EcN suspension treated and control pigs, respectively (Table 1). Control pigs developed diarrhea ( at days post HRV challenge, continuing for days with mean cumulative fecal score (Table 1). Single administration of EcN biofilm microspheres completely protected the pigs from diarrhea (Table 1). However, administration of EcN suspension protected only 50% of the pigs from diarrhea. No significant differences were observed for mean days to diarrheal onset ( days), mean cumulative fecal score ( and the mean duration of diarrhea ( days) when they are compared with those in the control group (Table 1). These findings suggest that administration of EcN biofilm suppressed HRV infection greater than EcN administered in suspension. EcN biofilm treatment enhanced natural killer (NK) cell cytotoxicity in blood mononuclear cells (MNCs), increased the frequencies of activated pDCs in systemic and intestinal tissues, and increased activated cDCs in the blood and duodenum NK cell cytotoxicity in blood MNCs was significantly enhanced in EcN biofilm treatment compared with control pigs (Fig 1A). On the other hand, frequency of apoptotic MNCs were marginally decreased in EcN biofilm (3%) compared with EcN suspension (5%) and control ( pigs in blood (S2 Fig). Fig 1. EcN biofilm enhanced NK cell activity in blood mononuclear cells (MNCs) and significantly increased the frequencies of activated pDCs in systemic and intestinal tissues and increased activated cDCs in blood and duodenum (significantly).(a) Blood MNCs and carboxyfluorescein diacetate succinimidyl ester (CFSE) stained K562 tumor cells were used as effector and target cells, respectively, and co-cultured at set ratios to assess the NK cytotoxic function, (EcN biofilm vs control group). The effector: target cell co-cultures were stained with 7-Aminoactinomycin D (7AAD) after 12 hours of incubation at 37°C, and the frequencies of CFSE-7AAD double positive cells (lysed K562 target cells) were assessed by flow cytometry. Mean frequencies of activated (b) pDCs and (c) cDCs in systemic and intestinal tissues. Data represent means ± SEM. Significant differences (*p < **p < ***p < are indicated. Gnotobiotic pigs were transplanted with human infant fecal microbiota (HIFM) at 4 days of age, post-HIFM transplantation day (PTD) 0. Pigs were fed a deficient diet. Probiotic was given to the respective groups at PTD 11, followed by challenge with virulent human rotavirus (HRV) on PTD 13/post-challenge day (PCD) 0 and pigs were euthanized on PTD 27/PCD 14. biofilm treatment significantly increased the frequencies of activated pDC in systemic and intestinal tissues as compared with EcN suspension and the control pigs (Fig 1B). Moreover, EcN biofilm treatment significantly increased the frequencies of activated cDC in duodenum while numerically in blood (Fig 1C). There were no differences observed in other tissues. CD103+ cDC were increased (numerically) in spleen and intestinal tissues in EcN biofilm treated group as compared with EcN suspension and control pigs (S3 Fig). There were no differences observed in blood. EcN biofilm treatment significantly increased the frequencies of activated antibody secreting B cells in systemic tissues, resting antibody forming B cells in blood, and IgA+ B cells in spleen EcN biofilm treated malnourished pigs had significantly increased frequencies of activated antibody forming B cells in systemic tissues as compared with EcN suspension or the control pigs (S4A and S4B Fig). The frequency of IgA+ B cells in the spleen (significantly, S4C Fig) and blood (numerically, S4D Fig) increased in EcN biofilm treatment compared with EcN suspension and control pigs. Moreover, the frequency of resting/memory antibody forming B cells was significantly increased in blood in EcN biofilm compared with EcN suspension treated pigs (S4E Fig). These findings suggest that EcN biofilm treatment enhanced B cell immune response in systemic tissues, although no significant trends were observed in intestinal tissues. EcN biofilm treatment increased the number of HRV-specific Antibody Secreting Cells (ASCs) in systemic and intestinal tissues, and increased HRV-specific IgA antibody titers in serum and Small Intestinal Contents (SIC) Coinciding with decreased HRV shedding and protection from diarrhea, the mean numbers of HRV-specific IgA ASCs were increased in systemic and intestinal tissues of EcN biofilm treatment compared with EcN suspension and control group pigs (Fig 2A and 2B). A similar trend was observed with HRV-specific IgG ASCs (S5 Fig). HRV-specific IgM ASC numbers were below the detection limit in systemic and intestinal tissues. HRV-specific IgA antibody titers were increased in serum (significantly) and SIC (numerically) of EcN biofilm treated pigs compared with EcN suspension and control group pigs, coinciding with increased HRV-specific IgA ASCs (Fig 2C and 2D). Similar trends were observed with HRV-specific IgG antibody titers in serum (S6 Fig). In addition, total IgA concentration was increased (numerically) in serum samples of EcN biofilm treated pigs compared with EcN suspension or control group pigs (S7 Fig). No significant trends were observed in total and HRV-specific IgG in SIC (S8 Fig). These results indicate that EcN biofilm treatment enhanced B cell formation and clonal expansion of antibody producing cells in malnourished, HIFM transplanted pigs infected with HRV. Fig 2. EcN biofilm significantly increased HRV-specific IgA Antibody Secreting Cells (ASCs) in systemic and intestinal tissues and increased HRV-specific IgA antibody titers in serum and Small Intestinal Contents (SIC).(a) HRV-specific IgA ASCs in systemic cells; (b) HRV-specific IgA ASCs in intestinal cells; (c) HRV-specific IgA antibody titers in serum and (d) SIC. No significant differences were observed in intestinal tissues. Data represent means ± SEM. Significant differences (*p < **p < ***p < are indicated. Gnotobiotic pigs were transplanted with human infant fecal microbiota (HIFM) at 4 days of age, post-HIFM transplantation day (PTD) 0. Pigs were fed a deficient diet. Probiotic was given to respective groups at PTD 11, followed by challenge with virulent human rotavirus (HRV) on PTD 13/post-challenge day (PCD) 0 and pigs were euthanized on PTD 27/PCD 14. EcN biofilm treatment significantly upregulated the expression of CgA and SOX9 mRNA levels in jejunal epithelial cells Gene expression levels of CgA, SOX9, villin, MUC2, and PCNA were assessed from jejunal epithelial cells. The relative mRNA levels of CgA, SOX9, and villin genes were increased in jejunal epithelial cells of EcN biofilm compared with EcN suspension and control treated malnourished pigs (Fig 3A–3C). This coincided with the decreased severity of HRV shedding and diarrhea. There were no differences in gene expression levels for MUC2 and PCNA in jejunal epithelial cells of EcN biofilm and EcN suspension treated pigs (S9 Fig). Fig 3. EcN biofilm upregulated the expression of various cell components in jejunal epithelial cells.(a) Relative mRNA levels of enteroendocrine cells chromogramin A (CgA), (b) intestinal epithelial stem cells (SOX9), and (c) enterocytes (villin) in EcN biofilm, EcN suspension groups measured by real-time quantitative RT-PCR (RT-PCR), normalized to β-actin gene. Graphs represent means ± SEM. Significant difference (*p < **p < relative to control) are indicated. Gnotobiotic pigs were transplanted with human infant fecal microbiota (HIFM) at 4 days of age, post-HIFM transplantation day (PTD) 0. Pigs were fed a deficient diet. Probiotic was given to respective groups at PTD 11, followed by challenge with virulent human rotavirus (VirHRV) on PTD 13/post-challenge day (PCD) 0 and pigs were euthanized on PTD 27/PCD 14. DiscussionUsing a malnourished and HIFM transplanted pig model, we showed that compared with EcN administered as suspension, EcN administered as a biofilm on dextranomer microspheres enhanced multiple aspects of the immune response. EcN biofilm treated pigs had significantly reduced titers of virus shedding and diarrhea following VirHRV challenge compared with EcN suspension treated and control pigs. The presence of HRV-specific IgA antibodies in pigs is strongly correlated with protection from HRV infection [46, 51, 52]. Moreover, our study demonstrated for the first time that EcN biofilm treatment enhanced HRV specific-IgA and IgG ASCs in circulation and gut, enhanced HRV-specific IgA and IgG antibody titers in serum and HRV-specific IgA antibody titers in SIC, which collectively coincided with reduced diarrhea and virus shedding. Total IgA concentration was marginally increased in serum of EcN biofilm treated malnourished pigs (data not shown). Although not examined in this study, EcN biofilm treatment might have increased colonization in the gut, inhibiting competition by other members of the gut microbiota [53, 54]. It is possible that the observed effects of EcN biofilm treatment on systemic IgA responses could be mediated by direct modulation of host immune responses, suggesting that biofilm microspheres maybe more stable and persistent compared to probiotics in suspension in the host’s gastrointestinal system. Innate immune responses are critical as a first line of defense, limiting RV replication and disease severity in the host [16, 55]. EcN biofilm treatment enhanced innate immune responses. For example, blood NK cell cytotoxicity was higher in EcN biofilm treatment compared to EcN suspension treated and control groups. This suggests that EcN as a biofilm promoted innate immune responses, improving protection against HRV infection in vivo. Also the frequency of apoptotic blood MNCs was slightly reduced in EcN biofilm treated pigs compared with EcN suspension treatment and control pigs (data not shown). DCs play a key role in probiotic bacteria stimulation of the innate immune system [56, 57] and pDCs were shown to contribute to RV clearance in a murine model [58]. Moreover, DC MHC II expression is a marker for maturation [59]. In our study, higher frequencies of activated pDCs in systemic and intestinal tissues and activated cDCs in the blood and duodenum were observed in EcN biofilm treated pigs compared with EcN suspension treated piglets. These results suggest that the biofilm provided stability to the probiotic and thus enhanced maturation of systemic and intestinal activated DC, promoting pDC development and increased IgA antibody responses in probiotic biofilm treated piglets compared with probiotic suspension treated pigs [60, 61]. Enhancing the protective effects of pDCs via an EcN biofilm may be critical for protection against enteric pathogens [16]. Expression of CD103 (αEβ7 integrin) has been demonstrated to influence cellular intraepithelial morphogenesis and motility [62], which are critical for the proper communication among pathogen, DCs, and T and B lymphocytes. We observed that EcN biofilm treatment increased CD103 expression by DCs and this could have further enhanced innate immune responses against HRV and reduced HRV infection. Consequently, enhancement of signaling between DCs and T/B lymphocytes could have contributed to improved antigen presentation to the lymphocytes resulting in increased HRV-specific IgA ASCs, IgA antibody titers, and increased NK cell activity in EcN biofilm treated pigs. The increased frequencies of activated and resting/memory B cells were enhanced in EcN biofilm treated pigs that coincided with increased frequencies of pDCs in the intestine. These results are similar to our previous studies where EcN protected against HRV infection [34, 37]. The frequency of IgA+ B cells were increased in EcN biofilm treated pigs in systemic tissues, suggesting that EcN as a biofilm may potentiate systemic IgA responses. These responses and the increased HRV-specific IgA antibody responses in serum and SIC coincided with reduced HRV diarrhea and shedding. An upregulation of the enteroendocrine CgA gene in EcN biofilm treated piglets could be reflective of greater protection of the epithelial intestinal barrier. Other studies have shown that enteroendocrine cells that produce hormones promoting repair of intestinal epithelium are activated after treatment with probiotics [63, 64]. In our investigations, we observed an upregulation of stem cell specific-gene SOX9 in the EcN biofilm treated pigs greater than in EcN suspension treated pigs. SOX9 plays an important role in the proliferative capacity of stem cells to replenish different lineages of IECs [65]. Moreover, we demonstrated that EcN biofilm treatment increased mRNA levels of the enterocyte-specific gene villin. It is likely that biofilm microspheres supported a greater number of villin cells and epithelial cells and probiotic adherence. This likely modulated the effects of HRV infection by increasing villin gene expression of enterocytes, repairing/restoring functional enterocytes and increasing barrier and absorptive functions during HRV-induced diarrhea. Our results suggest that using a microsphere biofilm as a novel delivery system for EcN compared to EcN as a suspension may have increased survival of the probiotics at low pH in the stomach and supported increased adherence to intestinal epithelial cells [24], thereby promoting probiotic longevity, survival, and persistence in the malnourished host. Additionally, the EcN biofilm enhanced innate and B cell immune responses in the HRV infected HIFM neonatal pigs. Our results support previous work demonstrating protection against experimental necrotizing enterocolitis in a rat model after treatment with Lactobacillus reuteri adhered to dextranomer microspheres [66]. Recently, Shelby et al. 2020 and colleagues have demonstrated that a single dose of Lactobacillus reuteri in its biofilm state reduces the severity and incidence of experimental C. difficile infection and necrotizing enterocolitis when administered as both prophylactic and treatment therapy [67, 68]. Moreover, Navarro and colleagues demonstrated that probiotic bacterium L. reuteri delivered in association with dextranomar microspheres adhered in greater numbers, conferred resistance to clearance, transported nutrients that promote bacterial growth, promoted the production of the antimicrobial reuterin or histamine, resisted acid-mediated killing, and better supported adherence to intestinal epithelial cells, thereby promoting persistence in the gut [24]. Thus, we this agreed with our hypothesis that EcN adhered to dextranomer microspheres acted similarly during HRV infection in the neonatal malnourished HIFM pig model. In the future, we have plan to increase to number of piglets and study different age groups to further investigate the biofilm impact. Thus, our results suggest that low cost, stable, and efficient dietary supplementation of EcN coupled with a dextranomer microsphere biofilm can protect against HRV infection in a physiologically relevant malnourished HIFM pig model. Similar studies are warranted in children to moderate the symptoms of other gastrointestinal infections and disorders including gastritis and chronic inflammatory bowel disease. Supporting information Acknowledgments We thank Marcia Lee and Rosario Candelero-Rueda for their technical assistance and Dr. Juliette Hanson, Ronna Wood, Jeffery Ogg, Megan Strother and Sara Tallmadge for animal care assistance. References1. Tate JE, Burton AH, Boschi-Pinto C, Steele AD, Duque J, et al. (2012) 2008 estimate of worldwide rotavirus-associated mortality in children younger than 5 years before the introduction of universal rotavirus vaccination programmes: a systematic review and meta-analysis. Lancet Infect Dis 12: 136–141. pmid:22030330 View Article PubMed/NCBI Google Scholar 2. Clark A, Black R, Tate J, Roose A, Kotloff K, et al. (2017) Estimating global, regional and national rotavirus deaths in children aged <5 years: Current approaches, new analyses and proposed improvements. PLoS One 12: e0183392. pmid:28892480 View Article PubMed/NCBI Google Scholar 3. Nelson EA, Glass RI (2010) Rotavirus: realising the potential of a promising vaccine. Lancet 376: 568–570. pmid:20692032 View Article PubMed/NCBI Google Scholar 4. UNICEF W (2017) World Bank Group Joint Child Malnutrition Estimates Levels and trends in child malnutrition. 5. Vlasova AN, Paim FC, Kandasamy S, Alhamo MA, Fischer DD, et al. (2017) Protein Malnutrition Modifies Innate Immunity and Gene Expression by Intestinal Epithelial Cells and Human Rotavirus Infection in Neonatal Gnotobiotic Pigs. mSphere 2. pmid:28261667 View Article PubMed/NCBI Google Scholar 6. Fischer DD, Kandasamy S, Paim FC, Langel SN, Alhamo MA, et al. (2017) Protein Malnutrition Alters Tryptophan and Angiotensin-Converting Enzyme 2 Homeostasis and Adaptive Immune Responses in Human Rotavirus-Infected Gnotobiotic Pigs with Human Infant Fecal Microbiota Transplant. Clin Vaccine Immunol 24. pmid:28637803 View Article PubMed/NCBI Google Scholar 7. Liu J, Bolick DT, Kolling GL, Fu Z, Guerrant RL (2016) Protein Malnutrition Impairs Intestinal Epithelial Cell Turnover, a Potential Mechanism of Increased Cryptosporidiosis in a Murine Model. Infect Immun 84: 3542–3549. pmid:27736783 View Article PubMed/NCBI Google Scholar 8. Hughes SM, Amadi B, Mwiya M, Nkamba H, Tomkins A, et al. (2009) Dendritic cell anergy results from endotoxemia in severe malnutrition. J Immunol 183: 2818–2826. pmid:19625645 View Article PubMed/NCBI Google Scholar 9. Iyer SS, Chatraw JH, Tan WG, Wherry EJ, Becker TC, et al. (2012) Protein energy malnutrition impairs homeostatic proliferation of memory CD8 T cells. J Immunol 188: 77–84. pmid:22116826 View Article PubMed/NCBI Google Scholar 10. Rytter MJ, Kolte L, Briend A, Friis H, Christensen VB (2014) The immune system in children with malnutrition—a systematic review. PLoS One 9: e105017. pmid:25153531 View Article PubMed/NCBI Google Scholar 11. Szajewska H, Mrukowicz JZ (2001) Probiotics in the treatment and prevention of acute infectious diarrhea in infants and children: a systematic review of published randomized, double-blind, placebo-controlled trials. J Pediatr Gastroenterol Nutr 33 Suppl 2: S17–25. pmid:11698781 View Article PubMed/NCBI Google Scholar 12. Sanders ME, Guarner F, Guerrant R, Holt PR, Quigley EM, et al. (2013) An update on the use and investigation of probiotics in health and disease. Gut 62: 787–796. pmid:23474420 View Article PubMed/NCBI Google Scholar 13. Kruis W, Fric P, Pokrotnieks J, Lukas M, Fixa B, et al. (2004) Maintaining remission of ulcerative colitis with the probiotic Escherichia coli Nissle 1917 is as effective as with standard mesalazine. Gut 53: 1617–1623. pmid:15479682 View Article PubMed/NCBI Google Scholar 14. Schultz M (2008) Clinical use of E. coli Nissle 1917 in inflammatory bowel disease Clinical use of E. coli Nissle 1917 in inflammatory bowel disease. Inflamm Bowel Dis 14: 1012–1018. pmid:18240278 View Article PubMed/NCBI Google Scholar 15. Kleta S, Steinrück H., Breves G., Duncker S., Laturnus C., Wieler Schierack P. (2006) Detection and distribution of probiotic Escherichia coli Nissle 1917 clones in swine herds in Germany. J Appl Microbiol 101:1357–66 101 1357–1366. pmid:17105567 View Article PubMed/NCBI Google Scholar 16. Vlasova AN, Shao L, Kandasamy S, Fischer DD, Rauf A, et al. (2016) Escherichia coli Nissle 1917 protects gnotobiotic pigs against human rotavirus by modulating pDC and NK-cell responses. Eur J Immunol 46: 2426–2437. pmid:27457183 View Article PubMed/NCBI Google Scholar 17. Kandasamy S, Vlasova AN, Fischer D, Kumar A, Chattha KS, et al. (2016) Differential Effects of Escherichia coli Nissle and Lactobacillus rhamnosus Strain GG on Human Rotavirus Binding, Infection, and B Cell Immunity. J Immunol 196: 1780–1789. pmid:26800875 View Article PubMed/NCBI Google Scholar 18. Paim FC, Langel SN, Fischer DD, Kandasamy S, Shao L, et al. (2016) Effects of Escherichia coli Nissle 1917 and Ciprofloxacin on small intestinal epithelial cell mRNA expression in the neonatal piglet model of human rotavirus infection. Gut Pathog 8: 66. pmid:27999620 View Article PubMed/NCBI Google Scholar 19. Ding WK, Shah NP (2007) Acid, bile, and heat tolerance of free and microencapsulated probiotic bacteria. J Food Sci 72: M446–450. pmid:18034741 View Article PubMed/NCBI Google Scholar 20. Li XGC Sun Park Cha (2011) Preparation of alginate/chitosan/carboxymethyl chitosan complex microcapsules and application in Lactobacillus casei ATCC 393. Carbohydr Polym: 1479–1485. View Article Google Scholar 21. Muthukumarasamy PA-W P., Holley (2006) Stability of Lactobacillus reuteri in different types of microcapsules. J Food Sci 71: 20–24. View Article Google Scholar 22. Cook MT, Tzortzis G, Charalampopoulos D, Khutoryanskiy VV (2012) Microencapsulation of probiotics for gastrointestinal delivery. J Control Release 162: 56–67. pmid:22698940 View Article PubMed/NCBI Google Scholar 23. Kailasapathy K (2014) Microencapsulation for gastrointestinal delivery of probiotic bacteria,” in Nano Microencapsulation Foods. WileyBlackwell: 167–197. View Article Google Scholar 24. Navarro JB, Mashburn-Warren L, Bakaletz LO, Bailey MT, Goodman SD (2017) Enhanced Probiotic Potential of Lactobacillus reuteri When Delivered as a Biofilm on Dextranomer Microspheres That Contain Beneficial Cargo. Front Microbiol 8: 489. pmid:28396655 View Article PubMed/NCBI Google Scholar 25. Underwood MA, Arriola J, Gerber CW, Kaveti A, Kalanetra KM, et al. (2014) Bifidobacterium longum subsp. infantis in experimental necrotizing enterocolitis: alterations in inflammation, innate immune response, and the microbiota. Pediatr Res 76: 326–333. pmid:25000347 View Article PubMed/NCBI Google Scholar 26. Bahadoria PS, Mahapatra (2001) Prospects, technological aspects, and limitations of probiotics- a world wide review. Eur J Food Res Rev 1: 23–42. View Article Google Scholar 27. Ng EW, Yeung M, Tong PS (2011) Effects of yogurt starter cultures on the survival of Lactobacillus acidophilus. Int J Food Microbiol 145: 169–175. pmid:21196060 View Article PubMed/NCBI Google Scholar 28. Cogan TM, Beresford TP, Steele J, Broadbent J, Shah NP, et al. (2007) Invited review: Advances in starter cultures and cultured foods. J Dairy Sci 90: 4005–4021. pmid:17699017 View Article PubMed/NCBI Google Scholar 29. Guerrant RL, Oria RB, Moore SR, Oria MO, Lima AA (2008) Malnutrition as an enteric infectious disease with long-term effects on child development. Nutr Rev 66: 487–505. pmid:18752473 View Article PubMed/NCBI Google Scholar 30. Prendergast AJ, Kelly P (2016) Interactions between intestinal pathogens, enteropathy and malnutrition in developing countries. Curr Opin Infect Dis 29: 229–236. pmid:26967147 View Article PubMed/NCBI Google Scholar 31. Ibrahim MK, Zambruni M, Melby CL, Melby PC (2017) Impact of Childhood Malnutrition on Host Defense and Infection. Clin Microbiol Rev 30: 919–971. pmid:28768707 View Article PubMed/NCBI Google Scholar 32. Nahaisi MH RS, Noratto GD (2014) Probiotics as a Strategy to Improve Overall Human Health in Developing Countries. J Prob Health 2: 1–9. View Article Google Scholar 33. Maldonado Galdeano C, Cazorla SI, Lemme Dumit JM, Velez E, Perdigon G (2019) Beneficial Effects of Probiotic Consumption on the Immune System. Ann Nutr Metab 74: 115–124. pmid:30673668 View Article PubMed/NCBI Google Scholar 34. Kandasamy S, Chattha KS, Vlasova AN, Rajashekara G, Saif LJ (2014) Lactobacilli and Bifidobacteria enhance mucosal B cell responses and differentially modulate systemic antibody responses to an oral human rotavirus vaccine in a neonatal