Loading metrics Open Access Peer-reviewed Research Article Anurag Singh, Ulrike Dringenberg, Regina Engelhardt, Ursula Seidler, Wiebke Hansen, André Bleich, Dunja Bruder, Anke Franzke, Gerhard Rogler, Sebastian Suerbaum, Jan Buer, Florian Gunzer, Astrid M. Westendorf Probiotic Escherichia coli Nissle 1917 Inhibits Leaky Gut by Enhancing Mucosal Integrity Sya N. Ukena, Anurag Singh, Ulrike Dringenberg, Regina Engelhardt, Ursula Seidler, Wiebke Hansen, André Bleich, Dunja Bruder, Anke Franzke, Gerhard Rogler x Published: December 12, 2007 Figures Abstract Background Probiotics are proposed to positively modulate the intestinal epithelial barrier formed by intestinal epithelial cells (IECs) and intercellular junctions. Disruption of this border alters paracellular permeability and is a key mechanism for the development of enteric infections and inflammatory bowel diseases (IBDs). Methodology and Principal Findings To study the in vivo effect of probiotic Escherichia coli Nissle 