gnotobiotic pig disease model. Gut Microbes 5: 639–651. pmid:25483333 View Article PubMed/NCBI Google Scholar 35. Chattha KS, Vlasova AN, Kandasamy S, Esseili MA, Siegismund C, et al. (2013) Probiotics and colostrum/milk differentially affect neonatal humoral immune responses to oral rotavirus vaccine. Vaccine 31: 1916–1923. pmid:23453730 View Article PubMed/NCBI Google Scholar 36. Chattha KS, Vlasova AN, Kandasamy S, Rajashekara G, Saif LJ (2013) Divergent immunomodulating effects of probiotics on T cell responses to oral attenuated human rotavirus vaccine and virulent human rotavirus infection in a neonatal gnotobiotic piglet disease model. J Immunol 191: 2446–2456. pmid:23918983 View Article PubMed/NCBI Google Scholar 37. Vlasova AN, Chattha KS, Kandasamy S, Liu Z, Esseili M, et al. (2013) Lactobacilli and bifidobacteria promote immune homeostasis by modulating innate immune responses to human rotavirus in neonatal gnotobiotic pigs. PLoS One 8: e76962. pmid:24098572 View Article PubMed/NCBI Google Scholar 38. Meyer RC, Bohl EH, Kohler EM (1964) Procurement and Maintenance of Germ-Free Seine for Microbiological Investigations. Appl Microbiol 12: 295–300. pmid:14199016 View Article PubMed/NCBI Google Scholar 39. Gonzalez LM, Williamson I, Piedrahita JA, Blikslager AT, Magness ST (2013) Cell lineage identification and stem cell culture in a porcine model for the study of intestinal epithelial regeneration. PLoS One 8: e66465. pmid:23840480 View Article PubMed/NCBI Google Scholar 40. Nossol C, Diesing AK, Walk N, Faber-Zuschratter H, Hartig R, et al. (2011) Air-liquid interface cultures enhance the oxygen supply and trigger the structural and functional differentiation of intestinal porcine epithelial cells (IPEC). Histochem Cell Biol 136: 103–115. pmid:21681518 View Article PubMed/NCBI Google Scholar 41. Collado-Romero M, Arce C, Ramirez-Boo M, Carvajal A, Garrido JJ (2010) Quantitative analysis of the immune response upon Salmonella typhimurium infection along the porcine intestinal gut. Vet Res 41: 23. pmid:19941811 View Article PubMed/NCBI Google Scholar 42. Kandasamy S, Chattha KS, Vlasova AN, Saif LJ (2014) Prenatal vitamin A deficiency impairs adaptive immune responses to pentavalent rotavirus vaccine (RotaTeq(R)) in a neonatal gnotobiotic pig model. Vaccine 32: 816–824. pmid:24380684 View Article PubMed/NCBI Google Scholar 43. Chattha KS, Kandasamy S, Vlasova AN, Saif LJ (2013) Vitamin A deficiency impairs adaptive B and T cell responses to a prototype monovalent attenuated human rotavirus vaccine and virulent human rotavirus challenge in a gnotobiotic piglet model. PLoS One 8: e82966. pmid:24312675 View Article PubMed/NCBI Google Scholar 44. Ward LA, Yuan L, Rosen BI, To TL, Saif LJ (1996) Development of mucosal and systemic lymphoproliferative responses and protective immunity to human group A rotaviruses in a gnotobiotic pig model. Clin Diagn Lab Immunol 3: 342–350. pmid:8705681 View Article PubMed/NCBI Google Scholar 45. Zhang W, Azevedo MS, Wen K, Gonzalez A, Saif LJ, et al. (2008) Probiotic Lactobacillus acidophilus enhances the immunogenicity of an oral rotavirus vaccine in gnotobiotic pigs. Vaccine 26: 3655–3661. pmid:18524434 View Article PubMed/NCBI Google Scholar 46. Yuan L, Ward LA, Rosen BI, To TL, Saif LJ (1996) Systematic and intestinal antibody-secreting cell responses and correlates of protective immunity to human rotavirus in a gnotobiotic pig model of disease. J Virol 70: 3075–3083. pmid:8627786 View Article PubMed/NCBI Google Scholar 47. Annamalai T, Saif LJ, Lu Z, Jung K (2015) Age-dependent variation in innate immune responses to porcine epidemic diarrhea virus infection in suckling versus weaned pigs. Vet Immunol Immunopathol 168: 193–202. pmid:26433606 View Article PubMed/NCBI Google Scholar 48. Cario E (2010) Toll-like receptors in inflammatory bowel diseases: a decade later. Inflamm Bowel Dis 16: 1583–1597. pmid:20803699 View Article PubMed/NCBI Google Scholar 49. Pan D, Das A, Liu D, Veazey RS, Pahar B (2012) Isolation and characterization of intestinal epithelial cells from normal and SIV-infected rhesus macaques. PLoS One 7: e30247. pmid:22291924 View Article PubMed/NCBI Google Scholar 50. Schmittgen TD, Livak KJ (2008) Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc 3: 1101–1108. pmid:18546601 View Article PubMed/NCBI Google Scholar 51. Azevedo MS, Yuan L, Iosef C, Chang KO, Kim Y, et al. (2004) Magnitude of serum and intestinal antibody responses induced by sequential replicating and nonreplicating rotavirus vaccines in gnotobiotic pigs and correlation with protection. Clin Diagn Lab Immunol 11: 12–20. pmid:14715539 View Article PubMed/NCBI Google Scholar 52. To TL, Ward LA, Yuan L, Saif LJ (1998) Serum and intestinal isotype antibody responses and correlates of protective immunity to human rotavirus in a gnotobiotic pig model of disease. J Gen Virol 79 (Pt 11): 2661–2672. View Article Google Scholar 53. Louis P, O’Byrne CP (2010) Life in the gut: microbial responses to stress in the gastrointestinal tract. Sci Prog 93: 7–36. pmid:20222354 View Article PubMed/NCBI Google Scholar 54. Kamada N, Chen GY, Inohara N, Nunez G (2013) Control of pathogens and pathobionts by the gut microbiota. Nat Immunol 14: 685–690. pmid:23778796 View Article PubMed/NCBI Google Scholar 55. Holloway G, Coulson BS (2013) Innate cellular responses to rotavirus infection. J Gen Virol 94: 1151–1160. pmid:23486667 View Article PubMed/NCBI Google Scholar 56. Foligne B, Zoumpopoulou G, Dewulf J, Ben Younes A, Chareyre F, et al. (2007) A key role of dendritic cells in probiotic functionality. PLoS One 2: e313. pmid:17375199 View Article PubMed/NCBI Google Scholar 57. Sugimura T, Takahashi H, Jounai K, Ohshio K, Kanayama M, et al. (2015) Effects of oral intake of plasmacytoid dendritic cells-stimulative lactic acid bacterial strain on pathogenesis of influenza-like illness and immunological response to influenza virus. Br J Nutr 114: 727–733. pmid:26234407 View Article PubMed/NCBI Google Scholar 58. Kruis W, Chrubasik S, Boehm S, Stange C, Schulze J (2012) A double-blind placebo-controlled trial to study therapeutic effects of probiotic Escherichia coli Nissle 1917 in subgroups of patients with irritable bowel syndrome. Int J Colorectal Dis 27: 467–474. pmid:22130826 View Article PubMed/NCBI Google Scholar 59. Winzler C, Rovere P, Rescigno M, Granucci F, Penna G, et al. (1997) Maturation stages of mouse dendritic cells in growth factor-dependent long-term cultures. J Exp Med 185: 317–328. pmid:9016880 View Article PubMed/NCBI Google Scholar 60. Deal EM, Lahl K, Narvaez CF, Butcher EC, Greenberg HB (2013) Plasmacytoid dendritic cells promote rotavirus-induced human and murine B cell responses. J Clin Invest 123: 2464–2474. pmid:23635775 View Article PubMed/NCBI Google Scholar 61. Tezuka H, Abe Y, Asano J, Sato T, Liu J, et al. (2011) Prominent role for plasmacytoid dendritic cells in mucosal T cell-independent IgA induction. Immunity 34: 247–257. pmid:21333555 View Article PubMed/NCBI Google Scholar 62. Schlickum S, Sennefelder H, Friedrich M, Harms G, Lohse MJ, et al. (2008) Integrin alpha E(CD103)beta 7 influences cellular shape and motility in a ligand-dependent fashion. Blood 112: 619–625. pmid:18492951 View Article PubMed/NCBI Google Scholar 63. Hajela N, Nair GB, Abraham P, Ganguly NK (2012) Health impact of probiotics—vision and opportunities. Gut Pathog 4: 1. pmid:22410274 View Article PubMed/NCBI Google Scholar 64. Skipper M, Lewis J (2000) Getting to the guts of enteroendocrine differentiation. Nat Genet 24: 3–4. pmid:10615112 View Article PubMed/NCBI Google Scholar 65. Carulli AJ, Samuelson LC, Schnell S (2014) Unraveling intestinal stem cell behavior with models of crypt dynamics. Integr Biol (Camb) 6: 243–257. pmid:24480852 View Article PubMed/NCBI Google Scholar 66. Olson JK, Rager TM, Navarro JB, Mashburn-Warren L, Goodman SD, et al. (2016) Harvesting the benefits of biofilms: A novel probiotic delivery system for the prevention of necrotizing enterocolitis. J Pediatr Surg 51: 936–941. pmid:27032609 View Article PubMed/NCBI Google Scholar 67. Shelby RD, Janzow GE, Mashburn-Warren L, Galley J, Tengberg N, et al. (2020) A novel probiotic therapeutic in a murine model of Clostridioides difficile colitis. Gut Microbes 12: 1814119. pmid:32954922 View Article PubMed/NCBI Google Scholar 68. Olson JK, Navarro JB, Allen JM, McCulloh CJ, Mashburn-Warren L, et al. (2018) An enhanced Lactobacillus reuteri biofilm formulation that increases protection against experimental necrotizing enterocolitis. Am J Physiol Gastrointest Liver Physiol 315: G408–G419. pmid:29848024 View Article PubMed/NCBI Google Scholar

Here, Escherichia coli Nissle 1917 (ECN), a kind of oral probiotic, was genetically engineered to overexpress catalase and superoxide dismutase (ECN-pE) for the treatment of intestinal inflammation. To improve the bioavailability of ECN-pE in the gastrointestinal tract, chitosan and sodium alginate, effective biofilms, were used to coat ECN-pE

AbstractAllergic asthma is characterized by a strong Th2 and Th17 response with inflammatory cell recruitment, airways hyperreactivity and structural changes in the lung. The protease allergen papain disrupts the airway epithelium triggering a rapid eosinophilic inflammation by innate lymphoid cell type 2 (ILC2) activation, leading to a Th2 immune response. Here we asked whether the daily oral administrations of the probiotic Escherichia coli strain Nissle 1917 (ECN) might affect the outcome of the papain protease induced allergic lung inflammation in BL6 mice. We find that ECN gavage significantly prevented the severe allergic response induced by repeated papain challenges and reduced lung inflammatory cell recruitment, Th2 and Th17 response and respiratory epithelial barrier disruption with emphysema and airway hyperreactivity. In conclusion, ECN administration attenuated severe protease induced allergic inflammation, which may be beneficial to prevent allergic asthma. IntroductionAllergic asthma is one of the most common chronic respiratory diseases with a significant impact on public health1,2. In recent years, the incidence of allergic asthma in developed countries has dramatically increased and it is predicted that the number of affected people worldwide will increase by 100 million by 20253. Risk alleles have been identified for the development of asthma4 but the rapidity of its increased incidence does not support solely a genetic basis and suggest the involvement of environmental factors. Long-term observations support the notion that urban life is associated with increased prevalence of chronic immunological disorders including asthma incidence as compared to children living in farms5. Early in life microbial exposure might modulate allergic disorders6. In addition, such favorable socioeconomic factors, like enriched dietary habits or increased level of hygiene are presumably important factors for a considerable shift in the gut microbiota and increased asthma susceptibility. Epidemiological and clinical studies indicate an association between alteration of intestinal microbial communities and increased incidence of allergic asthma7. Several studies revealed changes in gut microbiota composition in adults suffering from allergic diseases at distant body sites (eczema, rhinitis, asthma)8,9, which precede the development of allergic diseases10,11. Gut bacteria outnumber the human body cells and the microbiome encode approximately 100 times more genes than the human genome12. This impressive genetic capacity contribute to essential functions for the host including nutrients supply like short-chain fatty acids (SCFAs)13,14, vitamins and hormones15, energy balance16,17,18, metabolic signaling19, resistance to pathogens colonization20,21,22 and has a key role in promoting the postnatal maturation of the intestinal mucosal barrier23,24, etiology is complex, but exposure to allergens or air pollution, are clearly important factors for the pathogenesis5. Sensitization to allergen is one of the first steps involved in asthma. Various allergens, including house dust mite (HDM), fungi, cockroach and pollen have proteolytic activities26. Protease properties of allergens cause injury of the airway epithelium with increased permeability, airway remodeling, type 2 cytokine and chemokine production and cell recruitment27. Papain, a cysteine protease, induces a type 2 response characterized by interleukin (IL)-5 and IL-13 production, mediated by an IL-2-dependent IL-9 production28 and specific IgE production29,30. There is evidence that the commensal microflora is critical in the maintenance of systemic immune tolerance, which is instrumental in protecting against allergic asthma. Escherichia coli strain Nissle 1917 (Mutaflor®, ECN) is successfully used for the treatment of intestinal inflammation, especially in patients suffering from ulcerative colitis31. In the present study, we investigated the impact of the colonization by ECN on the allergic lung inflammatory response induced by single or repeated challenges to the protease allergen papain. We show here that chronic ECN administration reduces severe allergic lung inflammation, improves the respiratory epithelial barrier function and modulates emphysema in response to repeated papain colonization has a dual effect in acute papain-induced lung inflammationTo study the impact of the administration of the ECN strain on the development of allergic inflammation, we compared the susceptibility ECN treated mice to acute papain-induced lung inflammation in comparison to non-treated controls according to the protocol shown in Fig. 1a. ECN was administered by gavage over 6 days (108 cfu of live ECN/day) then the mice were challenged twice by intranasal instillation ( of the protease allergen papain (25 µg on day 7 and 8 and the inflammatory response was analyzed 24 h later as described before32. Microscopic examinations of the lungs revealed focal inflammatory cell infiltration around bronchi, capillaries and in alveoli, as well as mucus hypersecretion (Fig. 1b). The lung inflammation as assessed by a semi-quantitative score of microscopic lesions was not reduced in ECN fed mice (Fig. 1b,c), except for the production of mucus (Fig. 1d).Figure 1ECN colonization as a dual effect in acute papain-induced lung inflammation. (a) Experimental settings of acute papaïn-induced lung inflammation and ECN treatment. (b) Lung tissues were histologically examined 24 h after the last papaïn challenge. Lung sections stained with HE from controls (NaCl/NaCl), papaïn (NaCl/Papaïn) and ECN (ECN/Papaïn)-treated mice are represented. (c) Histological score of lung inflammation infiltration was performed on paraffin embedded section after HE staining. (d) Histological score of lung mucus production was performed on paraffin embedded section after PAS staining. (e) Total cells and differential cell count of eosinophils, neutrophils, lymphocytes and macrophages were determined in BALF by numeration of MGG stained cytospin. Lung homogenate level of (F) CCL11, (g) CCL17 and (h) CXCL1 were measured by ELISA. Data are expressed as mean + SEM from a single experiment representative of 2 experiments with n = 5 mice per group. The parametric one-way or two-way ANOVA test with multiple Bonferroni’s comparison test was used. *, ** and *** refer to P < P < and P < size imagePapain-induced lung inflammation is associated with enhanced cell recruitment in the lung, involving especially eosinophils32. Cell recruitment into the broncho-alveolar lavage fluid (BALF) was modulated with increased total cells, especially neutrophils upon ECN treatment as compared to control mice (Fig. 1e) with increased myeloperoxidase (MPO) (Supplementary Figure 1) and neutrophil chemoattractant CXCL1 levels (Fig. 1h). By contrast, the recruitment of eosinophils in the BALF was significantly decreased in ECN-treated animals as compared to papain controls (Fig. 1e). This was correlated with a lowered production of CCL17 (Fig. 1g) while CCL11 levels was not modified (Fig. 1f).Interestingly, mice treated with a non-probiotic K12 E. coli strain MG1655 and tested in the acute papain model (Supplementary Figure 2A) develop a similar lung neutrophilia as compared to ECN-treated animals (Supplementary Figure 2B–D), suggesting that this effect is probably mediated an E. coli genus dependent molecular determinant. On the contrary, MG1655 treatment has no protective effect on eosinophilia as observed with cell count and chemokine production (Supplementary Figure 2B,E,F). Taken together, these results suggest that gut colonization by ECN may modulate lung inflammation by enhancing neutrophil, but importantly reducing eosinophil cell recruitment in BALF and tissue. This data motivated studies in a chronic model of lung allergic lung inflammation induced by repeated papain challenges is attenuated by ECN administrationTo determine whether ECN modulates chronic airway inflammation induced by a protease allergen papain, BL6 mice were immunized with papain (25 µg on days 6, 7 by intranasal route), followed by two intranasal challenges at day 20 and 25 (25 µg). Control mice received vehicle (NaCl). In addition, mice were orally administered with 108 cfu of live ECN (Fig. 2a). 24 h after the last papain challenge, the mice were sacrificed and the extent of the lung inflammation was assessed. Histological analysis revealed a prominent lung inflammation characterized by perivascular, peribronchial and alveolar infiltration of eosinophils, neutrophils and air space enlargement with epithelial damage and disruption of alveolar septa, a hallmark of emphysema upon papain challenge (Fig. 2b,c). ECN-treated mice largely prevented lung inflammation, epithelial injury and emphysema (Fig. 2b–d). Finally, the extensive goblet cell hyperplasia and mucus production observed in primed/challenged mice was lowered in ECN probiotic treated mice (Fig. 2b,e). Diminished mucus expression was confirmed at the mRNA level for Muc5ac in lung (Fig. 2f). Interestingly, mice treated with E. coli strain MG1655 and tested in the chronic papain model develop a similar lung inflammation as compared to untreated animals, as revealed by the histological analysis (Supplementary Figure 3A–E), suggesting that the protective effect observed with ECN is due to intrinsic probiotic properties rather than a non-specific effect due to daily gavage E. coli species on the gut microbiota. The absence of protection with MG1655 is unlikely related to the lack of gut colonization, as we quantified equivalent Enterobacteria and E. coli colony counts in both ECN- and MG1655-treated animals along the treatment (Supplementary Figure 4).Figure 2Repeated papain challenges causing severe lung inflammation is attenuated by ECN administration. (a) Experimental settings of chronic papaïn-induced lung inflammation and ECN treatment. (b) Lung tissues were histologically examined 24 h after the last papaïn challenge. Lung sections stained with HE from controls (NaCl/NaCl), papaïn (NaCl/Papaïn) and ECN (ECN/Papaïn)-treated mice are represented. (c) Histological score of lung inflammation infiltration was performed on paraffin embedded section after HE staining. (d) Histological score of airway remodeling was performed on paraffin embedded section after HE staining. (e) Histological score of lung mucus production was performed on paraffin embedded section after PAS staining. (f) Muc5ac relative gene expression levels in lung tissues was measured by qPCR. Data are expressed as mean + SEM from a single experiment representative of 2 experiments with n = 5 mice per group. The parametric one-way or two-way ANOVA test with multiple Bonferroni’s comparison test was used. *, ** and *** refer to P < P < and P < size imageECN-treated mice develop reduced airway eosinophilia and Th2-driven airway inflammation upon papain chronic challengesPapain-induced chronic inflammation is characterized by a type 2 inflammatory response28. To determine whether ECN inhibited inflammatory cell recruitment, BALF cell counts were assessed for cell phenotyping. Saline sensitized and challenged mice present negligible leukocyte numbers in BALF, whereas papain-treated mice presented a dramatic increase of total cells, eosinophils and fewer neutrophils and macrophages (Fig. 3a). By contrast, ECN-treated mice had ~ less total BALF cell counts with a 2-fold reduction in eosinophils, neutrophils and macrophages. This was consistent with significant lower levels of eosinophils attracting chemokines CCL24 and CCL11 (Fig. 3b,d), EPO levels (Supplementary Figure 5) and neutrophils/monocytes chemoattractant CXCL1 (Fig. 3e), while CCL17 was unchanged in the lungs of ECN-treated mice as compared to controls. Moreover, Th2 cytokines such as IL-5 and to a lesser extent IL4 were significantly reduced in the lung of ECN-treated mice as compared to papain controls (Fig. 3f,g). The production of IFNγ was reduced, while IL17A level was unchanged in ECN probiotic-treated mice (Fig. 3h,i).Figure 3ECN-treated mice develop reduced airway eosinophilia and Th2-driven airway inflammation upon papaïn chronic challenges. (a) Total cells and differential cell count of eosinophils, neutrophils, lymphocytes and macrophages were determined in BALF by numeration of MGG stained cytospin. Lung homogenate level of (b) CCL24, (C) CCL17, (D) CCL11, (e) CXCL1, (f) IL-4, (g) IL-5, (h) IL-17 and (i) IFNγ were measured by ELISA. Data are expressed as mean + SEM from a single experiment representative of 2 experiments with n = 5 mice per group. The parametric one-way or two-way ANOVA test with multiple Bonferroni’s comparison test was used. *, ** and *** refer to P < P < and P < size imageTaking together, these data indicate that ECN gut colonization reduces papain induced Th2 immune airways hyperreactivity and respiratory barrier injury is attenuatedA hallmark of allergic lung inflammation is airways hyperreactivity (AHR), which is due functional changes of the respiratory barrier. AHR was assessed by invasive plethysmography in untreated and ECN-treated mice upon chronic papain exposure. Airway resistance and compliance in response to methacholine as a measure of AHR and were increased upon papain challenge. ECN administration reduced airway resistance and compliance indicating a significant amelioration of the lung function (Fig. 4a,b).Figure 4Papaïn-induced pulmonary dysfunction is attenuated by ECN. (a) Airway hyper-responsiveness to increasing doses of methacholine (Mch; 0−200 mg/ml) was measured by recording changes in lung resistance and (b) airway compliance. The pulmonary epithelial integrity was assessed by the leak of (c) Evans blue and (d) total protein in BAL. (e) Immunofluorescent staining for E-cadherin (green) on lung cryosections. (f) Quantitative evaluation of E-cadherin expression on lung sections. Data are expressed as mean + SEM from a single experiment representative of 2 experiments with n = 5 mice per group. The parametric one-way or two-way ANOVA test with multiple Bonferroni’s comparison test was used. *, ** and *** refer to P < P < and P < size imageThe protease papain induces inflammation and injury of the lung epithelium and capillaries with increased vascular permeability. The probiotic ECN has the ability to strengthen the epithelial barrier33. We used Evans Blue (EB), which binds to serum albumin, as a tracer of the capillary leak of macromolecules from the circulation into the BALF. Our data reveal that ECN treatment reduced the acute lung capillary/epithelial leak of intravenous administered EB upon papain exposure (Fig. 4c). Furthermore, total protein in BALF was also reduced (Fig. 4d). To get further insights into the role of ECN in the improvement of lung epithelial barrier function during allergic asthma, lung histological sections were analyzed for the expression of E-cadherin, a critical component of the epithelial barrier, which is crucial in the maintenance of the immunologic tolerance during airway allergic sensitization34. Immunofluorescence analysis revealed reduced E-cadherin expression concomitant with epithelial cell injury upon papain exposure, while ECN feeding attenuated the reduction of E-cadherin expression (Fig. 4e), which was confirmed by a semi-quantitative assessment of E-cadherin immunostaining (Fig. 4f).Therefore ECN colonization attenuated papain protease induced allergic lung inflammation with reduced Th2 response and airways hyperreactivity. Importantly the protease induced injury of the alveolar septae reflected by emphysema and of the respiratory barrier were significantly diminished by the probiotic strain mice has reduced Th2 lymphocytes and ILC2 activation upon papain chronic challengesTh2 lymphocytes and ILC2 accumulate in lungs after papaïn exposure and produce IL-5 and IL-1335. We determine the relative contribution of ECN on Th2 and ILC2 activation 24 h after the last allergen challenge. Lung cells were restimulated by papain and the production of cytokines was analyzed. IL-5 (Fig. 5a) and to a lesser extent IL-13 (Fig. 5b) was significantly reduced upon ECN treatment while IL-33 levels remain unchanged (Fig. 5c). Total Th2 and ILC2 producing IL-5 and IL-13 were analyzed by flow cytometry (Supplementary Figures 6 and 7). The frequency of CD3+ CD4+ IL5+ or IL13+ cells were significantly reduced in ECN-treated mice as compared to untreated controls (Fig. 5d–f). This was associated with a similar decrease of ILC2+ and ILC2+ IL13+ (Fig. 5g–i). These data indicate that ECN was able to dampen Th2 and ILC2 activation and the production of the prototypal pro-allergenic IL-5 and 5ECN-treated mice has reduced Th2 lymphocytes and ILC2 activation upon papain chronic challenges. IL-5 (a), IL-13 (b) and IL-33 (c) levels after lung mononuclear cell restimulation with papaïn for 72 h. Frequency of CD3+ CD4+ lymphocytes (d) producing IL-5 (e) or IL-13 (f) are shown. Frequency of ILC2 (g) producing IL-5 (h) or IL-13 (i) are shown. Data are expressed as mean + SEM from a single experiment with n = 5 mice per group. The parametric one-way or two-way ANOVA test with multiple Bonferroni’s comparison test was used. * and ** refer to P < and P < size imageDiscussionAllergic asthma is a major health issue with increasing incidence especially in developed countries with an epidemic feature36. Asthma etiology is complex including both genetic and environmental factors, such as exposure to allergens and/or air pollution, are important for the pathogenesis5. Data regarding the use of probiotics in the prevention of allergic diseases and asthma are conflicting37. Several different bacterial strains or combinations have been used in clinical trials to assess protective effects in the context of allergic asthma with significant reduction of both incidence and severity of allergic diseases38 which were not confirmed by others39. A meta-analysis concluded that probiotic are not efficient for the prevention of allergy40. This discrepancy may be related to the dose and duration of probiotic administration, immunomodulatory differences41 among strains, mostly Lactobacillus or Bifidobacterium probiotics42. Here we evaluated the probiotic potential of the Gram negative ECN to prevent allergic lung inflammatory allergic response induced by the protease papain. ECN drastically reduced the severity of chronic lung inflammation through the modulation of the Th2 inflammatory response, injury of the respiratory barrier and airways hyperreactivity. The beneficial effects of ECN has been demonstrated before in intestinal inflammatory disorders, especially in ulcerative colitis43. Two previous studies investigated ECN in experimental asthma. Bickert et al. using the inert protein allergen OVA observed a protection upon oral administration of ECN, but no inhibition of the Th2 immune response44. Adam et al. evaluated the prophylactic potential of ECN on recombinant house mite antigen Derp1 as mucosal antigen. ECN strongly reduced the antigen specific humoral response45. Here, using oral prophylactic administration of ECN we demonstrate for the first time a reduction of papain-induced lung inflammation and amelioration of AHR. In contrast, mice administered K12 E. coli strain MG1655 were as sensitive to lung inflammation as untreated papain challenged mice suggesting that the genetic background of the strain is of particular importance and determines its ability to act as a probiotic. Nevertheless, we observed that both E. coli strains has the ability to induce a potent lung neutrophilia. These results are in line with several papers demonstrating that ECN capsule antigen K5 was an important contributor the recruitment of neutrophil46,47. More generally, it has also been suggested that the presence of capsular antigen may induce an increased influx of pulmonary neutrophils48,49. The mechanisms by which capsular antigen modulate neutrophil response are not completely understood but may include direct effect such an upregulation of shed bacterial formylmethionyl-leucyl-phenylalanine50, a potent neutrophil chemotactic factor; or indirect by modulating the host’s generation of chemokines, including CXCL1 or IL-8 which was observed upon ECN or MG1655 of the best-characterized features contributing to the effectiveness of ECN is its ability to strengthen the epithelial barrier function51. This probiotic property of ECN has been extensively demonstrated in the context of intestinal inflammatory diseases. Asthma is often associated with mucosal barrier dysfunction52. We found that respiratory barrier dysfunction due to papain-induced inflammation and injury is alleviated by ECN with reduced protein leak and upregulation of E-cadherin. Recent studies suggests that this adhesion molecule contributes to the structural and immunological function of the airway epithelium, acting as a rheostat through the regulation of epithelial junctions and production of pro-inflammatory mediators34. Alterations of the airway epithelium enhance both allergic sensitization and airway remodeling including goblet cell hyperplasia, mucus hyperproduction and subepithelial fibrosis53 thus contributing to severe airways hyperreactivity. ECN conferred a significant reduction of inflammatory cell recruitment in BALF, lung tissue inflammation and disruption of alveolar septa with epithelial cells participate in the innate immune response of the lung and have barrier function. Barrier dysfunction favors the access of noxious or immunogenic protein or chemicals to the mucosa-associated lymphoid tissues. Thus, regulation of airway epithelial barrier function is an important checkpoint of the immune response during asthma54. In the present study, we show that ECN treatment affects a prevalent Th2 response known for papain induced lung inflammation28. We observed a significant reduction of eosinophils and eosinophil-related chemokines/cytokines associated with diminished recruitment of neutrophils and CXCL1 and IFN-γ levels. The data are consistent with previous studies showing that colonization by ECN lead to a modification of the cytokines repertoire55,56. In addition, we show for the first time that ECN treatment reduce Th2 CD4+ lymphocytes as well as ILC2 activation, resulting in decreased IL-5 and IL-13 production. The latter population is known to precede Th2 activation which is the cardinal feature of allergic asthma, culminating in airway hyperresponsiveness and Th2 cytokines and chemokines. In this setting, we investigated IL-33, which is known to be involved in ILC2 activation35 but we did not find any difference upon ECN treatment, which was also the case in another reduced allergic asthma molecular rationale behind the immunomodulatory properties of ECN has not yet been elucidated and is under investigation58. The beneficial effect of ECN could rely on the improvement of the intestinal barrier function and the resulting prevention of a continuous stimulation of the host innate immune system by the gut components. Indeed, we have recently demonstrated that ECN was able to prevent CNS inflammation through the improvement of the intestinal permeability59 showing that modulation of the gut microbiota with ECN exerts remote immunological imprinting. ECN genome encodes the production of specialized molecules that may modulate immune functions60,61,62. The intestinal mucosa represents an interface between bacterial-derived metabolites and mucosal immune processes that will influence immunological processes on the host conclusion, our findings indicate that ECN is able to prevent papain-induced lung inflammation after high dose per os administration supporting a gut-lung mucosal communication64. In addition, our results suggest that the prevention of the respiratory barrier dysfunction by probiotic treatment may be important to control allergic lung inflammation. Therefore, ECN might be considered as a valuable prophylactic or diet supplement to prevent allergic (B6) mice were bred in our specific pathogen free animal facility at TAAM-CNRS, Orleans, France (agreement D-45-234-6 delivered on March, 10 of 2014). Mice were maintained in a temperature-controlled (23 °C) facility with a strict 12 h light/dark cycle and were given free access to food and water. The experiments were performed with female mice aged 8–10 weeks using 5 mice per group, and the experiments were repeated at least twice. All animal experimental protocols were carried out in accordance with the French ethical and animal experiments regulations (see Charte Nationale, Code Rural R 214-122, 214-124 and European Union Directive 86/609/EEC) and were approved by the “Ethics Committee for Animal Experimentation of CNRS Campus Orleans” (CCO), registered (N°3) by the French National Committee of Ethical Reflexion for Animal Experimentation (CLE CCO 2013-1006).Bacterial preparation, growth conditions and administrationThe strains used in this study are the probiotic Escherichia coli Nissle 1917 (ECN) and the archetypal K12 E. coli strain MG1655. Both strains were engineered to exhibit a mutation in the rpsL gene, which is known to confer resistance to streptomycin62. Before oral administrations, ECN and MG1655 strains were grown for 6 h in LB broth supplemented with streptomycin (50 µg/mL) at 37 °C with shaking. This culture was diluted 1:100 in LB broth without antibiotics and cultured overnight at 37 °C with shaking. Bacterial pellets from this overnight culture were diluted in sterile PBS to the concentration of 109 colony forming units (cfu)/ml. Mice were treated by oral gavage with 108 cfu of ECN or MG1655 in 100 µl of PBS or 100 µl of PBS as negative lung inflammation model in miceMice were anesthetized by an iv injection of ketamine/xylazine followed by an intranasal administration of 25 µg of papain (Calbiochem, Darmstadt, Germany) in 40 µL of saline solution. Mice were euthanized by CO2 inhalation 24 h after papain administration and BALF was collected. After a hearth perfusion with ISOTON II (Acid free balanced electrolyte solution Beckman Coulter, Krefeld, Germany) lung were collected and sampled for alveolar lavage (BAL)BAL was performed by 4 lavages of lung with 500 µL of saline solution via a cannula introduced into mice trachea. BAL fluids were centrifuged at 400 g for 10 min at 4 °C, the supernatants were stored at −20 °C for ELISA analysis and pellets were recovered to prepare cytospin (Thermo scientific, Waltham, USA) glass slides followed by a Diff-Quik (Merz & Dade Dudingen, Switzerland) staining. Differential cell counts were performed with at least 400 eosinophil peroxidase (EPO) activityEPO activity was determined in order to estimate the recruitment of eosinophil counts in lung parenchyma as expressionTotal RNA was isolated from homogenized mouse lung using Tri Reagent (Sigma) and quantified by NanoDrop (Nd-1000). Reverse transcription was performed withSuperScript III Kit according to manufacturers’ instructions (Invitrogen). cDNA was subjected to quantitative PCR using primers for Muc5ac (sense 5′-CAGCCGAGAGGAGGGTTTGATCT-3′ and anti-sense 5′-AGTCTCTCTCCGCTCCTCTCA-3′; Sigma). Relative transcript expression of a gene is given as 2−ΔCt(ΔCt = Cttarget−Ctreference), and relative changes compared with control are 2−ΔΔCtvalues (ΔΔCt = ΔCttreated−ΔCtcontrol) {John, 2014 #340}.Enzyme-linked Immunosorbent assay (ELISA)Homogenized lung or cell supernatant were tested for MPO, CXCL1, CCL24, CCL11, CCL17, IL-4, IL17A and IFNγ (R&D systems Abingdon, UK), IL-13, IL-5, IL-33 (eBiosciences, San-5, Diego, USA) using commercial ELISA kits according to the manufacturer’s left lobe of lung was fixed in 4% buffered formaldehyde and paraffin embedded under standard conditions. Tissue sections (3 µm) were stained with PAS. Histological changes such as inflammation and emphysema were assessed by a semi-quantitative score from 0 to 5 for cell infiltration (with increasing severity) as described before66. The slides were examined by two blinded investigators with a Leica microscope (Leica, Germany).Determination of bronchial hyperresponsiveness (AHR)For invasive measurement of dynamic resistance, mice were anesthetized with intra-peritoneal injection of solution containing ketamine (100 mg/kg, Merial) and xylasine (10 mg/kg, Bayer), paralyzed using D-tubocuranine ( Sigma), and intubated with an 18-gauge catheter. Respiratory frequency was set at 140 breaths per min with a tidal volume of ml and a positive end-expiratory pressure of 2 ml H2O. Increasing concentrations of aerosolized methacholine ( 75 and 150 mg/ml) were administered. Resistance was recorded using an invasive plethysmograph (Buxco, London, UK). Baseline resistance was restored before administering the subsequent doses of immunofluorescence stainingLungs were fixed for 3 days in 4% PFA and submerged in 20% sucrose for 1 week. Lungs were embedded in OCT (Tissue-Teck) and 10 µM sections were prepared with cryotome (Leica). Slides were incubated 30 min in citrate buffer at 80 °C, washed in TBS-Tween and then incubated overnight with mouse-anti-mouse-E-cadherin (1 µg/ml, ab76055, Abcam). After washing with slides were treated with 0,05% pontamin sky blue (Sigma) for 15 min and then incubated with secondary AF-546 goat anti-mouse antibody (Abcam) for 30 min at room temperature. After washing, slides were incubated with DAPI (Fisher Scientific) and mounted in fluoromount® (SouthernBiotech). Lung sections were observed on a fluorescence microscope Leica (Leica, CTR6000) at x200 magnification. The slides were analyzed and semi-quantitatively scored and the MFI was epithelial barrier functionTotal protein in BAL fluid and Evans blue EB leak in BAL fluid was determined as described mononuclear cell isolation and stimulationLung mononuclear cells were isolated from mice 24 h after the last challenge as described previously67. Briefly the aorta and the inferior vena cava were sectioned and the lungs were perfused with 10 mL of saline. The lobes of the lungs were sliced into small cubes and then incubated for 45 min in 1 ml of RPMI 1640 solution and digested in 1,25 mg/ml of Liberase TL (Roche Diagnostics) and 1 mg/ml DNAse 1 (Sigma) during 1 h at 37 °C. Red blood cells were lysed with lysing buffer (BD Pharm LyseTM – BD Pharmingen). Isolated lung mononuclear single live cells were plated in round bottom 96-well plates (1 × 106/ml) and restimulated 3 h at 37 °C with PMA (50 ng/mL) and ionomicyn (750 ng/mL) in the presence of Brefeldin A (1 μl/1 × 106 cells, BD Biosciences) for intracellular flow cytometry analysis. Lung mononuclear cell (1 × 106 cells) were restimulated with 25 µg of papain in RPMI and 10% SVF at 37 °C in a 96 well plate for 3 days. Supernatants were analyzed for the presence of IL-5, IL-13 and IL-33 by ELISA (invitrogen).Flow cytometry analysis on lung mononuclear cellsLung mononuclear cells were stained with V450-conjugated anti-CD45 (clone 30F11), PerCp anti-CD3e (clone 145-2C11), FITC-conjugated anti-CD4 (clone RM4-5), PE-Cy7 -conjugated anti-ICOS (clone FITC-conjugated anti-ST2 (clone U29-93), anti B220 (clone RA3-6B2), anti FcεRI (clone MAR-1), anti CD11b (clone M1/70), anti Siglec-F (clone E50-2440) and Fixable Viability Dye eFluor™ 780 (eBioscience). After washing, cells were permeabilized for 20 min with cytofix/cytoperm kit (BD Biosciences) and stained with, eFluor 660-conjugated anti-IL13 (clone eBio13A, eBiosciences) and PE-conjugated anti-IL-5 (clone All antibodies used in this were from BD Biosciences, unless otherwise specified. Data were acquired using FACS Canto II flow cytometer and analyzed using Diva and FlowJo analysisData were analyzed using Prism version 5 (Graphpad Software, San Diego, USA). The parametric one-way ANOVA test with multiple Bonferroni’s comparison test was used. Values are expressed as mean ± SEM. Statistical significance was defined at a p-value < ReferencesAccordini, S. et al. The cost of persistent asthma in Europe: an international population-based study in adults. International archives of allergy and immunology 160, 93–101, (2013).Article PubMed Google Scholar Barnett, S. B. & Nurmagambetov, T. A. Costs of asthma in the United States: 2002–2007. The Journal of allergy and clinical immunology 127, 145–152, (2011).Article PubMed Google Scholar Masoli, M., Fabian, D., Holt, S. & Beasley, R., Global Initiative for Asthma, P. The global burden of asthma: executive summary of the GINA Dissemination Committee report. Allergy 59, 469–478, (2004).Article PubMed Google Scholar Ober, C. & Yao, T. C. The genetics of asthma and allergic disease: a 21st century perspective. Immunological reviews 242, 10–30, (2011).Article PubMed PubMed Central CAS Google Scholar Ege, M. J. et al. Exposure to environmental microorganisms and childhood asthma. The New England journal of medicine 364, 701–709, (2011).Article PubMed CAS Google Scholar Round, J. L. & Mazmanian, S. K. The gut microbiota shapes intestinal immune responses during health and disease. Nature reviews. Immunology 9, 313–323, (2009).Article PubMed PubMed Central CAS Google Scholar Okada, H., Kuhn, C., Feillet, H. & Bach, J. F. The ‘hygiene hypothesis’ for autoimmune and allergic diseases: an update. Clin Exp Immunol 160, 1–9 (2010).Article PubMed PubMed Central CAS Google Scholar Penders, J. et al. Gut microbiota composition and development of atopic manifestations in infancy: the KOALA Birth Cohort Study. Gut 56, 661–667 (2007).Article PubMed CAS Google Scholar Hong, P. Y. et al. Comparative analysis of fecal microbiota in infants with and without eczema. PLoS One 5, e9964 (2010).Article PubMed PubMed Central ADS CAS Google Scholar Vael, C., Vanheirstraeten, L., Desager, K. N. & Goossens, H. Denaturing gradient gel electrophoresis of neonatal intestinal microbiota in relation to the development of asthma. BMC Microbiol 11, 68 (2011).Article PubMed PubMed Central CAS Google Scholar Nakayama, J. et al. Aberrant structures of fecal bacterial community in allergic infants profiled by 16S rRNA gene pyrosequencing. FEMS Immunol Med Microbiol 63, 397–406 (2011).Article PubMed CAS Google Scholar Savage, D. C. Microbial ecology of the gastrointestinal tract. Annual review of microbiology 31, 107–133, (1977).Article PubMed CAS Google Scholar Roediger, W. E. Role of anaerobic bacteria in the metabolic welfare of the colonic mucosa in man. Gut 21, 793–798 (1980).Article PubMed PubMed Central CAS Google Scholar Flint, H. J. & Bayer, E. A. Plant cell wall breakdown by anaerobic microorganisms from the Mammalian digestive tract. Annals of the New York Academy of Sciences 1125, 280–288, (2008).Article PubMed ADS CAS Google Scholar Yatsunenko, T. et al. Human gut microbiome viewed across age and geography. Nature 486, 222–227, (2012).Article PubMed PubMed Central ADS CAS Google Scholar Turnbaugh, P. J. et al. An obesity-associated gut microbiome with increased capacity for energy harvest. Nature 444, 1027–1031, (2006).Article PubMed ADS Google Scholar Martens, E. C., Chiang, H. C. & Gordon, J. I. Mucosal glycan foraging enhances fitness and transmission of a saccharolytic human gut bacterial symbiont. Cell host & microbe 4, 447–457, (2008).Article CAS Google Scholar Backhed, F. et al. The gut microbiota as an environmental factor that regulates fat storage. Proceedings of the National Academy of Sciences of the United States of America 101, 15718–15723, (2004).Article PubMed PubMed Central ADS CAS Google Scholar Xu, J. & Gordon, J. I. Honor thy symbionts. Proceedings of the National Academy of Sciences of the United States of America 100, 10452–10459, (2003).Article PubMed PubMed Central ADS CAS Google Scholar Chung, H. et al. Gut immune maturation depends on colonization with a host-specific microbiota. Cell 149, 1578–1593, (2012).Article PubMed PubMed Central CAS Google Scholar Heczko, U., Abe, A. & Finlay, B. B. Segmented filamentous bacteria prevent colonization of enteropathogenic Escherichia coli O103 in rabbits. The Journal of infectious diseases 181, 1027–1033, (2000).Article PubMed CAS Google Scholar Ivanov, I. I. et al. Induction of intestinal Th17 cells by segmented filamentous bacteria. Cell 139, 485–498, (2009).Article PubMed PubMed Central CAS Google Scholar Hooper, L. V. & Macpherson, A. J. Immune adaptations that maintain homeostasis with the intestinal microbiota. Nature reviews. Immunology 10, 159–169, (2010).Article PubMed CAS Google Scholar Hooper, L. V., Littman, D. R. & Macpherson, A. J. Interactions between the microbiota and the immune system. Science 336, 1268–1273, (2012).Article PubMed PubMed Central ADS CAS Google Scholar Stappenbeck, T. S., Hooper, L. V. & Gordon, J. I. Developmental regulation of intestinal angiogenesis by indigenous microbes via Paneth cells. Proceedings of the National Academy of Sciences of the United States of America 99, 15451–15455, (2002).Article PubMed PubMed Central ADS CAS Google Scholar Hammad, H. & Lambrecht, B. N. Dendritic cells and epithelial cells: linking innate and adaptive immunity in asthma. Nat Rev Immunol 8, 193–204, (2008).Article PubMed CAS Google Scholar Jacquet, A. Interactions of airway epithelium with protease allergens in the allergic response. Clin Exp Allergy 41, 305–311, (2011).Article PubMed CAS Google Scholar Wilhelm, C. et al. An IL-9 fate reporter demonstrates the induction of an innate IL-9 response in lung inflammation. Nat Immunol 12, 1071–1077, (2011).Article PubMed PubMed Central CAS Google Scholar Sokol, C. L., Barton, G. M., Farr, A. G. & Medzhitov, R. A mechanism for the initiation of allergen-induced T helper type 2 responses. Nat Immunol 9, 310–318, (2008).Article PubMed PubMed Central CAS Google Scholar Kamijo, S. et al. IL-33-mediated innate response and adaptive immune cells contribute to maximum responses of protease allergen-induced allergic airway inflammation. J Immunol 190, 4489–4499, (2013).PubMed CAS Article Google Scholar Kruis, W. et al. Maintaining remission of ulcerative colitis with the probiotic Escherichia coli Nissle 1917 is as effective as with standard mesalazine. Gut 53, 1617–1623, (2004).Article PubMed PubMed Central CAS Google Scholar Agoro, R. et al. IL-1R1-MyD88 axis elicits papain-induced lung inflammation. European journal of immunology 46, 2531–2541, (2016).Article PubMed CAS Google Scholar Ukena, S. N. et al. Probiotic Escherichia coli Nissle 1917 inhibits leaky gut by enhancing mucosal integrity. PloS one 2, e1308, (2007).Article PubMed PubMed Central ADS CAS Google Scholar Nawijn, M. C., Hackett, T. L., Postma, D. S., van Oosterhout, A. J. & Heijink, I. H. E-cadherin: gatekeeper of airway mucosa and allergic sensitization. Trends in immunology 32, 248–255, (2011).Article PubMed CAS Google Scholar Halim, T. Y. et al. Group 2 innate lymphoid cells are critical for the initiation of adaptive T helper 2 cell-mediated allergic lung inflammation. Immunity 40, 425-435, doi:S1074-7613(14)00068-5 (2014).Eder, W., Ege, M. J. & von Mutius, E. The asthma epidemic. N Engl J Med 355, 2226–2235 (2006).Article PubMed CAS Google Scholar Yao, T. C., Chang, C. J., Hsu, Y. H. & Huang, J. L. Probiotics for allergic diseases: realities and myths. Pediatric allergy and immunology: official publication of the European Society of Pediatric Allergy and Immunology 21, 900–919, (2010).Article Google Scholar Kalliomaki, M., Salminen, S., Poussa, T., Arvilommi, H. & Isolauri, E. Probiotics and prevention of atopic disease: 4-year follow-up of a randomised placebo-controlled trial. Lancet 361, 1869–1871, (2003).Article PubMed Google Scholar Gruber, C. et al. Randomized, placebo-controlled trial of Lactobacillus rhamnosus GG as treatment of atopic dermatitis in infancy. Allergy 62, 1270–1276, (2007).Article PubMed CAS Google Scholar Osborn, D. A. & Sinn, J. K. Probiotics in infants for prevention of allergic disease and food hypersensitivity. The Cochrane database of systematic reviews, CD006475, (2007).de Roock, S. et al. Lactic acid bacteria differ in their ability to induce functional regulatory T cells in humans. Clinical and experimental allergy: journal of the British Society for Allergy and Clinical Immunology 40, 103–110, (2010).CAS Article Google Scholar Boyle, R. J. & Tang, M. L. The role of probiotics in the management of allergic disease. Clinical and experimental allergy: journal of the British Society for Allergy and Clinical Immunology 36, 568–576, (2006).Article CAS Google Scholar Jacobi, C. A. & Malfertheiner, P. Escherichia coli Nissle 1917 (Mutaflor): new insights into an old probiotic bacterium. Digestive diseases 29, 600–607, (2011).Article PubMed Google Scholar Bickert, T. et al. Probiotic Escherichia coli Nissle 1917 suppresses allergen-induced Th2 responses in the airways. International archives of allergy and immunology 149, 219–230, (2009).Article PubMed CAS Google Scholar Adam, E. et al. Probiotic Escherichia coli Nissle 1917 activates DC and prevents house dust mite allergy through a TLR4-dependent pathway. European journal of immunology 40, 1995–2005, (2010).Article PubMed CAS Google Scholar Sabharwal, H., Cichon, C., Olschlager, T. A., Sonnenborn, U. & Schmidt, M. A. Interleukin-8, CXCL1, and MicroRNA miR-146a Responses to Probiotic Escherichia coli Nissle 1917 and Enteropathogenic E. coli in Human Intestinal Epithelial T84 and Monocytic THP-1 Cells after Apical or Basolateral Infection. Infection and immunity 84, 2482–2492, (2016).Article PubMed PubMed Central CAS Google Scholar Hafez, M. et al. The K5 capsule of Escherichia coli strain Nissle 1917 is important in mediating interactions with intestinal epithelial cells and chemokine induction. Infection and immunity 77, 2995–3003, (2009).Article PubMed PubMed Central CAS Google Scholar Russo, T. A. et al. Human neutrophil chemotaxis is modulated by capsule and O antigen from an extraintestinal pathogenic Escherichia coli strain. Infection and immunity 71, 6435–6445 (2003).Article PubMed PubMed Central CAS Google Scholar Russo, T. A. et al. Capsular polysaccharide and O-specific antigen divergently modulate pulmonary neutrophil influx in an Escherichia coli model of gram-negative pneumonitis in rats. Infection and immunity 68, 2854–2862 (2000).Article PubMed PubMed Central CAS Google Scholar Le, Y., Murphy, P. M. & Wang, J. M. Formyl-peptide receptors revisited. Trends in immunology 23, 541–548 (2002).Article PubMed CAS Google Scholar Guzy, C. et al. The probiotic Escherichia coli strain Nissle 1917 induces gammadelta T cell apoptosis via caspase- and FasL-dependent pathways. International immunology 20, 829–840, (2008).Article PubMed CAS Google Scholar Xiao, C. et al. Defective epithelial barrier function in asthma. The Journal of allergy and clinical immunology 128(549–556), e541–512, (2011).CAS Article Google Scholar Davies, D. E. The role of the epithelium in airway remodeling in asthma. Proceedings of the American Thoracic Society 6, 678–682, (2009).Article PubMed PubMed Central Google Scholar Schleimer, R. P., Kato, A., Kern, R., Kuperman, D. & Avila, P. C. Epithelium: at the interface of innate and adaptive immune responses. The Journal of allergy and clinical immunology 120, 1279–1284, (2007).Article PubMed PubMed Central CAS Google Scholar Cross, M. L., Ganner, A., Teilab, D. & Fray, L. M. Patterns of cytokine induction by gram-positive and gram-negative probiotic bacteria. FEMS immunology and medical microbiology 42, 173–180, (2004).Article PubMed CAS Google Scholar Sturm, A. et al. Escherichia coli Nissle 1917 distinctively modulates T-cell cycling and expansion via toll-like receptor 2 signaling. Infection and immunity 73, 1452–1465, (2005).Article PubMed PubMed Central CAS Google Scholar Madouri, F. et al. Protein kinase Ctheta controls type 2 innate lymphoid cell and TH2 responses to house dust mite allergen. The Journal of allergy and clinical immunology 139, 1650–1666, (2017).Article PubMed CAS Google Scholar Secher, T., Brehin, C. & Oswald, E. Early settlers: which E. coli strains do you not want at birth? American journal of physiology. Gastrointestinal and liver physiology 311, G123–129, (2016).Article PubMed Google Scholar Secher, T. et al. Oral Administration of the Probiotic Strain Escherichia coli Nissle 1917 Reduces Susceptibility to Neuroinflammation and Repairs Experimental Autoimmune Encephalomyelitis-Induced Intestinal Barrier Dysfunction. Frontiers in immunology 8, 1096, (2017).Article PubMed PubMed Central Google Scholar Vizcaino, M. I., Engel, P., Trautman, E. & Crawford, J. M. Comparative metabolomics and structural characterizations illuminate colibactin pathway-dependent small molecules. Journal of the American Chemical Society 136, 9244–9247, (2014).Article PubMed PubMed Central CAS Google Scholar Payros, D. et al. Maternally acquired genotoxic Escherichia coli alters offspring’s intestinal homeostasis. Gut microbes 5, 313–325, (2014).Article PubMed PubMed Central Google Scholar Olier, M. et al. Genotoxicity of Escherichia coli Nissle 1917 strain cannot be dissociated from its probiotic activity. Gut microbes 3, 501–509, (2012).Article PubMed PubMed Central Google Scholar Dorrestein, P. C., Mazmanian, S. K. & Knight, R. Finding the missing links among metabolites, microbes, and the host. Immunity 40, 824–832, (2014).Article PubMed PubMed Central CAS Google Scholar Tulic, M. K., Piche, T. & Verhasselt, V. Lung-gut cross-talk: evidence, mechanisms and implications for the mucosal inflammatory diseases. Clinical and experimental allergy: journal of the British Society for Allergy and Clinical Immunology 46, 519–528, (2016).Article CAS Google Scholar Besnard, A. G. et al. NLRP3 inflammasome is required in murine asthma in the absence of aluminum adjuvant. Allergy 66, 1047–1057, (2011).Article PubMed CAS Google Scholar Yu, H. S., Angkasekwinai, P., Chang, S. H., Chung, Y. & Dong, C. Protease allergens induce the expression of IL-25 via Erk and p38 MAPK pathway. J Korean Med Sci 25, 829–834, (2010).Article PubMed PubMed Central CAS Google Scholar Hachem, P. et al. Alpha-galactosylceramide-induced iNKT cells suppress experimental allergic asthma in sensitized mice: role of IFN-gamma. European journal of immunology 35, 2793–2802, (2005).Article PubMed CAS Google Scholar Download referencesAcknowledgementsWe thank Corinne Panek, Pascal Mauny and Nathalie Froux for excellent technical assistance in maintaining mouse colonies. The authors are grateful to Dieudonnée Togbé for helpful discussions and suggestions. This work was supported by ANR (ANR-GUI-AAP-06-Coliforlife), le Centre National de la Recherche Scientifique, the University of Orléans, la Région Centre (2013-00085470), European funding in Region Centre-Val de Loire (FEDER N° 2016-00110366), le Ministère de l’Education Nationale, de la Recherche et de la Technologie to RA as PhD fellowship, l’Institut National de la Santé et de la Recherche Médicale to ACM as a postdoctoral informationAuthor notesThomas SecherPresent address: INSERM, UMR 1100, Research Center for Respiratory Diseases, and University of Tours, Tours, FranceAuthors and AffiliationsIRSD, Université de Toulouse, INSERM, INRA, ENVT, UPS, Toulouse, FranceThomas Secher, Michèle Boury & Eric OswaldCNRS, UMR7355, Experimental and Molecular Immunology and Neurogenetics, Orleans, FranceIsabelle Maillet, Claire Mackowiak, Jessica Le Bérichel, Amandine Philippeau, Corinne Panek, Francois Erard, Marc Le Bert, Valérie Quesniaux, Aurélie Couturier-Maillard & Bernhard RyffelCHU Toulouse, Hôpital Purpan, Service de Bactériologie-Hygiène, Toulouse, FranceEric OswaldCentre de Physiopathologie de Toulouse Purpan (CPTP), Université de Toulouse, UPS, Inserm, CNRS, Toulouse, FranceAbdelhadi SaoudiUniversity of Orleans, Orleans, FranceValérie Quesniaux & Bernhard RyffelUniversity of Cape Town, IDM, Cape Town, Republic of South AfricaBernhard RyffelAuthorsThomas SecherYou can also search for this author in PubMed Google ScholarIsabelle MailletYou can also search for this author in PubMed Google ScholarClaire MackowiakYou can also search for this author in PubMed Google ScholarJessica Le BérichelYou can also search for this author in PubMed Google ScholarAmandine PhilippeauYou can also search for this author in PubMed Google ScholarCorinne PanekYou can also search for this author in PubMed Google ScholarMichèle BouryYou can also search for this author in PubMed Google ScholarEric OswaldYou can also search for this author in PubMed Google ScholarAbdelhadi SaoudiYou can also search for this author in PubMed Google ScholarFrancois ErardYou can also search for this author in PubMed Google ScholarMarc Le BertYou can also search for this author in PubMed Google ScholarValérie QuesniauxYou can also search for this author in PubMed Google ScholarAurélie Couturier-MaillardYou can also search for this author in PubMed Google ScholarBernhard RyffelYou can also search for this author in PubMed Google ScholarContributionsConceived and designed the experiments: and Performed the experiments: and Analyzed the data: Wrote the paper: and authorsCorrespondence to Thomas Secher or Bernhard declarations Competing Interests The authors declare no competing interests. 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To view a copy of this license, visit Reprints and PermissionsAbout this articleCite this articleSecher, T., Maillet, I., Mackowiak, C. et al. The probiotic strain Escherichia coli Nissle 1917 prevents papain-induced respiratory barrier injury and severe allergic inflammation in mice. Sci Rep 8, 11245 (2018). citationReceived: 12 September 2017Accepted: 16 July 2018Published: 26 July 2018DOI: CommentsBy submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.
In vitro studies showed the probiotic Escherichia coli strain Nissle 1917 (EcN) to efficiently inhibit the production of Stx. Life threatening EHEC strains as for example the serotype O104:H4, responsible for the great outbreak in 2011 in Germany, evolutionary developed from certain E. coli strains which got infected by stx2 -encoding lambdoid
Maintaining remission of ulcerative colitis with the probiotic Escherichia coli Nissle 1917 is as effective as with standard mesalazine Free W Kruis1, P Frič2, J Pokrotnieks3, M Lukáš4, B Fixa5, M Kaščák6, M A Kamm7, J Weismueller8, C Beglinger9, M Stolte10, C Wolff11, J Schulze111Evangelisches Krankenhaus Kalk, University of Cologne, Germany2Ustředná vojenská nemocnice, II interní oddělení, Praha, Czech Republic3Paula Stradina Clinical University Hospital, Riga, Latvia4IV Interni Klinika, Charles University, Praha, Czech Republic52nd Department of Medicine, Charles University Prague, Medical Faculty, Hradec Kralove, Czech Republic6Interné oddelenie NsP, Trenčín, Slovak Republic7St Mark’s Hospital, London, UK8Private Practice, Koblenz, Germany9Division of Gastroenterology, University Hospital, Basel, Switzerland10Institut für Pathologie, Klinikum Bayreuth, Germany11Ardeypharm, Herdecke, GermanyCorrespondence to: Dr W Kruis Evangelisches Krankenhaus Kalk, Buchforststr 2, 51103 Cologne, Germany; Abstract Background and aim: Evidence exists for the pathogenic role of the enteric flora in inflammatory bowel disease. Probiotics contain living microorganisms which exert health effects on the host. We compared the efficacy in maintaining remission of the probiotic preparation Escherichia coli Nissle 1917 and established therapy with mesalazine in patients with ulcerative colitis. Patients and methods: In total, 327 patients were recruited and assigned to a double blind, double dummy trial to receive either the probiotic drug 200 mg once daily (n = 162) or mesalazine 500 mg three times daily (n = 165). The study lasted for 12 months and patients were assessed by clinical and endoscopic activity indices (Rachmilewitz) as well as by histology. The primary aim of the study was to confirm equivalent efficacy of the two drugs in the prevention of relapses. Results: The per protocol analysis revealed relapses in 40/110 ( patients in the E coli Nissle 1917 group and 38/112 ( in the mesalazine group (significant equivalence p = Subgroup analyses showed no differences between the treatment groups in terms of duration and localisation of disease or pretrial treatment. Safety profile and tolerability were very good for both groups and were not different. Conclusions: The probiotic drug E coli Nissle 1917 shows efficacy and safety in maintaining remission equivalent to the gold standard mesalazine in patients with ulcerative colitis. The effectiveness of probiotic treatment further underlines the pathogenetic significance of the enteric flora. UC, ulcerative colitisIBD, inflammatory bowel diseaseEcN, Escherichia coli Nissle 1917GCP, good clinical practiceCAI, clinical activity indexEI, endoscopic indexITT, intention to treat populationPP, per protocol population5-ASA, 5-aminosalicylic acidulcerative colitismaintenance therapyprobioticsEscherichia coli Nissle Statistics from Request Permissions If you wish to reuse any or all of this article please use the link below which will take you to the Copyright Clearance Center’s RightsLink service. You will be able to get a quick price and instant permission to reuse the content in many different ways. UC, ulcerative colitisIBD, inflammatory bowel diseaseEcN, Escherichia coli Nissle 1917GCP, good clinical practiceCAI, clinical activity indexEI, endoscopic indexITT, intention to treat populationPP, per protocol population5-ASA, 5-aminosalicylic acidulcerative colitismaintenance therapyprobioticsEscherichia coli Nissle Ulcerative colitis (UC) is a chronic relapsing disease. The aims of treatment are induction of remission and prevention of relapses. Guidelines1,2 recommend aminosalicylates for maintenance treatment. Aminosalicylates exert various effects on leukotrienes, cytokines, and oxygen Their mode of action in UC remains unclear. It is suggested that the sum of their anti-inflammatory activities constitutes their therapeutic principle. Thus maintenance treatment with aminosalicylates is only effective when inflammation starts, but not in the non-inflamed gut. Growing evidence exists for a role of the intestinal microflora in the pathogenesis of inflammatory bowel disease (IBD). Findings from genetically engineered animal models as well as clinical observations have elucidated the importance of commensal Antibacterial treatment showed some beneficial effects7,8 but the use of antibiotics is limited. Therefore, treatment with probiotics has been proposed. Probiotics are viable non-pathogenic microorganisms that confer health benefits to the host by improving the microbial balance of the indigenous Apart from anecdotal experience, two controlled studies with the probiotic bacterial strain Escherichia coli Nissle 1917 (EcN) in UC already These trials showed no difference between the relapse preventing effects of EcN and standard mesalazine. However, some criticism was raised as to the validity of these The present study was undertaken to confirm that the relapse preventing effects of probiotic therapy with EcN and standard mesalazine are equivalent. MATERIALS AND METHODS The study was conducted according to the Helsinki Declaration (revised version of Hong Kong) and adhered to good clinical practice (GCP) guidelines. The study was approved by the Ethikkommission der Ärztekammer Nordrhein, Germany, as well as by the local ethics committees of the participating centres. All patients received material in their own language and gave written informed consent. Patients were included in the study if aged 18–70 years and diagnosed with UC in remission (clinical activity index (CAI) ⩽4, endoscopic index (EI) ⩽4, and no signs of acute inflammation on histological examination). In addition, inclusion criteria comprised at least two acute attacks of UC prior to the study and a duration of the current remission of no longer than 12 months. Exclusion criteria were: active UC; proctitis with up to 10 cm proximal spread; Crohn’s disease; infectious colitis; severe accompanying illnesses or major colonic surgery; use of antibiotics, sulphonamides, steroids, or other therapies for UC at entry into the trial; administration of EcN within the previous six months before trial entry; as well as known intolerance to salicylates. Study medication The investigational drug was a bacterial preparation for oral use containing non-pathogenic Escherichia coli of strain Nissle 1917 (serotype O6:K5:H1). Capsules were enteric coated to protect the microorganisms from gastric juice and contained viable bacteria (Mutaflor 100 mg; Ardeypharm GmbH, Herdecke, Germany). The control preparation was mesalazine, consisting of eudragit L coated 5-aminosalicylic acid (Salofalk500 mg; Dr Falk Pharma GmbH, Freiburg, Germany). The test group received one capsule of Mutaflor 100 mg once daily and one tablet of placebo three times daily from day 1 to day 4, and two capsules of Mutaflor 100 mg once daily and one tablet of placebo three times daily from day 5 to the end of the study. The control group received one capsule of placebo once daily and one tablet of Salofalk 500 mg three times daily from day 1 to day 4, and two capsules of placebo once daily and one tablet of Salofalk 500 mg three times daily from day 5 to the end of the study. No concomitant medication for UC was allowed throughout the study. Study design This was a randomised, double blind, double dummy trial comparing the relapse preventing effects and safety of a bacterial preparation containing viable EcN and mesalazine for 12 months in patients with UC in remission. The study was conducted in 60 hospitals and private settings in 10 European countries (see list of participating investigators in the appendix). Randomisation was carried out in a double blind manner in blocks of four patients using 1:1 allocation to the two treatment groups. Only complete blocks of random numbers were used for each centre. If patients were eligible for study entry, they were assigned to random numbers ( = patient numbers) in ascending order within each centre according to the chronological order of their randomisation and were given the corresponding study medication. Evaluation Clinic visits were required at the start and end of the study as well as after 1, 2, 3, 6, and 9 months of treatment. The primary objective of the study was to compare the number of patients experiencing a relapse of UC during the 12 month observation period between the two treatment groups. Patients were classified as suffering a relapse when all three of the following criteria were met: CAI >6 or an increase in CAI of at least 3 points with CAI = 4 being exceeded at the same time; EI >4; and histological signs of acute inflammation. CAI was defined according to At trial entry and at the end of the study, patients underwent colonoscopy where biopsies were taken. Endoscopic activity was assessed using a four point index14: granularity, vascular pattern, vulnerability of mucosa, and mucosal damage. All biopsies were examined by a single pathologist using a four point Secondary efficacy variables were the physician’s and patient’s assessment of general well being and calculation of a quality of life Additionally, time to relapse, CAI, EI, and histological findings were documented. Laboratory assessments, including erythrocyte sedimentation rate, C reactive protein, orosomucoids, blood counts, liver enzymes, creatinine, serum iron, and serum albumin were performed at trial entry and at the end of the study. Incidence and severity of adverse events were reported according to GCP for clinical trials of medication in the European Community (91/507/EWG, CPMP/ICH/135/95). Tolerance of the study medication was assessed on a four point scale (very good, good, fair, poor), and patient compliance was ascertained by pill counting. Statistical analysis The aim of the study was to statistically confirm one sided equivalent efficacy of EcN and mesalazine in preventing relapses of UC. Relapse rates were compared using the one sided test of Farrington and Manning17: this tests the null hypothesis that the difference between treatment groups is greater than or equal to the upper equivalence margin Δ of 20% versus the alternative that the true difference is less than 20% (α = upper confidence limit 95%). Assuming a 12 month relapse rate of 30% under mesalazine treatment and 35% under EcN treatment, to reach a statistical power of 80% at least n = 127 patients were required in each treatment group according to the sample size term for comparative binomial trials with the null hypothesis of non-zero risk Two sets of patients were analysed: an intention to treat population (ITT), including all patients who took at least one dose of the study medication, and a per protocol population (PP). According to generally accepted standards for equivalence and non-inferiority trials,18 primary analysis of the main objective (difference in relapse rates) was based on the PP population. Assuming 25% protocol violators, a total number of 160 patients in each treatment group was therefore planned. Baseline comparability and statistical analysis of secondary objectives was assessed using Fisher’s exact test (two sided; α = In addition, Kaplan-Meier curves were plotted. If no CAI or other parameter was documented at the individual study end, the “last observation carried forward” method was applied. Results are given as mean (SD). Statistical tests were executed using SPSS software package version under the Microsoft Windows NT operating system. For exploratory comparisons (tables 2, 3), the Student’s t test was used. RESULTS Patient characteristics In total, 327 patients were enrolled and randomised to either the EcN preparation (n = 162) or mesalazine (n = 165). The two patient groups were matched with regard to demographic, clinical, and pretreatment characteristics (table 1). The time gap between the end of the last relapse before the study and entry into the study was not longer than four weeks in of patients receiving EcN and in receiving mesalazine, and not longer than three months in and of EcN and mesalazine patients, respectively. All 327 randomised patients received at least one dose of the study medication and thus were included in the ITT and safety analysis this table:View inline Table 1 Demographic data and prestudy clinical characteristics Before unblinding the study, a steering committee assessed protocol violations in 105/327 ( patients. Major protocol deviations comprised violation of inclusion criteria (CAI ⩽4, EI ⩽4, and no signs of acute inflammation on histological examination) (32 patients in both groups), premature discontinuation of the study without relapse (see below), and unknown or not unequivocally assessed end point (EcN 29 patients, mesalazine 24 patients). Accordingly, the PP analysis set comprised 222 patients (EcN 110, mesalazine 112). Mean duration of the study observation period was 250 (144) (median 357) days in the EcN group and 287 (125) (median 360) days in the mesalazine group. The number of patients in the study at the scheduled visits is shown in fig 1. Premature discontinuation of the study for reasons other than relapse of disease occurred in 39/327 ( patients (in 19/162 ( patients in the EcN group and in 20/165 ( patients in the mesalazine group) (table 2). Newly emerged exclusion criteria during the study were start of concomitant medication in four patients on EcN. One patient on mesalazine became afraid of 5-aminosalicylic acid (5-ASA) and another patient underwent cardiac this table:View inline Table 2 Reasons for premature discontinuation of the study Relapse (primary objective) PP analysis revealed relapse in 40/110 ( patients in the EcN group and in 38/112 ( patients in the mesalazine group (fig 2), resulting in significant equivalence between the two groups (p = The corresponding one sided upper 95% confidence limit for the difference in treatment was (that is, within the equivalence range of 20%). Figure 3 depicts the probability of remaining in remission by Kaplan-Meier curves. Median time to relapse in the EcN group could not be calculated due to the large number of late censorings. In the mesalazine group it was 386 days. ITT analysis confirmed these results, showing a relapse rate of in the EcN group and in the mesalazine group (significant equivalence p = The upper limit of the 95% confidence interval for the difference in treatment was Subgroup analyses (secondary objectives) All subgroup analyses were performed in the ITT population. CAI increased in all patients by ( points over the study period, showing a slightly larger increase in the EcN group ( ( than in the mesalazine group ( ( No differences were observed in EI or histology between the start and end of the study (fig 4). Table 3 lists relapse rates with regard to duration, localisation, and pretrial treatment. There were no significant differences between the treatment groups for any of these characteristics. Quality of life scores on admission were ( in the EcN group and ( in the mesalazine group. Respective values after 12 months were ( and ( No significant changes occurred during the 12 month observation this table:View inline Table 3 Relapse rates according to clinical characteristics (intention to treat population) Safety and tolerance As rated by the patients, overall tolerance was very good or good in the EcN group in and in the mesalazine group in According to the physician’s assessment, the respective values were and Discontinuation of the study medication due to adverse events (relapse included) occurred in 22 ( patients (11 ( in the EcN group and 11 ( in the mesalazine group). Most frequent reasons were gastrointestinal disorders such as bloody stools, nausea, diarrhoea, mucous secretion (EcN mesalazine and abdominal pain (EcN mesalazine Generally, no unexpected drug reactions occurred during the study. No deaths but 17 serious adverse events were reported in 13/327 (4%) patients (EcN 7, mesalazine 6). Each serious adverse event occurred only once. Adverse events were reported in 68/162 ( patients treated with EcN and in 58/165 ( patients treated with mesalazine. Many adverse events reflect symptoms common for active UC such as bloody stools ( diarrhoea ( and abdominal pain ( The most frequent non-intestinal adverse events were viral infections (EcN mesalazine nausea ( and headache ( Laboratory tests showed no significant alterations. DISCUSSION Most controlled trials are designed to test differences in efficacy. In contrast, our trial was aimed at proving equivalence. Indeed, we demonstrated that the probiotic EcN provides significantly equivalent efficacy in preventing relapses of UC and is not inferior to the established gold standard mesalazine. This result was not only confirmed by statistical analysis of the PP population, which is preferred in equivalence studies,18 but also by ITT analysis. Therapeutic efficacy is usually demonstrated by superiority in a placebo controlled trial. In serious disease however when effective therapy exists that has already been tested by comparison with placebo, additional placebo controlled trials may be considered A meta-analysis19 reviewed 16 studies of maintenance therapy involving 2341 patients with UC. In four of these 16 trials, preparations containing 5-ASA were compared with placebo; in the remaining 12 studies sulphasalazine was compared. 5-ASA was observed to be significantly more effective than placebo in all dosage subgroups (<1 g/day, 1– g/day, ⩾2 g/day). A dose dependent trend was not Indeed, some studies comparing at least two doses were performed showing mainly negative or conflicting results20: Pentasa 3 g/day was not superior to g/day; balsalazide 4 g/day was better than 2 g/day; balsalazide 6 g/day was better than 3 g/day in one study but in another trial was similarly effective; and two studies with olsalazine reached different conclusions. Thus superior efficacy of doses higher than g/day has not been It can be stated that mesalazine g/day presently reflects the standard in the prevention of UC relapses and thus it qualifies as a control in an equivalence trial. Previous studies on EcN were criticised12,13 for several reasons—for example, short observation period10 or heterogeneity of patients and outcome The present trial considered this critique and followed actual standards. The observation period was 12 months, only patients with UC in remission were included, and the clinical outcome was assessed by well established endoscopic and histological activity indices resulting in a low relapse rate for the mesalazine group comparable with previous A total of 327 patients were included to achieve a statistical power sufficient to test for equivalence in a one sided set. Most likely, IBD is caused by an unrestrained inflammatory response to as yet undefined agents. Although precise identification of the antigenic stimuli has not been determined, the intestinal microflora represents a likely To manipulate the resident gut bacteria therefore seems to offer a rational approach to maintaining remission in IBD. One way of doing this, which has gained credence over recent years, is by using Mechanisms which may account for probiotic activity include production of antimicrobial agents, inhibition of adhesion of pathogens, and influence on mucosal barrier It was reported that inhibition of nuclear factor κB could be mediated by probiotic The properties of EcN are well characterised25 and its genome has been extensively It carries non-pathogenic adhesion molecules. A specific lipopolysaccharide renders it immunogenic without showing any immunotoxic Immunomodulating activity was demonstrated for specific immune responses as well as for induction of non-specific natural immunity in preterm EcN develops antagonistic activity against enterobacteria such as Salmonella enteritidis, Shigella dysenteriae, Yersinia enterocolitica, and Vibrio It prevents invasion of Salmonella typhimurium into intestinal cells,31 inhibits adhesion and invasion of adherent invasive E coli,32 and reduces concentrations of mucosa associated colonic microflora constituents in EcN is safe. Molecular genetics as well as functional analyses have revealed that EcN does not produce any virulence factors or carry any genes for pathogenicity It does not bear genes for antibiotic resistance, transferable genes or plasmids, and does not take up foreign pathogenic DNA. No formation of enterotoxins, cytotoxins, or haemolysins has been observed and there is no serum Clinical studies have demonstrated a favourable safety profile for EcN compared with placebo,35,36 mesalazine,10,11 and Our study confirms this excellent safety and tolerance record. There are other controlled studies with different probiotics. Relapse prevention with Lactobacillus GG tested negatively for maintenance therapy in surgically induced remission of Crohn’s disease38 but a small study showed positive results when Saccharomyces boulardii was added to Inflammation of the ileal pouch constructed after proctocolectomy and ileoanal anastomosis in patients with UC is of particular interest because bacterial growth seems to be of pivotal pathophysiological significance. Cases successfully treated with EcN have been A formulation comprising eight different probiotic bacteria demonstrated convincing therapeutic effects in primary prevention41 and chronic In an uncontrolled study, this preparation was able to colonise the gut and maintain remission in patients with In conclusion, the use of probiotics in IBD is in accordance with its pathogenesis. They may prevent induction of inflammatory reactions. EcN shows therapeutic efficacy and safety in maintaining remission in UC. It can be considered as an alternative to mesalazine. APPENDIX The following institutions, local principal investigators, and local coordinators participated in this study: Austria: University Hospital, Graz: W Petritsch. Czech Republic: Nemocnice Milosrdnych sester sv Karla Boromejského, Prague: J Dosedel; University Hospital, Hradec Kralove: B Fixa; Central Military Hospital, Prague: P Frič; University Hospitals, Prague: M Kment, M Lukáš; University Hospital Plzen: J Koželuhová; University Hospital Brno: H Simonová; Masaryk Hospital, Ústí nad Labem: K Mareš, J Stehlík. Estonia: Central Hospital, Tallin: B Margus; University Hospital, Tartu: R Salupere. Germany: Private Practice, Essen: A Boekstegers; University Hospital, Jena: H Bosseckert; University Hospital, Regensburg: V Gross; DRK-Kliniken Westend, Berlin: R Büchsel; Charité-Campus Virchow, Berlin: A Dignass; Private Practice, Rottenburg aN: F Dreher; Private Practice, Frankenberg: R Engelhard; Private Practice, Bad Homburg: G Ermert; Private Practice, Karlsruhe: U Farack; Private Practice, Marburg: J Hein; Kreisklinik München-Pasing, München: J Heinkelein; Mittelrhein-Klinik Bad Salzig, Boppard: R Herz; Private Practice, Bautzen: I König; Ev Krankenhaus Kalk, Köln: W Kruis; Private Practice, Münster: Th Krummenerl; Private Practice, Cottbus: A Kühn; Israelitisches Krankenhaus, Hamburg: P Layer; University Hospital, Dresden: G Lobeck; Charité-Humboldt-University, Berlin: H Lochs; Private Practice, Neuenkirchen: R Moellmann; Private Practice, Cottbus: E Muehlberg; University Hospital Großhadern, München: Th Ochsenkühn; Städtisches Klinikum Friedrichstadt, Dresden: H Porst; Krankenhaus Tabea, Hamburg: A Raedler; University Hospital, Erlangen: M Raithel; Krankenhaus Nordwest, Frankfurt: W Rösch; University Hospital, Bonn: Ch Scheurlen; Private Practice, Gera: U Schindler; Private Practice, Reutlingen: W Schmeißer; Private Practice, Regensburg: E Schütz; Krankenhaus Speyerer, Heidelberg: R Singer; University Hospital Benjamin Franklin, Berlin: R Stange; University Hospital, Frankfurt: J Stein; Klinikum der RWTH, Aachen: Th Schönfelder; University Hospital, Mainz: R Wanitschke; Private Practice, Koblenz: A Lütke, J Weismüller; St Michael Krankenhaus, Völklingen: D Woerdehoff; Private Practice, Erlangen: J Zeus. Latvia: Paula Stradina Clinical University Hospital, Riga: J Pokrotnieks. Lithuania: University Hospital, Vilnius: A Irnius; Kauno Medicinos Akademija, Kaunas: L Kupcinskas. Slovak Republic: Comenius University Hospital, Bratislava: M Huorka; City Hospital, Trencíne: M Kaščák; University Hospital, Košice: T Hildebrand. Sweden: Sabbatsberg Naersjukhuset, Stockholm: P Benno; Karolinska Institutet: A Uribe. Switzerland: Kantonsspital-University, Basel: Ch Beglinger. UK: Leeds General Infirmary, Leeds: ATR Axon; St Mark’s Hospital, London: MA Kamm. REFERENCES↵ ↵ Stange EF, Riemann J, von Herbay A, et al. Diagnosis and therapy of ulcerative colitis—results of an evidence-based consensus conference of the German Society of Digestive and Metabolic Diseases. Z Gastroenterol2001;39:19–20. ↵ Travis SP, Jewell DP. Salicylates for ulcerative colitis—their mode of action. Pharmacol Ther1994;63:135–61. ↵ Shanahan F . Probiotics and inflammatory bowel disease: is there a scientific rationale? Inflamm Bowel Dis2000;6:107–15. D’Haens GR, Geboes K, Peeters M, et al. Early lesions of recurrent Crohn’s disease caused by infusion of intestinal contents in excluded ileum. Gastroenterology1998;114:262–7. ↵ Sartor RB. Postoperative recurrence of Crohn’s disease: the enemy is within the fecal stream. Gastroenterology1998;114:398–400. ↵ Rutgeerts P , Hiele M, Geboes K, et al. Controlled trial of metronidazole treatment for prevention of Crohn’s recurrence after ileal resection. Gastroenterology1995;108:1617–21. ↵ Hulten K , Almashhrawi A, El Zaatari FA, et al. Antibacterial therapy for Crohn’s disease: a review emphasizing therapy directed against mycobacteria. Dig Dis Sci2000;45:445–56. ↵ Hart AL, Stagg AJ, Kamm MA. Related articles, links use of probiotics in the treatment of inflammatory bowel disease. J Clin Gastroenterol2003;36:111–19. ↵ Kruis W , Schütz E, Fric P, et al. Double-blind comparison of an oral Escherichia coli preparation and mesalazine in maintaining remission of ulcerative colitis. Aliment Pharmacol Ther1997;11:853–8. ↵ Rembacken BJ, Snelling AM, Hawkey PM, et al. Non-pathogenic Escherichia coli versus mesalazine for the treatment of ulcerative colitis: a randomised trial. Lancet1999;354:635–9. ↵ Folwaczny C . Probiotics for prevention of ulcerative colitis recurrence: alternative medicine added to standard treatment? Z Gastroenterol2000;38:547–50. ↵ Faubion WA, Sandborn WJ. Probiotic therapy with E. coli for ulcerative colitis: take the good with the bad, Gastroenterology2000;118:630–1. ↵ Rachmilewitz D . Coated mesalazine (5-aminosalicylic acid) versus sulphasalazine in the treatment of active ulcerative colitis: a randomised trial. BMJ1989;298:82–6. ↵ Riley SA, Mani V, Goodman MJ, et al. Microscopic activity in ulcerative colitis: what does it mean? Gut1991;32:174–8. ↵ Guyatt G , Mitchell A, Irvine EJ, et al. A new measure of health status for clinical trials in inflammatory bowel disease. Gastroenterology1989;96:804–10. ↵ Farrington CP, Manning G. Test statistics and sample size formulae for comparative binomial trials with null hypothesis of non-zero risk difference of non-unity relative risk. Stat Med1990;9:1447–54. ↵ ↵ Sutherland LR, Roth DE, Beck PL. Alternatives to sulfasalazine: A meta-analysis of 5-ASA in the treatment of ulcerative colitis. Inflamm Bowel Dis1997;3:65–78. ↵ Riley SA. What dose of 5-aminosalicylic acid (mesalazine) in ulcerative colitis? Gut1998;42:761–3. ↵ Sartor RB. Enteric microflora in IBD: pathogens or commensals? Inflamm Bowel Dis1997;3:230–5. ↵ Campieri M , Gionchetti P. Bacteria as the cause of ulcerative colitis. Gut2001;48:132–5. ↵ Hamilton-Miller JMT. A review of clinical trials of probiotics in the management of inflammatory bowel disease. Infect Dis Rev2001;3:83–7. ↵ Neish AS, Gewirtz AT, Zeng H, et al. Prokaryotic regulation of epithelial responses by inhibition of IkappaB-alpha ubiquitination. Science2000;289:1560–3. ↵ Blum G , Marre R, Hacker J. Properties of Escherichia coli strains of serotype O6. Infection1995;23:234–6. ↵ Blum-Oehler G . The scanned bacterium: Analysis of the microbial genome. 4th Interdisciplinary Symposium: Internal microflora in symbiosis and pathogenicity, Berlin 2000. Hagen: Alfred-Nissle-Gesellschaft eV 2001:23–31. ↵ Grozdanov L , Zaehringer U, Blum-Oehler G, et al. A single-nucleotide exchange in the wzy gene is responsible for the semi-rough O6 LPS phenotype and serum sensitivity of Escherichia coli strain Nissle 1917. J Bacteriol2002;184:5912–25. ↵ Cukrowska B , Lodinova-Zadnikova R, Enders C, et al. Specific proliferative and antibody responses of premature infants to intestinal colonization with nonpathogenic probiotic E. coli strain Nissle 1917. Scand J Immunol2002;55:204–9. ↵ Schulze J , Sonnenborn U. Oral administration of a certain strain of live Escherichia coli for intestinal disorders? Infection1995;23:184–6. ↵ Schulze J , Lorenz A, Mandel L. Colonisation of Escherichia coli in different gnotobiotic animal models. Microb Ecol Health Dis1992;5:iv–v. ↵ Oelschläger TA, Altenhoefer A, Hacker J. Inhibition of Salmonella typhimurium invasion into intestinal cells by the probiotic E. coli strain Nissle 1917. Gastroenterology2001;120 (suppl) :A326. ↵ Boudeau J , Rich C, France CF, et al. Escherichia coli strain Nissle 1917 inhibits adhesion to and invasion of intestinal epithelial cells by adherent-invasive E. coli isolated from a Crohn’s disease patient. Gastroenterology2001;120 (suppl) :A190. ↵ Swidsinski A , Swidsinski S, Godzun A, et al. Therapy with E. coli Nissle reduces concentrations of mucosa associated colonic flora in patients with ulcerative colitis. Gastroenterology2000;118 (suppl) :A1138. ↵ Schulze J , Sonnenborn U. The role of the gut flora in inflammatory bowel diseases. In: Shimoyama T, Axon A, Lee A, et al, eds. In: Helicobacter meets inflammatory bowel disease. Tokyo: Medical Tribune Inc, 2002:393–417. ↵ Möllenbrink M , Bruckschen E. Treatment of chronic constipation with physiologic Escherichia coli bacteria. Results of a clinical study of the effectiveness and tolerance of microbiological therapy with the E. coli Nissle 1917 strain (Mutaflor). Med Klin1994;89:587–93. ↵ Malchow HA. Crohn’s disease and Escherichia coli. A new approach in therapy to maintain remission of colonic Crohn’s disease? J Clin Gastroenterol1997;25:653–8. ↵ Bruckschen E , Horosiewicz H. Chronic constipation. Comparision of microbiological therapy and lactulose. MMW1994;136:241–5. ↵ Prantera C , Scribano ML, Falasco G, et al. Ineffectiveness of probiotics in preventing recurrence after curative resection for Crohn’s disease: a randomised controlled trial with Lactobacillus GG. Gut2002;51:405–9. ↵ Guslandi M , Mezzi G, Sorghi M, et al. Saccharomyces boulardii in maintenance treatment of Crohn’s disease. Dig Dis Sci2000;45:1462–4. ↵ Kuzela L , Kaščák M, Vavrecka A. Induction and maintenance of remission with nonpathogenic Escherichia coli in patients with pouchitis. Am J Gastroenterol2001;96:3218–19. ↵ Gionchetti P , Rizzello F, Helwig U, et al. Prophylaxis of pouchitis onset with probiotic therapy: a double-blind, placebo-controlled trial. Gastroenterology2003;124:1202–9. ↵ Gionchetti P , Rizzello F, Venturi A, et al. Oral bacteriotherapy as maintenance treatment in patients with chronic pouchitis: a double-blind, placebo-controlled trial. Gastroenterology2000;119:305–9. ↵ Venturi A , Gionchetti P, Rizzello F, et al. Impact on the composition of the faecal flora by a new probiotic preparation: preliminary data on maintenance treatment of patients with ulcerative colitis. Aliment Pharmacol Ther1999;13:1103–8. 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Escherichia coli Nissle 1917 (EcN) is an intestinal probiotic that is effective for the treatment of intestinal disorders, such as inflammatory bowel disease and ulcerative colitis. EcN is a representative Gram-negative probiotic in biomedical research and is an intensively studied probiotic. Loading metrics Open Access Peer-reviewed Research Article Sandeep Kumar, Lesley A. Ogilvie, Bhavik A. Patel, Cinzia Dedi, Wendy M. Macfarlane, Brian V. Jones Disruption of Escherichia coli Nissle 1917 K5 Capsule Biosynthesis, through Loss of Distinct kfi genes, Modulates Interaction with Intestinal Epithelial Cells and Impact on Cell Health Jonathan Nzakizwanayo, Sandeep Kumar, Lesley A. Ogilvie, Bhavik A. Patel, Cinzia Dedi, Wendy M. Macfarlane, Brian V. Jones x Published: March 19, 2015 Figures AbstractEscherichia coli Nissle 1917 (EcN) is among the best characterised probiotics, with a proven clinical impact in a range of conditions. Despite this, the mechanisms underlying these "probiotic effects" are not clearly defined. Here we applied random transposon mutagenesis to identify genes relevant to the interaction of EcN with intestinal epithelial cells. This demonstrated mutants disrupted in the kfiB gene, of the K5 capsule biosynthesis cluster, to be significantly enhanced in attachment to Caco-2 cells. However, this phenotype was distinct from that previously reported for EcN K5 deficient mutants (kfiC null mutants), prompting us to explore further the role of kfiB in EcN:Caco-2 interaction. Isogenic mutants with deletions in kfiB (EcNΔkfiB), or the more extensively characterised K5 capsule biosynthesis gene kfiC (EcNΔkfiC), were both shown to be capsule deficient, but displayed divergent phenotypes with regard to impact on Caco-2 cells. Compared with EcNΔkfiC and the EcN wild-type, EcNΔkfiB exhibited significantly greater attachment to Caco-2 cells, as well as apoptotic and cytotoxic effects. In contrast, EcNΔkfiC was comparable to the wild-type in these assays, but was shown to induce significantly greater COX-2 expression in Caco-2 cells. Distinct differences were also apparent in the pervading cell morphology and cellular aggregation between mutants. Overall, these observations reinforce the importance of the EcN K5 capsule in host-EcN interactions, but demonstrate that loss of distinct genes in the K5 pathway can modulate the impact of EcN on epithelial cell health. Citation: Nzakizwanayo J, Kumar S, Ogilvie LA, Patel BA, Dedi C, Macfarlane WM, et al. (2015) Disruption of Escherichia coli Nissle 1917 K5 Capsule Biosynthesis, through Loss of Distinct kfi genes, Modulates Interaction with Intestinal Epithelial Cells and Impact on Cell Health. PLoS ONE 10(3): e0120430. Editor: Markus M. Heimesaat, Charité, Campus Benjamin Franklin, GERMANYReceived: December 9, 2014; Accepted: January 22, 2015; Published: March 19, 2015Copyright: © 2015 Nzakizwanayo et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are creditedData Availability: All relevant data are within the paper and its Supporting Information Support is provided by the Medical Research Council (G0901553) awarded to BVJ; University of Brighton Studentship to JN; Society of Applied Microbiology; BVJ is also supported by the Queen Victoria Hospital Charitable Trust. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the interests: The authors have declared that no competing interests exist. IntroductionDue to the intimate role of the gut microbiome in human health and disease processes, this predominantly bacterial community is increasingly viewed as an important target for the development of novel approaches to diagnose, prevent, or treat a wide range of disorders [1–4]. In this context, probiotics are among the most promising tools for manipulation of the gut microbiome, and have been defined as “live microorganisms which when administered in adequate amounts confer a health benefit on the host” [5]. The majority of probiotics are Gram-positive bacterial species, and considerable evidence is accumulating regarding the efficacy of these organisms in treating or preventing a variety of gastrointestinal (GI) diseases, and potentially also extra-intestinal disorders [1–4]. Among the probiotics currently available, Escherichia coli Nissle 1917 (EcN; serotype O6:K5:H1) is of particular interest. Not only is this one of the most extensively characterized probiotic organisms (in terms of phenotype, genotype, and clinical efficacy), but is currently the only Gram-negative species in use [6]. EcN was first isolated from the faeces of a World War I soldier who, in contrast to comrades in his trench, was not affected by an outbreak of dysentery [7]. This gastroprotective strain is the active component of Mutaflor (Ardeypharm GmbH, Herdecke, Germany), a microbial drug that is marketed and used in several countries. Clinical trials have shown EcN to be effective for maintaining remission of ulcerative colitis (UC) [8–11], stimulation of the immune system in premature infants [12], treatment of infectious diarrhoea [13], and protection of human intestinal epithelial cells (IECs) against pathogens [14, 15]. These benefits are largely attributed to the immuno-modulatory effects elicited by EcN, which encompass both innate and adaptive elements of the immune system. For example, colonisation with EcN has been indicated to alter the host cytokine profile, and also chemokine production in cultured IECs [16–19]; stimulate the production of mucosal peptide based defences [20]; influence the clonal expansion of T-Cell populations, and modulate antibody responses [12, 21, 22]. Notably, the modulation of T-cell functions mediated by EcN may also extend to γδ T-cells, potentially enabling EcN to coordinate modulation of both innate and adaptive responses [22]. EcN has also been indicated to alter COX-2 expression in intestinal epithelial cells [23], which is an important target in the treatment or prevention of several GI diseases including IBD and colorectal cancer [24–27]. Although most closely related to uropathogenic strains of E. coli (UPEC), EcN is considered non-pathogenic. Genomic characterisation has highlighted the absence of genes encoding the typical UPEC virulence factors, but the retention or accumulation of factors proposed to facilitate general adaptability, colonisation of the GI tract, and the probiotic effects of EcN [28, 29]. These include a range of surface associated structures that are likely to provide the primary interface between host and microbe in the GI tract, such as flagella, fimbriae, a special truncated lipopolysaccharide (LPS) variant, and a K5 type polysaccharide capsule [6, 29–31]. In particular, structures such as flagellin, peptidoglycan and LPS, are recognised by immune regulating Toll-like receptors (TLRS) expressed by IECs, which have been established as key routes of host-microbe communication in the gut, with TLR signalling integral to epithelial homoeostasis and defence [32–34]. Signaling by several TLRs is known to be modulated either directly or indirectly by EcN derived ligands [6, 17–20, 30, 35], which include surface associated structures absent in most or all other probiotic organisms. The K5 capsule produced by EcN in particular is notable in this context, and although not a ligand for known TLRs, the EcN capsule has been implicated in the interaction of this organism with IECs, and impact on chemokine expression and TLR signalling [18,19]. Nevertheless, as with other probiotics, the detailed mechanisms underlying the clinical effectiveness of EcN remain poorly understood overall, with a greater comprehension required to fully realise the potential of this important probiotic species. Here we describe the application of random transposon mutagenesis to identify genes and surface structures involved in the interaction of EcN with human intestinal epithelial cells, and provide new insight into the mechanisms through which EcN interacts with epithelial cells. Results Isolation and genetic characterisation of EcN mutants with disruptions in genes related to cell surface structures Because cell surface structures are a primary point of contact between EcN and IECs, and processes such as biofilm formation and attachment to abiotic surfaces also depends on many of the same structures, we reasoned that selection of mutants with alterations in biofilm formation would enrich for those defective in cell surface associated features also likely to be involved in EcN-IEC interaction. Therefore, we initially subjected a total of 4,116 EcN mini-Tn5 mutants to a preliminary high throughput screen for alterations in biofilm formation (both enhancements and reductions), in order to enrich for mutants attenuated in cell surface features. In this precursor biofilm screen 21 mutants were found to be significantly different in their ability to form biofilms as compared to the EcN wild-type (EcN WT), but unaltered in general growth rate. The majority of these (n = 15) exhibited a biofilm formation enhanced (BFE) phenotype, whereas six exhibited biofilm formation deficient (BFD) phenotype as compared to the WT (Table 1). Identities of genes disrupted in these mutants indicated that the majority were associated with synthesis of cell surface structures, or aspects of cell envelope biogenesis, previously linked to host-IEC interaction or intestinal colonisation (Table 1; [18, 35, 37–40]). A subset of 6 mutants disrupted in genes predicted to encode for cell surface structures, and encompassing both BFD and BFE phenotypes, were subsequently selected for further characterisation of their interaction with cultured IECs. Fig 1. Adherence of EcN mini-Tn5 mutants to Caco-2 cells. A subset of mutants recovered from biofilm screens with disruptions in genes predicted to be involved in generation of surface tstructures, were assessed for their ability to attach to Caco-2 cells in in vitro co-culture models. Caco-2 cell monolayers (~80% confluence) were exposed to bacterial suspensions from mid-log-phase cultures at an MOI of 1:1 for 4 h at 37°C, 5% CO2. Genes disrupted in mutants tested are noted in parentheses and details can be found in Table 1. Data are expressed as the mean of three replicates, and error bars show SE of the mean. Significant differences between attachment of EcN WT and mutants is indicated by ** (P ≤ or **** (P were confirmed biofilm altered mutants and defined as biofilm enhanced (BFE) or biofilm deficient (BFD) mutants. Mutants biofilm formation index was calculated as the percentage of CV (OD595) measured in the EcN WT. Genetic characterisation of biofilm-altered mutants Genes disrupted in mutants of interest were identified using a “cloning free” arbitrary PCR-based approach to amplify DNA segments flanking the transposon insertion, as described by Manoil [55] using primers listed in S2 Table. The resulting amplicons were sequenced by GATC Biotech Ltd. (London, UK) using transposon end primer pLR27Primer 3. The putative function of disrupted genes was assigned by mapping sequence data flanking the mini-Tn5 insert site to the E. coli Nissle Draft genomes sequence [28], and the previously published genomic islands [29]. Sequence reads from mutants were trimmed to remove the 5’ low quality regions (typically ~30–50 nt), and the immediate ~40 nt flanking sections correlated with the EcN genome. Where EcN genome annotations did not provide any clear indication of putative function wider searches of the nr dataset using BlastX and/or the conserved domain database were employed. Construction of kfiB and kfiC deletion mutants Deletion mutants EcNΔkfiB and EcNΔkfiC were constructed by homologous recombination using the Xer-ciseTM chromosomal modification system (Cobra Biologics, Keele, UK) according to manufacturer’s instructions and protocols described by Bloor and Cranenburgh [56]. The system comprises plasmids pTOPO-DifCAT and pLGBE, for construction of target gene specific integration cassette and provision of the Red λ recombination functions, respectively. Briefly, kfiB or kfiC integration cassettes consisting of the difE. coli-cat-difE. coli region from pTOPO-DifCAT plasmid flanked by 50 nt regions homologous to the 3’ and 5' ends of the target gene, were generated by PCR using 70-nt primers, or (listed in S2 Table). EcN WT was first transformed with the Tc-selectable plasmid pLGBE and transformants EcN-pLGBE were used to generate electrocompetent cells, which were subsequently transformed with the PCR product of the difE. coli-cat-difE. coli integration cassette constructs. Integrants were selected on LB agar supplemented with 20 μg ml–1 Chloramphenicol. Loss of pLGBE and generation of chloramphenicol-sensitive clones, indicating resolution of difE. coli-cat-difE. coli marker genes by native recombinases and generation of markerless deletion mutants (mutants EcNΔkfiB and EcNΔkfiC) was achieved by sub-culturing the integrants in LB broth in the absence of antibiotics. Loss of pLGBE was verified by plasmid extraction, and by PCR for marker cassettes kfiB or kfiC specific primers EcNkfiB _F/R or EcNkfiC _F/R, respectively, and confirmed by PCR. Examination of polar effects in EcNΔkfiB and EcNΔkfiC mutants The effect of gene deletion or disruptions in kfiB and kfiC mutants, on the expression of downstream genes (polar effects) was assessed using RT-PCR. Total RNA was extracted from mid-log-phase bacterial cells using the RNeasy Protect Cell Mini Kit (Qiagen) according to manufacturer’s instructions, and treated using the Ambion TURBO DNA-free system (Ambion-Life technologies, Paisley, UK) to remove any potential DNA contamination. The treated RNA was used to generate cDNA using the One Step RT-PCR kit (Qiagen) according to the manufacturer’s instructions, utilising 15 ng RNA per reaction as template. Resulting cDNA was used as template in standard PCRs for detection of gene transcripts with specific primers detailed in S2 Table. Confirmation of K5 capsule absence in EcNΔkfiB and EcNΔkfiC mutants The K5 capsule-specific bacteriophage (ΦK5) [57] was used in this study to determine if the K5 capsule was expressed by EcN WT and deletion mutants. The bacteriophage was diluted and maintained in phage dilution buffer (PDB) (100 mM NaCl, 8 mM MgSO4, gelatine, 50 mM Tris pH Cultures of mutants EcNΔkfiB and EcNΔkfiC, controls EcN WT and MG1655 were grown in LB with shaking at 37°C to an OD600 of then pelleted by centrifugation (10,000 × g for 10 min) and resuspended in ice-cold 10 mM MgSO4. Aliquots of cell suspension (100 μl) were mixed with 100 μl of the appropriate bacteriophage dilution (ranging from 101 to 109 PFU ml–1 from stock suspension of × 109 PFU ml–1) in sterile mL Eppendorf tube then incubated at RT for 30 min, statically. The phage-bacteria mixture was added to a volume of 3 ml of soft agar (1% NaCl, yeast extract, 1% tryptone, agar) held at 42°C in 15 ml sterile glass tube, and the content of the tubes were mixed gently by swirling. The inoculated soft agar was poured on top of LB agar and incubated for 16 h at 37°C to allow formation of plaques. Intestinal epithelial cell culture and co-culture conditions Caco-2 cells (passage 51–79) were grown at 37°C with 5% CO2 in Dulbecco's modified Eagle's medium (DMEM, g glucose l–1) supplemented with 10% fetal bovine serum and 1× non-essential amino acids (PAA Laboratories, Somerset, UK). Cells were seeded into 6-well or 96-well plates, grown up to ~ 60–80% confluence, and used in co-culture experiments with bacteria. Mid-log-phase bacteria (OD600 of were washed with PBS and suspended in DMEM to the required final count, corresponding to the appropriate multiplicity of infection (MOI) and added to Caco-2 monolayers before plates were incubated at 37°C and 5% CO2. Bacterial adherence to Caco-2 cells Adherence was calculated according to the strategy employed by Hafez et al. [18]. Mid-log phase bacteria cultures were suspended in DMEM then added to monolayers of Caco-2 grown in 6-well plates (80% confluence) at an MOI of 1:1 and incubated at 37°C and 5% CO2 for 4 h. The monolayers were washed 3 times with PBS to remove non-adherent cells then treated with lysis solution, 1% wt / vol saponin (Sigma Aldrich) in trypsin-EDTA (PAA Laboratories, Somerset, UK) for 10 min to allow permeabilisation of Caco-2 cells and recovery of total cell-associated bacteria. Cells were mixed gently by pipetting, serially diluted in sterile PBS, plated onto LB agar, and incubated at 37°C overnight. The obtained viable count represented the total number of cell associated bacteria (adherent and internalised). Internalised bacteria were calculated using the same protocol but Caco-2 cells were treated with gentamicin for 2h (200 μg ml-1) to kill external bacteria prior to lysis and enumeration. The number of adherent bacteria was taken as the difference between total cell associated bacteria and internalised bacteria. The effect of EcN mutants on induction of apoptosis in Caco-2 cells The effect of EcN mutants on induction of apoptosis Caco-2 cells was assessed by measuring the activity of caspase 3/7 using the Caspase-Glo 3/7 kit (Promega, Southampton, UK), according to manufacturer’s instructions. Cells were seeded in 96-well plates with 5,000 cells/well and cultured to achieve ~ 60% confluence then treated with bacteria or bacterial supernatants in co-culture. Media was replaced with serum-free DMEM for 12 h prior to the treatment. Bacterial suspensions were prepared in serum-free DMEM from mid-log-phase cultures then added to Caco-2 cells at an MOI of 10:1 (bacteria:Caco-2) in a final volume of 100 μl/ well. The plates were incubated for 2 h at 37°C and 5% CO2 then media was replaced with fresh serum-free DMEM supplemented with gentamicin at 200 μg ml–1 to stop bacterial growth, and plates were incubated for another 10 h. Bacterial supernatants were obtained from cells grown in 5 mL serum-free DMEM at 37°C overnight, with shaking, and recovered by centrifugation (1,500 × g for 10 min), pH adjusted to and filter-sterilised ( The supernatants were diluted in fresh serum-free DMEM at a ratio of 1:1, and used in place of cell suspensions as described above. Caspase 3/7 activity was measured as relative light units (RLUs) using a Synergy Multi-Mode Plate Reader (BioTek, Potton, UK) operated with BioTek software. Analysis of cytotoxicity The effect of EcN strains on induction of cytotoxicity in Caco-2 cells was assessed by measuring the amount of lactate dehydrogenase (LDH) released into the co-culture media, using the CytoTox 96 Non-Radioactive Cytotoxicity Assay kit (Promega). Caco-2 cells were treated with bacteria and controls as described for the analysis of apoptosis (above) and both assays were performed in parallel. After treatment of Caco-2 cells, supernatants were collected from plate wells using a multichannel pipette then transferred to fresh 96-well at 50 μl/well. The supernatant was diluted further in serum-free culture media then mixed with the CytoTox 96 substrate at a ration of 1:1. Plates were incubated in the dark at room temperature for 30 min and absorbance at 490 nm (OD490) was recorded. The percentage of cytotoxicity was calculated as LDH released in treated cells (OD490)/maximum LDH release (OD490) × 100. Maximum release was determined as the amount released by total lysis of untreated Caco-2 cells with the CytoTox 96 lysis Solution (10X). Analysis of cellular and nuclear morphology Membrane integrity and nuclear morphology of Caco-2 cells were analysed by fluorescent phalloidin (F-actin) and Dapi (DNA) stainings. Cells were grown on sterile glass cover slips in 6-well plates then treated with EcN strains and controls (MG1655 and mM camptothecin; Sigma) as described above (analysis of apoptosis). After the treatments, the cells on coverslips were washed with PBS then fixed with 4% formaldehyde (Sigma) in PBS for 20 min at RT. The fixed cells were washed three times with PBS and permeabilised with Triton X-100 (Sigma) in PBS for 5 min at RT. The cells were washed three times with PBS, 5 min per wash with gentle rocking, then treated with a μg ml–1 solution of fluorescein isothiocyanate-phalloidin (Sigma- Aldrich) in PBS for 1 h at RT in the dark. The cells were washed twice with PBS and were mounted with the Fluoroshield DAPI medium (Sigma) and examined under a Leica TCS SP5 Confocal Laser Scanning microscope (Leica Microsystems, Wetzlar, Germany). Analysis of COX-2 expression The expression of COX-2 protein in Caco-2 co-cultures was analysed by western blotting using standard methods. Briefly, Caco-2 cells were seeded in 6 wells plates, and at ~ 60% confluence, were treated with EcN K5 mutants and controls as described above (analysis of apoptosis). Lipopolysaccharide (LPS, final concentration, 5 μg ml–1) from Salmonella enterica (Sigma, UK) and human tumour necrosis factor alpha (TNF-α, 10 ng ml–1) (Sigma, UK) were used as pro-inflammatory stimulator positive controls. Treated Caco-2 cell monolayers were washed 3 times with PBS, trypsinised then resuspended in 100 μl of hypotonic buffer (10 mM HEPES, 10 mM KCl, mM EDTA, mM EGTA, 1 mM DTT in SDW, pH containing Sigma protease inhibitor cocktail (1:20), for 15 min at 4°C. Cells were lysed in 25 μl 10% Triton X-100 for 30 min and total protein obtained by centrifugation (10,000 g for 1 min at 4°C). Protein concentration was determined by the Bradford method (Bio-Rad) and equivalent amounts of protein lysates (10 μg) separated by electrophoresis on SDS—PAGE (10%), and then transferred onto a nitrocellulose membrane (GE Healthcare, Giles, UK). The blots were blocked at RT with 10% skimmed milk powder in TBST buffer (10 mM Tris, pH M NaCl, Tween 20), and incubated with primary antibody, anti-COX-2 rabbit polyclonal (Abcam, Cambridge, UK) 1:1,000 in TBST, overnight at 4°C. Blots were washed with TBST then incubated with anti-rabbit HRP-conjugated secondary antibody (Sigma, UK) 1:5,000 in TBST, for 1h at RT. Membranes were washed further then visualised by incubation with the ECL chemiluminescent reagent (Amersham, Little Chalfont, UK) and exposed to Kodak Image Station 440 for signal detection. Blots were then stripped and reprobed with loading control anti-GAPDH mouse monoclonal (Ambion, Cambridge, UK); anti-mouse IgG HRP-conjugated (Sigma, UK) as secondary antibody. The bands of COX-2 densitometry readings were normalized to the GAPDH control. Analysis of cell morphology and aggregation Bacteria were grown statically in 5 mL LB in 50 mL sterile polystyrene tube at 37°C for 16 h. The cultures were mix gently by swirling and 3 μL of each was directly transferred onto glass slide, allowed to rest for 1 min then covered with a cover slip and visualised using ×40 magnification phase contrast microscopy. For each culture 10 randomly selected fields of view across each slide were captured using the Olympus Cell Sense software, and subsequently reviewed. Representative images were selected and adjusted only for brightness and contrast. Statistical analysis All statistical analysis was performed using Prism For Mac OS X (Graphpad Software inc. USA; Data was analysed using either Student’s t-test, or ANOVA with the Bonferroni correction for multiple comparisons. Supporting InformationS1 Fig. Overview of K5 capsule biosynthesis in E. coli, and associated genes disrupted in this show the genetic organisation of the K5 gene cluster in E. coli Nissle 1917 based on data from Cress et al. [28]; Grozdanov et al. [29], and an overview of the current model for K5 capsule biosynthesis and assembly adapted from Griffiths et al. [36]; Whitfield [41]; Petit et al. [42]; Bliss et al. [43]; Hodson et al. [44]; Corbett and Roberts [45]; Whitfield and Roberts [46]; Rigg et al. [47]; Whitfield and Willis [58]. A) Physical map of the EcN K5 capsular polysaccharide gene cluster. Region I (kpsF,E,D,U,C,S) and Region III (kpsM,T) encode elements of synthesis and export machinery, and are conserved among E. coli strains generating Group 2 polysaccharide capsules. Region II encodes K5 specific polysaccharide synthesis machinery (kfiA,B,C,D). Genes disrupted by transposon mutagenesis (kfiB, kpsT) and/or subject to gene knockout (kfiB,C) in this study are identified. HP—denote hypothetical proteins of unknown function B) Representation of main stages and associated K5 biosynthetic machinery (stages 1–3). K5 assembly is localised to the cytoplasmic face of the inner membrane, and is underpinned by the formation of a biosynthetic complex which catalyses synthesis and export polysaccharide precursors for incorporation in the maturing capsule on the cell surface. During K5 assembly it is believed that a unified biosynthetic complex is developed which progressively catalyses main stages [1–3]. However, for clarity here we have separated each main stage of K5 synthesis and associated membrane complexes. Stage 1) Proteins encoded by kpsF,U,C,S are believed to be responsible for the initial generation of the phospatyidyl acceptor and Kdo linker (keto-3-deoxy-manno-2-octulosonic acid), upon which the polysaccharide chain is synthesised. Stage 2) Proteins encoded by kfiA-D are responsible for synthesis of the polysaccharide chain through addition of alternating units of GlcA (glucuronic acid) and GlcNAc (N-acetyl-glucosamine) from UDP-sugar precursors. Stage 3) Proteins generated by kpsD,E,M,T form an ABC transporter complex that translocates completed polysaccharide chains to the cell surface, in an energy dependant process. Acknowledgments We wish to thank Prof Jun Zhu (University of Pennsylvania, School of Medicine) and Prof Ian Roberts (University of Manchester, Faculty of Life Sciences) for gifts of pRL27::mini-Tn5 system and ΦK5 bacteriophage, respectively. We also thank Dr Rocky Cranenburgh (Cobra Biologics) for generous provision of the Xer-cise system, and technical support during its application. We also thank Joseph Hawthorn, Rowena Berterelli, Heather Catty, Christopher Morris and Maurizio Valeri for excellent technical support, and Dr Claire Rosten and Dr Caroline Jones for constructive comments and criticism. Author ContributionsConceived and designed the experiments: BVJ JN. Performed the experiments: JN SK. Analyzed the data: BVJ JN LAO BAP WMM. Wrote the paper: JN LAO BAP CD WMM Foxx-Orenstein AE, Chey W. Manipulation of the gut microbiota as a novel treatment strategy for gastrointestinal disorders. Am J Gastroenterol Suppl. 2012; 1: 41–46. View Article Google Scholar 2. Shanahan F. Therapeutic implications of manipulating and mining the microbiota. J Physiol. 2009; 587: 4175–4179. pmid:19505978 View Article PubMed/NCBI Google Scholar 3. O'Sullivan GC, Kelly P, O'Halloran S, Collins C, Collins JK, Dunne C, et al. Probiotics: an emerging therapy. Curr Pharm Des. 2005; 11: 3–10. pmid:15641939 View Article PubMed/NCBI Google Scholar 4. Ringel Y, Quigley EMM, Li HC. Using probiotics in gastrointestinal disorders. Am J Gastroenterol Suppl. 2012; 1: 34–40. View Article Google Scholar 5. FAO/WHO. Guidelines for the evaluation of probiotics in food. Report of a Joint FAO/WHO Working Group on Drafting Guidelines for the Evaluation of Probiotics in Food; Ontario, Canada. April 30, May 1, 2002. 6. Sonnenborn U, Schulze J. The non-pathogenic Escherichia coli strain Nissle 1917—features of a versatile probiotic. Microb Ecol Health Dis. 2009; 21: 122–158. View Article Google Scholar 7. Nissle A. Die antagonistische Behandlung chronischer Darmstörungen mit Colibakterien. Med Klin. 1918; 2: 29–33. View Article Google Scholar 8. Schultz M. Clinical use of E. coli Nissle 1917 in inflammatory bowel disease. Inflamm Bowel Dis. 2008; 14: 1012–1018. pmid:18240278 View Article PubMed/NCBI Google Scholar 9. Kruis W, Schütz E, Fric P, Fixa B, Judmaier G, Stolte M. Double-blind comparison of an oral Escherichia coli preparation and mesalazine in maintaining remission of ulcerative colitis. Aliment Pharmacol Ther. 1997; 11: 853–858. pmid:9354192 View Article PubMed/NCBI Google Scholar 10. Kruis W, Fric P, Pokrotnieks J, Lukás M, Fixa B, Kascák M, et al. Maintaining remission of ulcerative colitis with the probiotic Escherichia coli Nissle 1917 is as effective as with standard mesalazine. Gut 2004; 53:1617–1623. pmid:15479682 View Article PubMed/NCBI Google Scholar 11. Matthes H, Krummenerl T, Giensch M, Wolff C, Schulze J. Clinical trial: probiotic treatment of acute distal ulcerative colitis with rectally administered Escherichia coli Nissle (EcN). BMC Complement Altern Med. 2010; 10:13. pmid:20398311 View Article PubMed/NCBI Google Scholar 12. Cukrowska B, LodInová-ZádnIková R, Enders C, Sonnenborn U, Schulze J, Tlaskalová-Hogenová H. Specific proliferative and antibody responses of premature infants to intestinal colonization with nonpathogenic probiotic E. coli strain Nissle 1917. Scand J Immunol. 2002; 55: 204–209. pmid:11896937 View Article PubMed/NCBI Google Scholar 13. Henker J, Laass M, Blokhin BM, Bolbot YK, Maydannik VG, Elze M, et al. The probiotic Escherichia coli strain Nissle 1917 (EcN) stops acute diarrhoea in infants and toddlers. Eur J Pediatr. 2007; 166: 311–318. pmid:17287932 View Article PubMed/NCBI Google Scholar 14. Boudeau J, Glasser AL, Julien S, Colombel JF, Darfeuille-Michaud A. Inhibitory effect of probiotic Escherichia coli strain Nissle 1917 on adhesion to and invasion of intestinal epithelial cells by adherent-invasive E. coli strains isolated from patients with Crohn's disease. Aliment Pharmacol Ther. 2003; 18: 45–56. pmid:14531740 View Article PubMed/NCBI Google Scholar 15. Lodinová-Zádniková R, Sonnenborn U. Effect of preventive administration of a nonpathogenic Escherichia coli strain on the colonization of the intestine with microbial pathogens in newborn infants. Biol Neonate 1997; 71: 224–232. pmid:9129791 View Article PubMed/NCBI Google Scholar 16. Ukena SN, Westendorf AM, Hansen W, Rohde M, Geffers R, Coldewey S, et al. The host response to the probiotic Escherichia coli strain Nissle 1917: specific up-regulation of the proinflammatory chemokine MCP-1. BMC Med Genet. 2005; 6: 43. pmid:16351713 View Article PubMed/NCBI Google Scholar 17. Grabig A, Paclik D, Guzy C, Dankof A, Baumgart DC, Erckenbrecht J, et al. Escherichia coli strain Nissle 1917 ameliorates experimental colitis via toll-like receptor 2- and toll-like receptor 4-dependent pathways. Infect Immun. 2006; 74: 4075–4082. pmid:16790781 View Article PubMed/NCBI Google Scholar 18. Hafez M, Hayes K, Goldrick M, Warhurst G, Grencis R, Roberts IS. The K5 capsule of Escherichia coli strain Nissle 1917 is important in mediating interactions with intestinal epithelial cells and chemokine induction. Infect Immun. 2009; 77: 2995–3003. pmid:19380467 View Article PubMed/NCBI Google Scholar 19. Hafez M, Hayes K, Goldrick M, Grencis RK, Roberts IS. The K5 capsule of Escherichia coli strain Nissle 1917 is important in stimulating expression of Toll-like receptor 5, CD14, MyD88, and TRIF together with the induction of interleukin-8 expression via the mitogen-activated protein kinase pathway in epithelial cells. Infect Immun. 2010; 78: 2153–2162. pmid:20145095 View Article PubMed/NCBI Google Scholar 20. Schlee M, Wehkamp J, Altenhoefer A, Oelschlaeger TA, Stange EF, Fellermann K. Induction of Human β-Defensin 2 by the Probiotic Escherichia coli Nissle 1917 Is Mediated through Flagellin. Infect Immun. 2007; 75: 2399–2407. pmid:17283097 View Article PubMed/NCBI Google Scholar 21. Sturm A, Rilling K, Baumgart DC, Gargas K, Abou-Ghazalé T, Raupach B, et al. Escherichia coli Nissle 1917 distinctively modulates T-cell cycling and expansion via toll-like receptor 2 signaling. Infect Immun. 2005; 73: 1452–1465. pmid:15731043 View Article PubMed/NCBI Google Scholar 22. Guzy C, Paclik D, Schirbel A, Sonnenborn U, Wiedenmann B, Sturm A. The probiotic Escherichia coli strain Nissle 1917 induces γδ T cell apoptosis via caspase- and FasL-dependent pathways. Int Immunol. 2008; 20: 829–840. pmid:18448456 View Article PubMed/NCBI Google Scholar 23. Otte JM, Mahjurian-Namari R, Brand S, Werner I, Schmidt WE, Schmitz F. Probiotics regulate the expression of COX-2 in intestinal epithelial cells. Nutr Cancer 2009; 61: 103–113. pmid:19116880 View Article PubMed/NCBI Google Scholar 24. Wang D, Dubois RN. The role of COX-2 in intestinal inflammation and colorectal cancer. Oncogene 2010; 29: 781–788. pmid:19946329 View Article PubMed/NCBI Google Scholar 25. Wallace JL. Prostaglandin biology in inflammatory bowel disease. Gastroenterol Clin North Am. 2001; 30: 971–980. pmid:11764538 View Article PubMed/NCBI Google Scholar 26. Ritland SR, Gendler SJ. Chemoprevention of intestinal adenomas in the ApcMin mouse by piroxicam: kinetics, strain effects and resistance to chemosuppression. Carcinogenesis 1999; 20: 51–58. pmid:9934849 View Article PubMed/NCBI Google Scholar 27. Rhodes JM, Campbell BJ. Inflammation and colorectal cancer: IBD-associated and sporadic cancer compared. Trends Mol Med. 2002; 8: 10–16. pmid:11796261 View Article PubMed/NCBI Google Scholar 28. Cress BF, Linhardt RJ, Koffas MA. Draft Genome Sequence of Escherichia coli Strain Nissle 1917 (Serovar O6:K5:H1). Genome Announc. 2013; 1: e00047–13. View Article Google Scholar 29. Grozdanov L, Raasch C, Schulze J, Sonnenborn U, Gottschalk G, Hacker J, et al. Analysis of the genome structure of the nonpathogenic probiotic Escherichia coli strain Nissle 1917. J Bacteriol. 2004; 186: 5432–5441. pmid:15292145 View Article PubMed/NCBI Google Scholar 30. Grozdanov L, Zähringer U, Blum-Oehler G, Brade L, Henne A, Knirel YA, et al. A single nucleotide exchange in the wzy gene is responsible for the semirough O6 lipopolysaccharide phenotype and serum sensitivity of Escherichia coli strain Nissle 1917. J Bacteriol. 2002; 184: 5912–5925. pmid:12374825 View Article PubMed/NCBI Google Scholar 31. Vejborg RM, Friis C, Hancock V, Schembri MA, Klemm P. A virulent parent with probiotic progeny: comparative genomics of Escherichia coli strains CFT073, Nissle 1917 and ABU 83972. Mol Genet Genomics 2010; 283: 469–484. pmid:20354866 View Article PubMed/NCBI Google Scholar 32. Rakoff-Nahoum S, Paglino J, Eslami-Varzaneh F, Edberg S, Medzhitov R. Recognition of commensal microflora by toll-like receptors is required for intestinal homeostasis. Cell 2004; 118: 229–241. pmid:15260992 View Article PubMed/NCBI Google Scholar 33. Lundin A, Bok CM, Aronsson L, Björkholm B, Gustafsson JA, Pott S, et al. Gut flora, Toll-like receptors and nuclear receptors: a tripartite communication that tunes innate immunity in large intestine. Cell Microbiol. 2008; 10: 1093–1103. pmid:18088401 View Article PubMed/NCBI Google Scholar 34. Pålsson-McDermott EM, O'Neill LA. Signal transduction by the lipopolysaccharide receptor, Toll-like receptor-4. Immunology 2004; 113: 153–162. pmid:15379975 View Article PubMed/NCBI Google Scholar 35. Lasaro MA, Salinger N, Zhang J, Wang Y, Zhong Z, Goulian M, et al. F1C fimbriae play an important role in biofilm formation and intestinal colonization by the Escherichia coli commensal strain Nissle 1917. Appl Environ Microbiol. 2009; 75: 246–251. pmid:18997018 View Article PubMed/NCBI Google Scholar 36. Griffiths G, Cook NJ, Gottfridson E, Lind T, Lidholt K, Roberts IS. Characterization of the glycosyltransferase enzyme from the Escherichia coli K5 capsule gene cluster and identification and characterization of the glucuronyl active site. J Biol Chem. 1998; 273: 11752–11757. pmid:9565598 View Article PubMed/NCBI Google Scholar 37. Pratt LA, Kolter R. Genetic analysis of Escherichia coli biofilm formation: roles of flagella, motility, chemotaxis and type I pili. Mol Microbiol. 1998; 30: 285–293. pmid:9791174 View Article PubMed/NCBI Google Scholar 38. Chilcott GS, Hughes KT. Coupling of flagellar gene expression to flagellar assembly in Salmonella enterica serovar typhimurium and Escherichia coli. Microbiol Mol Biol Rev. 2000; 64: 694–708. pmid:11104815 View Article PubMed/NCBI Google Scholar 39. Troge A, Scheppach W, Schroeder BO, Rund SA, Heuner K, Wehkamp J, et al. More than a marine propeller—the flagellum of the probiotic Escherichia coli strain Nissle 1917 is the major adhesin mediating binding to human mucus. Int J Med Microbiol. 2012; 302: 304–314. pmid:23131416 View Article PubMed/NCBI Google Scholar 40. Klemm P, Schembri MA. Bacterial adhesins: function and structure. Int J Med Microbiol. 2000; 290: 27–35. pmid:11043979 View Article PubMed/NCBI Google Scholar 41. Whitfield C. Biosynthesis and assembly of capsular polysaccharides in Escherichia coli. Annu Rev Biochem. 2006; 75: 39–68. pmid:16756484 View Article PubMed/NCBI Google Scholar 42. Petit C, Rigg GP, Pazzani C, Smith A, Sieberth V, Stevens M, et al. Region 2 of the Escherichia coli K5 capsule gene cluster encoding proteins for the biosynthesis of the K5 polysaccharide. Mol Microbiol. 1995; 17: 611–620. pmid:8801416 View Article PubMed/NCBI Google Scholar 43. Bliss JM, Silver RP. Coating the surface: a model for expression of capsular polysialic acid in Escherichia coli K1. Mol Microbiol. 1996; 21: 221–231. pmid:8858578 View Article PubMed/NCBI Google Scholar 44. Hodson N, Griffiths G, Cook N, Pourhossein M, Gottfridson E, Lind T, et al. Identification that KfiA, a protein essential for the biosynthesis of the Escherichia coli K5 capsular polysaccharide, is an alpha-UDP-GlcNAc glycosyltransferase. The formation of a membrane-associated K5 biosynthetic complex requires KfiA, KfiB, and KfiC. J Biol Chem. 2000; 275: 27311–27315. pmid:10859322 View Article PubMed/NCBI Google Scholar 45. Corbett D, Roberts IS. Capsular polysaccharides in Escherichia coli. Adv Appl Microbiol. 2008; 65: 1–26. pmid:19026860 View Article PubMed/NCBI Google Scholar 46. Whitfield C, Roberts IS. Structure, assembly and regulation of expression of capsules in Escherichia coli. Mol Microbiol. 1999; 31: 1307–1319. pmid:10200953 View Article PubMed/NCBI Google Scholar 47. Rigg GP, Barrett B, Roberts IS. The localization of KpsC, S and T, and KfiA, C and D proteins involved in the biosynthesis of the Escherichia coli K5 capsular polysaccharide: evidence for a membrane-bound complex. Microbiology 1998; 144: 2905–2914. pmid:9802032 View Article PubMed/NCBI Google Scholar 48. Pelkonen S. Capsular sialyl chains of Escherichia coli K1 mutants resistant to K1 phage. Curr Microbiol. 1990; 21: 23–28. View Article Google Scholar 49. Pelkonen S, Aalto J, Finne J. Differential activities of bacteriophage depolymerase on bacterial polysaccharide: binding is essential but degradation is inhibitory in phage infection of K1-defective Escherichia coli. J Bacteriol. 1992; 174: 7757–7761. pmid:1447142 View Article PubMed/NCBI Google Scholar 50. Schembri MA, Dalsgaard D, Klemm P. Capsule shields the function of short bacterial adhesins. J Bacteriol. 2004; 186: 1249–1257. pmid:14973035 View Article PubMed/NCBI Google Scholar 51. Ulett GC, Valle J, Beloin C, Sherlock O, Ghigo JM, Schembri MA. Functional analysis of antigen 43 in uropathogenic Escherichia coli reveals a role in long-term persistence in the urinary tract. Infect Immun. 2007; 75: 3233–3244. pmid:17420234 View Article PubMed/NCBI Google Scholar 52. Hanna A, Berg M, Stout V, Razatos A. Role of capsular colanic acid in adhesion of uropathogenic Escherichia coli. Appl Environ Microbiol. 2003; 69:4474–4481. pmid:12902231 View Article PubMed/NCBI Google Scholar 53. Larsen RA, Wilson MM, Guss AM, Metcalf WW. Genetic analysis of pigment biosynthesis in Xanthobacter autotrophicus Py2 using a new, highly efficient transposon mutagenesis system that is functional in a wide variety of bacteria. Arch Microbiol. 2002; 178: 193–201. pmid:12189420 View Article PubMed/NCBI Google Scholar 54. O'Toole GA, Kolter R. Flagellar and twitching motility are necessary for Pseudomonas aeruginosa biofilm development. Mol Microbiol. 1998; 30: 295–304. pmid:9791175 View Article PubMed/NCBI Google Scholar 55. Manoil C. Tagging exported proteins using Escherichia coli alkaline phosphatase gene fusions. Methods Enzymol. 2000; 326: 35–47. pmid:11036633 View Article PubMed/NCBI Google Scholar 56. Bloor AE, Cranenburgh RM. An efficient method of selectable marker gene excision by Xer recombination for gene replacement in bacterial chromosomes. Appl Environ Microbiol. 2006; 72: 2520–2525. pmid:16597952 View Article PubMed/NCBI Google Scholar 57. Gupta DS, Jann B, Schmidt G, Golecki JR, Ørskov I, Ørskov F, et al. Coliphage K5, specific for E. coli exhibiting the capsular K5 antigen. FEMS Microbiol Lett. 1982; 14: 75–78. View Article Google Scholar 58. Willis LM, Whitfield C. KpsC and KpsS are retaining 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) transferases involved in synthesis of bacterial capsules. Proc Natl Acad Sci U S A. 2013; 110: 20753–20758. pmid:24302764 View Article PubMed/NCBI Google Scholar 59. Jones BV, Young R, Mahenthiralingam E, Stickler DJ. Ultrastructure of Proteus mirabilis swarmer cell rafts and role of swarming in catheter-associated urinary tract infection. Infect Immun. 2004; 72: 3941–3950. View Article Google Scholar
Escherichia coli Nissle1917 (EcN) is a non-pathogenic probiotic strain widely used to maintain gut health, treat gastrointestinal disorders, and modulate the gut microbiome due to its anti-inflammatory and competitive exclusion effects against pathogenic bacteria. Heparin, abundant on intestinal muc …
Authors: Pallavi Subhraveti1, Peter Midford1, Anamika Kothari1, Ron Caspi1, Peter D Karp1 1SRI International Summary: This Pathway/Genome Database (PGDB) was generated on 8-Mar-2022 from the annotated genome of Escherichia coli Nissle 1917, as obtained from RefSeq (annotation date: 26-MAY-2021). The PGDB was created computationally by the PathoLogic component of the Pathway Tools software (version [Karp16, Karp11] using MetaCyc version [Caspi20]. It has not undergone any manual curation or review, and may contain errors. Development of this PGDB was supported by grant GM080746 from the National Institutes of Health. Sequence Source: Taxonomic Lineage: cellular organisms, Bacteria , Proteobacteria, Gammaproteobacteria, Enterobacterales, Enterobacteriaceae, Escherichia, Escherichia coli, Escherichia coli Nissle 1917 Unification Links: BIOSAMPLE:SAMN07451663, NCBI BioProject:PRJNA224116, NCBI-Taxonomy:316435 Organism or Sample Properties Environment: stool Geographic Location: Germany Freiburg Altitude (m): Collection Date: 1917 Host: Homo sapiens Annotation Provider: NCBI RefSeq Annotation Date: 2021-5-25 17:34:29 Annotation Pipeline: NCBI Prokaryotic Genome Annotation Pipeline (PGAP) Annotation Pipeline Version: Annotation Comment: Best-placed reference protein set; GeneMarkS-2+ RepliconTotal GenesProtein GenesRNA GenesPseudogenesSize (bp)NCBI Link NZ_CP0226864,8114,5381141595,055,316NCBI-RefSeq:NZ_CP022686 pNissle116140211,499NCBI-RefSeq:NZ_CP022687 pMUT287015,514NCBI-RefSeq:NZ_CP023342 Total:4,8374,5591141625,072,329 Ortholog data available?Yes Genes:4,837 Pathways:423 Enzymatic Reactions:2,300 Transport Reactions:250 Polypeptides:4,561 Protein Complexes:26 Enzymes:1,777 Transporters:708 Compounds:1,565 Transcription Units:2,883 tRNAs:86 Protein Features:6,449 GO Terms:3,793 Genetic Code Number: 11 -- Bacterial, Archaeal and Plant Plastid (same as Standard, except for alternate initiation codons) PGDB Unique ID: 2K79 References Caspi20: Caspi R, Billington R, Keseler IM, Kothari A, Krummenacker M, Midford PE, Ong WK, Paley S, Subhraveti P, Karp PD (2020). "The MetaCyc database of metabolic pathways and enzymes - a 2019 update." Nucleic Acids Res 48(D1);D445-D453. PMID: 31586394 Karp11: Karp PD, Latendresse M, Caspi R (2011). "The pathway tools pathway prediction algorithm." Stand Genomic Sci 5(3);424-9. PMID: 22675592 Karp16: Karp PD, Latendresse M, Paley SM, Krummenacker M, Ong QD, Billington R, Kothari A, Weaver D, Lee T, Subhraveti P, Spaulding A, Fulcher C, Keseler IM, Caspi R (2016). "Pathway Tools version update: software for pathway/genome informatics and systems biology." Brief Bioinform 17(5);877-90. PMID: 26454094Report Errors or Provide Feedback Page generated by Pathway Tools version (software by SRI International) on Wed Jul 27, 2022, BIOCYC17B.
Towards this aim, we proposed an engineered Escherichia coli (E. coli) Nissle 1917 (EcN) strain with SARS-CoV-2 spike protein (SP)-coding plasmid, which was able to expose SP on its cellular surface by a hybridization with the adhesin involved in diffuse adherence 1 (AIDA1). In this study, we presented the effectiveness of a 16-week
Review Escherichiacoli Nissle 1917 as a Novel Microrobot for Tumor-Targeted Imaging and Therapy Qingyao Liu et al. Pharmaceutics. 2021. Free PMC article Abstract Highly efficient drug delivery systems with excellent tumor selectivity and minimal toxicity to normal tissues remain challenging for tumor treatment. Although great effort has been made to prolong the blood circulation and improve the delivery efficiency to tumor sites, nanomedicines are rarely approved for clinical application. Bacteria have the inherent properties of homing to solid tumors, presenting themselves as promising drug delivery systems. Escherichia coli Nissle 1917 (EcN) is a commonly used probiotic in clinical practice. Its facultative anaerobic property drives it to selectively colonize in the hypoxic area of the tumor for survival and reproduction. EcN can be engineered as a bacteria-based microrobot for molecular imaging, drug delivery, and gene delivery. This review summarizes the progress in EcN-mediated tumor imaging and therapy and discusses the prospects and challenges for its clinical application. EcN provides a new idea as a delivery vehicle and will be a powerful weapon against cancer. Keywords: E. coli Nissle 1917; bacteria-mediated tumor imaging; bacteria-mediated tumor therapy; microrobot; tumor colonization. Conflict of interest statement The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Figures Figure 1 EcN-mediated tumor imaging and therapy. (A) Schematic illustration of the ability of preferential tumor colonization in hypoxic regions. EcN can be designed to load drugs or integrate nanoparticles and express exogenous genes; (B) Schematic diagram of the strategies of various imaging modalities and treatment patterns for EcN, EcN minicell, and EcN ghost. Figure 2 (A) [18F]-FDS PET imaging in CT26-bearing mice treated with E. coli. (A) PET imaging was performed at day 0, 1, 3, 5 after intravenous injection of E. coli. The radioactivity uptake of the tumor was significantly higher at day 1, 3, 5 than in pre-treatment. (B) Positive correlation between SUVmax and the number of viable bacteria. (C) Schematic illustration of the synthesis of [18F]-FDS from [18F]-FDG. Reproduced with permission from Jung-Joon Min, Theranostics; published by Ivyspring International Publisher, 2020. Figure 3 (A) Schematic illustration of the synthesis of MTdox@EcN; (B) Schematic illustration of the mechanism of MTDOX@EcN as a biorocket for drug delivery in tumor; (C) Typical SEM and (D) CLSM images of MTDOX@EcN. (E) Tumor inhibition and (F) survival rates of MTdox@EcN treatment in tumor-bearing mice. Reproduced with permission from Xiaohong Li, Chemical Engineering Journal; published by Elsevier, 2020. Figure 4 (A) Schematic illustration of the engineering EcN strain named SYNB1891; (B) Tumor inhibition and (C) survival rates of SYNB1891 treatment in B16F10 tumor-bearing mice. Reproduced with permission from Jose M. Lora, Nature Communications; published by Springer Nature, 2020. ** p = (blue stars), **** p < (pink stars), ** p = (pink stars), ** p = (black stars), *** p = (black stars). Figure 5 The future application of nano-bacteria hybrid system. Similar articles Expressing cytotoxic compounds in Escherichia coli Nissle 1917 for tumor-targeting therapy. Li R, Helbig L, Fu J, Bian X, Herrmann J, Baumann M, Stewart AF, Müller R, Li A, Zips D, Zhang Y. Li R, et al. Res Microbiol. 2019 Mar;170(2):74-79. doi: Epub 2018 Nov 14. Res Microbiol. 2019. PMID: 30447257 Intestinal probiotics E. coli Nissle 1917 as a targeted vehicle for delivery of p53 and Tum-5 to solid tumors for cancer therapy. He L, Yang H, Tang J, Liu Z, Chen Y, Lu B, He H, Tang S, Sun Y, Liu F, Ding X, Zhang Y, Hu S, Xia L. He L, et al. J Biol Eng. 2019 Jun 28;13:58. doi: eCollection 2019. J Biol Eng. 2019. PMID: 31297149 Free PMC article. High density fermentation of probiotic E. coli Nissle 1917 towards heparosan production, characterization, and modification. Datta P, Fu L, Brodfuerer P, Dordick JS, Linhardt RJ. Datta P, et al. Appl Microbiol Biotechnol. 2021 Feb;105(3):1051-1062. doi: Epub 2021 Jan 22. Appl Microbiol Biotechnol. 2021. PMID: 33481068 Genetic engineering of probiotic Escherichia coli Nissle 1917 for clinical application. Ou B, Yang Y, Tham WL, Chen L, Guo J, Zhu G. Ou B, et al. Appl Microbiol Biotechnol. 2016 Oct;100(20):8693-9. doi: Epub 2016 Sep 17. Appl Microbiol Biotechnol. 2016. PMID: 27640192 Review. [Escherichia coli Nissle 1917 as safe vehicles for intestinal immune targeted therapy--a review]. Xia P, Zhu J, Zhu G. Xia P, et al. Wei Sheng Wu Xue Bao. 2013 Jun 4;53(6):538-44. Wei Sheng Wu Xue Bao. 2013. PMID: 24028055 Review. Chinese. Cited by Encoding with a fluorescence-activating and absorption-shifting tag generates living bacterial probes for mammalian microbiota imaging. Cao Z, Wang L, Liu R, Lin S, Wu F, Liu J. Cao Z, et al. Mater Today Bio. 2022 Jun 6;15:100311. doi: eCollection 2022 Jun. Mater Today Bio. 2022. PMID: 35711290 Free PMC article. Native and Engineered Probiotics: Promising Agents against Related Systemic and Intestinal Diseases. Shen H, Zhao Z, Zhao Z, Chen Y, Zhang L. Shen H, et al. Int J Mol Sci. 2022 Jan 6;23(2):594. doi: Int J Mol Sci. 2022. PMID: 35054790 Free PMC article. Review. References Wilhelm S., Tavares Dai Q., Ohta S., Audet J., Dvorak Chan Analysis of nanoparticle delivery to tumours. Nat. Rev. Mater. 2016;1:16014. doi: - DOI Heldin Rubin K., Pietras K., Östman A. High interstitial fluid pressure—an obstacle in cancer therapy. Nat. Rev. Cancer. 2004;4:806–813. doi: - DOI - PubMed Khawar Kim Kuh Improving drug delivery to solid tumors: Priming the tumor microenvironment. J. Control. Release. 2015;201:78–89. doi: - DOI - PubMed Malmgren Flanigan Localization of the vegetative form of Clostridium tetani in mouse tumors following intravenous spore administration. Cancer Res. 1955;15:473–478. - PubMed Starnes Coley’s toxins in perspective. Nature. 1992;357:11–12. doi: - DOI - PubMed Publication types LinkOut - more resources Full Text Sources Europe PubMed Central Multidisciplinary Digital Publishing Institute (MDPI) PubMed Central
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Patients and methods: In a post-marketing-surveillance study with the probiotic Escherichia Coli strain Nissle 1917 (EcN) data on the range of indications as well as on efficacy and tolerance were gathered prospectively in 446 centres. The intended treatment duration was limited to a maximum of 12 weeks.
AbstractBackgroundGenetically modified probiotics have potential for use as a novel approach to express bioactive molecules for the treatment of obesity. The objective of the present study was to investigate the beneficial effect of genetically modified Escherichia coli Nissle 1917 (EcN-GM) in obese C57BL/6J an obesity model in C57BL/6J mice was successfully established. Then, the obese mice were randomly assigned into three groups: obese mice (OB), obese mice + EcN-GM (OB + EcN-GM), and obese mice + orlistat (OB + orlistat) (n = 10 in each group). The three groups were gavaged with ml of 1010 CFU/ml control EcN, EcN-GM (genetically engineered EcN) and 10 ml/kg orlistat. Body weight, food consumption, fat pad and organ weight, hepatic biochemistry and hepatic histopathological alterations were measured. The effects of EcN-GM on the levels of endocrine peptides and the intestinal microbiota were also supplementation for 8 weeks, EcN-GM was associated with decreases in body weight gain, food intake, fat pad and liver weight, and alleviation hepatocyte steatosis in obese mice. EcN-GM also increased the level of GLP-1 in serum and alleviated leptin and insulin resistance. Moreover, supplementation with EcN-GM increased the α-diversity of the intestinal microbiota but did not significantly influence the relative abundance of Firmicutes and results indicated that EcN-GM, a genetically modified E. coli strain, may be a potential therapeutic approach to treat obesity. The beneficial effect of EcN-GM may be independent of the alteration of the diversity and composition of the intestinal microbiota in obese mice. This is a preview of subscription content Access options Subscribe to JournalGet full journal access for 1 year111,22 €only 9,27 € per issueAll prices are NET prices. VAT will be added later in the calculation will be finalised during articleGet time limited or full article access on ReadCube.$ prices are NET prices. Additional access options: Log in Learn about institutional subscriptions ReferencesKyle TK, Dhurandhar EJ, Allison DB. Regarding obesity as a disease: evolving policies and their implications. Endocrinol Metab Clin North Am. 2016;45:511– PubMed Central Google Scholar Obesity: preventing and managing the global epidemic. Report of a WHO consultation. World Health Organ Tech Rep Ser. 2000;894:1– GA, Kim KK, Wilding JPH, World Obesity F. Obesity: a chronic relapsing progressive disease process. A position statement of the World Obesity Federation. Obes Rev. 2017;18:715– Article Google Scholar O’Neil PM, Birkenfeld AL, McGowan B, Mosenzon O, Pedersen SD, Wharton S, et al. Efficacy and safety of semaglutide compared with liraglutide and placebo for weight loss in patients with obesity: a randomised, double-blind, placebo and active controlled, dose-ranging, phase 2 trial. Lancet. 2018;392:637– Google Scholar Apovian CM, Aronne LJ, Bessesen DH, McDonnell ME, Murad MH, Pagotto U, et al. Pharmacological management of obesity: an endocrine Society clinical practice guideline. J Clin Endocrinol Metab. 2015;100:342– Article Google Scholar Srivastava G, Apovian CM. Current pharmacotherapy for obesity. Nat Rev Endocrinol. 2018;14:12– Article Google Scholar Bessesen DH, Van Gaal LF. Progress and challenges in anti-obesity pharmacotherapy. Lancet Diabetes Endocrinol. 2018;6:237– Google Scholar Komaroff AL. The microbiome and risk for obesity and diabetes. JAMA. 2017;317:355– Google Scholar Heymsfield SB, Wadden TA. Mechanisms, pathophysiology, and management of obesity. N Engl J Med. 2017;376:254– Article Google Scholar Turnbaugh PJ, Ley RE, Mahowald MA, Magrini V, Mardis ER, Gordon JI. An obesity-associated gut microbiome with increased capacity for energy harvest. Nature. 2006;444:1027– Google Scholar Pedrolli DB, Ribeiro NV, Squizato PN, de Jesus VN, Cozetto DA, Team AQAUai. Engineering microbial living therapeutics: the synthetic biology toolbox. Trends Biotechnol. 2019;37:100– Article Google Scholar Reardon S. Genetically modified bacteria enlisted in fight against disease. Nature. 2018;558:497– Article Google Scholar Hendrikx T, Duan Y, Wang Y, Oh JH, Alexander LM, Huang W, et al. Bacteria engineered to produce IL-22 in intestine induce expression of REG3G to reduce ethanol-induced liver disease in mice. Gut. 2019;68:1504– Article Google Scholar Chen Z, Guo L, Zhang Y, Walzem RL, Pendergast JS, Printz RL, et al. Incorporation of therapeutically modified bacteria into gut microbiota inhibits obesity. J Clin Invest. 2014;124:3391– Article PubMed Central Google Scholar Wassenaar TM. Insights from 100 years of research with probiotic E. Coli. Eur J Microbiol Immunol. 2016;6:147– Google Scholar Grozdanov L, Raasch C, Schulze J, Sonnenborn U, Gottschalk G, Hacker J, et al. Analysis of the genome structure of the nonpathogenic probiotic Escherichia coli strain Nissle 1917. J Bacteriol. 2004;186:5432– Article PubMed Central Google Scholar Hancock V, Dahl M, Klemm P. Probiotic Escherichia coli strain Nissle 1917 outcompetes intestinal pathogens during biofilm formation. J Med Microbiol. 2010;59:392– Google Scholar Kurtz CB, Millet YA, Puurunen MK, Perreault M, Charbonneau MR, Isabella VM. et al. An engineered E. coli Nissle improves hyperammonemia and survival in mice and shows dose-dependent exposure in healthy humans. Sci Transl Med. 2019;11: Google Scholar Isabella VM, Ha BN, Castillo MJ, Lubkowicz DJ, Rowe SE, Millet YA, et al. Development of a synthetic live bacterial therapeutic for the human metabolic disease phenylketonuria. Nat Biotechnol. 2018;36:857– Article Google Scholar Hwang IY, Koh E, Wong A, March JC, Bentley WE, Lee YS, et al. Engineered probiotic Escherichia coli can eliminate and prevent Pseudomonas aeruginosa gut infection in animal models. Nat Commun. 2017;8: Article PubMed Central Google Scholar Ma J, Li C, Wang J, Gu J. Genetically engineered Escherichia coli Nissle 1917 secreting GLP-1 analog exhibits potential antiobesity effect in high-fat diet-induced obesity mice. Obesity. 2020;28:315– Article Google Scholar Yumuk V, Tsigos C, Fried M, Schindler K, Busetto L, Micic D, et al. European guidelines for obesity management in adults. Obes Facts. 2015;8:402– PubMed Central Google Scholar Collaborators GBDO, Afshin A, Forouzanfar MH, Reitsma MB, Sur P, Estep K, et al. Health effects of overweight and obesity in 195 countries over 25 years. N Engl J Med. 2017;377:13– Google Scholar Reid G, Sanders ME, Gaskins HR, Gibson GR, Mercenier A, Rastall R, et al. New scientific paradigms for probiotics and prebiotics. J Clin Gastroenterol. 2003;37:105– Google Scholar Yadav H, Lee JH, Lloyd J, Walter P, Rane SG. Beneficial metabolic effects of a probiotic via butyrate-induced GLP-1 hormone secretion. J Biol Chem. 2013;288:25088– Article PubMed Central Google Scholar Kang JH, Yun SI, Park MH, Park JH, Jeong SY, Park HO. Anti-obesity effect of Lactobacillus gasseri BNR17 in high-sucrose diet-induced obese mice. PLoS ONE. 2013;8: Article PubMed Central Google Scholar Valsecchi C, Carlotta Tagliacarne S, Castellazzi A. Gut microbiota and obesity. J Clin Gastroenterol. 2016;50:S157–8. Suppl 2Proceedings from the 8th Probiotics, Prebiotics & New Foods for Microbiota and Human Health meeting held in Rome, Italy on September 13–15, Article Google Scholar Wei P, Yang Y, Li T, Ding Q, Sun H. A engineered Bifidobacterium longum secreting a bioative penetratin-Glucagon-like peptide 1 fusion protein enhances Glucagon-like peptide 1 absorption in the intestine. J Microbiol Biotechnol. 2015. Epub ahead of FF, Liu JH, March JC. Engineered commensal bacteria reprogram intestinal cells into glucose-responsive insulin-secreting cells for the treatment of diabetes. Diabetes. 2015;64:1794– Article PubMed Central Google Scholar Rao S, Hu S, McHugh L, Lueders K, Henry K, Zhao Q, et al. Toward a live microbial microbicide for HIV: commensal bacteria secreting an HIV fusion inhibitor peptide. Proc Natl Acad Sci USA. 2005;102:11993– Article PubMed Central Google Scholar Federico A, Dallio M, Rosa DIS, Giorgio V, Miele L. Gut microbiota, obesity and metabolic disorders. Minerva Gastroenterol Dietol. 2017;63:337– Google Scholar Bäckhed F, Ding H, Wang T, Hooper LV, Koh GY, Nagy A, et al. The gut microbiota as an environmental factor that regulates fat storage. Proc Natl Acad Sci USA. 2004;101:15718– PubMed Central Google Scholar Boulange CL, Neves AL, Chilloux J, Nicholson JK, Dumas ME. Impact of the gut microbiota on inflammation, obesity, and metabolic disease. Genome Med. 2016;8: PubMed Central Google Scholar Patterson E, Ryan PM, Cryan JF, Dinan TG, Ross RP, Fitzgerald GF, et al. Gut microbiota, obesity and diabetes. Postgrad Med J. 2016;92:286– Article Google Scholar Madsen MSA, Holm JB, Palleja A, Wismann P, Fabricius K, Rigbolt K, et al. Metabolic and gut microbiome changes following GLP-1 or dual GLP-1/GLP-2 receptor agonist treatment in diet-induced obese mice. Sci Rep. 2019;9: PubMed Central Google Scholar Zhang N, Tao J, Gao L, Bi Y, Li P, Wang H, et al. Liraglutide attenuates nonalcoholic fatty liver disease by modulating gut microbiota in rats administered a high-fat diet. Biomed Res Int. 2020;2020: PubMed Central Google Scholar Liu Q, Cai BY, Zhu LX, Xin X, Wang X, An ZM, et al. Liraglutide modulates gut microbiome and attenuates nonalcoholic fatty liver in db/db mice. Life Sci. 2020;261: Article Google Scholar Ehrlich SD. Probiotics—little evidence for a link to obesity. Nat Rev Microbiol. 2009;7: Article Google Scholar Daniel H, Gholami AM, Berry D, Desmarchelier C, Hahne H, Loh G, et al. High-fat diet alters gut microbiota physiology in mice. ISME J. 2014;8:295– Article Google Scholar Kanoski SE, Hayes MR, Skibicka KP. GLP-1 and weight loss: unraveling the diverse neural circuitry. Am J Physiol Regul Integr Comp Physiol. 2016;310:R885– PubMed Central Google Scholar Download referencesAuthor informationAuthors and AffiliationsDepartment of Research and Development, Weichuang Tianyi Biotechnology Co., Ltd, Chengdu, Sichuan, PR ChinaJie MaDepartment of Research and Development, LiTong Bio-Medical Science, Chengdu, Sichuan, PR ChinaJie Ma & Lu XuSavaid Medical School, University of Chinese Academy of Sciences, Beijing, PR ChinaJunrui WangDepartment of Orthopaedics, Chengdu Second People’s Hospital, Chengdu, Sichuan, PR ChinaJunrui WangCollege of Comprehensive Health Management, Xihua University, Chengdu, Sichuan, PR ChinaYuanqi LiuDepartment of Neurosurgery, PLA Strategic Support Force Characteristic Medical Center, Beijing, PR ChinaJianwen GuAuthorsJie MaYou can also search for this author in PubMed Google ScholarJunrui WangYou can also search for this author in PubMed Google ScholarLu XuYou can also search for this author in PubMed Google ScholarYuanqi LiuYou can also search for this author in PubMed Google ScholarJianwen GuYou can also search for this author in PubMed Google ScholarContributionsAll authors contributed to this work. JM, JW, and JG designed the experiment. JM and JW performed the experiment. LX and YL analyzed the data. JM and JW drafted the manuscript. JM, LX, and YL prepared the figures. JM, JW, LX, and JG critically revised the manuscript. All the listed authors reviewed and approved the submitted authorsCorrespondence to Jie Ma or Jianwen declarations Competing interests The authors declare no competing interests. Additional informationPublisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional and permissionsAbout this articleCite this articleMa, J., Wang, J., Xu, L. et al. The beneficial effects of genetically engineered Escherichia coli Nissle 1917 in obese C57BL/6J mice. Int J Obes 46, 1002–1008 (2022). citationReceived: 17 June 2021Revised: 07 January 2022Accepted: 12 January 2022Published: 25 January 2022Issue Date: May 2022DOI:

Bacterial cancer therapy was developed using probiotic Escherichia coli Nissle 1917 (EcN) for medical intervention of colorectal cancer. EcN was armed with HlyE, a small cytotoxic protein, under

Review Escherichia coli Nissle 1917 in Ulcerative Colitis Treatment: Systematic Review and Meta-analysis Giuseppe Losurdo et al. J Gastrointestin Liver Dis. 2015 Dec. Free article Abstract Background and aims: Escherichia coli Nissle 1917 (EcN) has been recommended as a therapeutic tool for ulcerative colitis (UC) treatment. However, to date, no meta-analysis has been performed on this topic. Methods: We performed a literature search on PubMed, MEDLINE, Science Direct and EMBASE. We evaluated success rates for induction of remission, relapse rates and side effects, expressed as Intention-To-Treat. Odd ratios (OR), pooled OR and 95% confidence intervals (CI) were calculated, based on the Mantel-Haenszel method. Heterogeneity was assessed by using the χ2 and I2 statistics and, if present, a random-effects model was adopted. Results: We selected six eligible trials, with 719 patients, 390 assigned to the study group and 329 to the control group. EcN induced remission in of cases, while in the control group (mesalazine) the remission was achieved in of cases, with a mean difference of The pooled OR was (95% CI p= A single study showed a better performance of EcN than the placebo. A relapse of the disease occurred in in the EcN group and in in the control group (mesalazine), with a mean difference of OR= with a 95% CI of (p= Side effects were comparable (OR= 95% CI p= Conclusions: EcN is equivalent to mesalazine in preventing disease relapse, thus confirming current guideline recommendations. EcN seems to be as effective as controls in inducing remission and therefore, its use cannot be recommended as in one study the comparison was performed against placebo. Further studies may be helpful for this subject. Similar articles Role and mechanisms of action of Escherichia coli Nissle 1917 in the maintenance of remission in ulcerative colitis patients: An update. Scaldaferri F, Gerardi V, Mangiola F, Lopetuso LR, Pizzoferrato M, Petito V, Papa A, Stojanovic J, Poscia A, Cammarota G, Gasbarrini A. Scaldaferri F, et al. World J Gastroenterol. 2016 Jun 28;22(24):5505-11. doi: World J Gastroenterol. 2016. PMID: 27350728 Free PMC article. Review. Non-pathogenic Escherichia coli versus mesalazine for the treatment of ulcerative colitis: a randomised trial. Rembacken BJ, Snelling AM, Hawkey PM, Chalmers DM, Axon AT. Rembacken BJ, et al. Lancet. 1999 Aug 21;354(9179):635-9. doi: Lancet. 1999. PMID: 10466665 Clinical Trial. Maintaining remission of ulcerative colitis with the probiotic Escherichia coli Nissle 1917 is as effective as with standard mesalazine. Kruis W, Fric P, Pokrotnieks J, Lukás M, Fixa B, Kascák M, Kamm MA, Weismueller J, Beglinger C, Stolte M, Wolff C, Schulze J. Kruis W, et al. Gut. 2004 Nov;53(11):1617-23. doi: Gut. 2004. PMID: 15479682 Free PMC article. Clinical Trial. [Maintaining remission of ulcerative colitis with the probiotic Escherichia Coli Nissle 1917 is as effective as with standard mesalazine]. Adam B, Liebregts T, Holtmann G. Adam B, et al. Z Gastroenterol. 2006 Mar;44(3):267-9. doi: Z Gastroenterol. 2006. PMID: 16514573 German. No abstract available. Probiotics for maintaining remission of ulcerative colitis in adults. Do VT, Baird BG, Kockler DR. Do VT, et al. Ann Pharmacother. 2010 Mar;44(3):565-71. doi: Epub 2010 Feb 2. Ann Pharmacother. 2010. PMID: 20124461 Review. Cited by Efficacy and Safety of Probiotics Combined With Traditional Chinese Medicine for Ulcerative Colitis: A Systematic Review and Meta-Analysis. Hu Y, Ye Z, She Y, Li L, Wu M, Qin K, Li Y, He H, Hu Z, Yang M, Lu F, Ye Q. Hu Y, et al. Front Pharmacol. 2022 Mar 7;13:844961. doi: eCollection 2022. Front Pharmacol. 2022. PMID: 35321324 Free PMC article. Review. Comment on Depoorter, L.; Vandenplas, Y. Probiotics in Pediatrics. A Review and Practical Guide. Nutrients 2021, 13, 2176. von Bünau R, Erhardt A, Stange E. von Bünau R, et al. Nutrients. 2022 Feb 9;14(4):724. doi: Nutrients. 2022. PMID: 35215374 Free PMC article. Review. A Probiotic Friend. Dubbert S, von Bünau R. Dubbert S, et al. mSphere. 2021 Dec 22;6(6):e0085621. doi: Epub 2021 Dec 22. mSphere. 2021. PMID: 34935447 Free PMC article. No abstract available. MicroRNA and Gut Microbiota: Tiny but Mighty-Novel Insights into Their Cross-talk in Inflammatory Bowel Disease Pathogenesis and Therapeutics. Casado-Bedmar M, Viennois E. Casado-Bedmar M, et al. J Crohns Colitis. 2022 Jul 14;16(6):992-1005. doi: J Crohns Colitis. 2022. PMID: 34918052 Free PMC article. Review. Efficient markerless integration of genes in the chromosome of probiotic E. coli Nissle 1917 by bacterial conjugation. Seco EM, Fernández LÁ. Seco EM, et al. Microb Biotechnol. 2022 May;15(5):1374-1391. doi: Epub 2021 Nov 9. Microb Biotechnol. 2022. PMID: 34755474 Free PMC article. Publication types MeSH terms Substances LinkOut - more resources Full Text Sources Iuliu Hatieganu Medical Publishing House Other Literature Sources The Lens - Patent Citations Medical Genetic Alliance MedlinePlus Health Information

E. coli Nissle 1917 (EcN) has been known to penetrate the barrier and proliferate in the interface between the viable and necrotic regions of tumors. This study aimed to fabricate a nanoscale minicell via genetic engineering of EcN for targeted delivery of chemotherapeutic drugs to the hypoxic regions of tumors for cancer therapy. The probiotic Escherichia coli strain Nissle 1917 interferes with invasion of human intestinal epithelial cells by different enteroinvasive bacterial pathogens Artur Altenhoefer et al. FEMS Immunol Med Microbiol. 2004. Free article Abstract The probiotic Escherichia coli strain Nissle 1917 (Mutaflor) of serotype O6:K5:H1 was reported to protect gnotobiotic piglets from infection with Salmonella enterica serovar Typhimurium. An important virulence property of Salmonella is invasion of host epithelial cells. Therefore, we tested for interference of E. coli strain Nissle 1917 with Salmonella invasion of INT407 cells. Simultaneous administration of E. coli strain Nissle 1917 and Salmonella resulted in up to 70% reduction of Salmonella invasion efficiency. Furthermore, invasion of Yersinia enterocolitica, Shigella flexneri, Legionella pneumophila and even of Listeria monocytogenes were inhibited by the probiotic E. coli strain Nissle 1917 without affecting the viability of the invasive bacteria. The observed inhibition of invasion was not due to the production of microcins by the Nissle 1917 strain because its isogenic microcin-negative mutant SK22D was as effective as the parent strain. Reduced invasion rates were also achieved if strain Nissle 1917 was separated from the invasive bacteria as well as from the INT407 monolayer by a membrane non-permeable for bacteria. We conclude E. coli Nissle 1917 to interfere with bacterial invasion of INT407 cells via a secreted component and not relying on direct physical contact with either the invasive bacteria or the epithelial cells. Similar articles Detection and distribution of probiotic Escherichia coli Nissle 1917 clones in swine herds in Germany. Kleta S, Steinrück H, Breves G, Duncker S, Laturnus C, Wieler LH, Schierack P. Kleta S, et al. J Appl Microbiol. 2006 Dec;101(6):1357-66. doi: J Appl Microbiol. 2006. PMID: 17105567 E. coli Nissle 1917 Affects Salmonella adhesion to porcine intestinal epithelial cells. Schierack P, Kleta S, Tedin K, Babila JT, Oswald S, Oelschlaeger TA, Hiemann R, Paetzold S, Wieler LH. Schierack P, et al. PLoS One. 2011 Feb 17;6(2):e14712. doi: PLoS One. 2011. PMID: 21379575 Free PMC article. Nonpathogenic Escherichia coli strain Nissle 1917 inhibits signal transduction in intestinal epithelial cells. Kamada N, Maeda K, Inoue N, Hisamatsu T, Okamoto S, Hong KS, Yamada T, Watanabe N, Tsuchimoto K, Ogata H, Hibi T. Kamada N, et al. Infect Immun. 2008 Jan;76(1):214-20. doi: Epub 2007 Oct 29. Infect Immun. 2008. PMID: 17967864 Free PMC article. Tumor-specific colonization, tissue distribution, and gene induction by probiotic Escherichia coli Nissle 1917 in live mice. Stritzker J, Weibel S, Hill PJ, Oelschlaeger TA, Goebel W, Szalay AA. Stritzker J, et al. Int J Med Microbiol. 2007 Jun;297(3):151-62. doi: Epub 2007 Apr 19. Int J Med Microbiol. 2007. PMID: 17448724 Effect of probiotic strains on interleukin 8 production by HT29/19A cells. Lammers KM, Helwig U, Swennen E, Rizzello F, Venturi A, Caramelli E, Kamm MA, Brigidi P, Gionchetti P, Campieri M. Lammers KM, et al. Am J Gastroenterol. 2002 May;97(5):1182-6. doi: Am J Gastroenterol. 2002. PMID: 12014725 Cited by The potential utility of fecal (or intestinal) microbiota transplantation in controlling infectious diseases. Ghani R, Mullish BH, Roberts LA, Davies FJ, Marchesi JR. Ghani R, et al. Gut Microbes. 2022 Jan-Dec;14(1):2038856. doi: Gut Microbes. 2022. PMID: 35230889 Free PMC article. Review. The microbial ecology of Escherichia coli in the vertebrate gut. Foster-Nyarko E, Pallen MJ. Foster-Nyarko E, et al. FEMS Microbiol Rev. 2022 May 6;46(3):fuac008. doi: FEMS Microbiol Rev. 2022. PMID: 35134909 Free PMC article. Review. Quantifying cumulative phenotypic and genomic evidence for procedural generation of metabolic network reconstructions. Moutinho TJ Jr, Neubert BC, Jenior ML, Papin JA. Moutinho TJ Jr, et al. PLoS Comput Biol. 2022 Feb 7;18(2):e1009341. doi: eCollection 2022 Feb. PLoS Comput Biol. 2022. PMID: 35130271 Free PMC article. Efficient markerless integration of genes in the chromosome of probiotic E. coli Nissle 1917 by bacterial conjugation. Seco EM, Fernández LÁ. Seco EM, et al. Microb Biotechnol. 2022 May;15(5):1374-1391. doi: Epub 2021 Nov 9. Microb Biotechnol. 2022. PMID: 34755474 Free PMC article. Escherichia coli Nissle 1917 secondary metabolism: aryl polyene biosynthesis and phosphopantetheinyl transferase crosstalk. Jones CV, Jarboe BG, Majer HM, Ma AT, Beld J. Jones CV, et al. Appl Microbiol Biotechnol. 2021 Oct;105(20):7785-7799. doi: Epub 2021 Sep 21. Appl Microbiol Biotechnol. 2021. PMID: 34546406 Publication types MeSH terms Substances LinkOut - more resources Full Text Sources Wiley Other Literature Sources The Lens - Patent Citations Research Materials NCI CPTC Antibody Characterization Program toFc9SO.
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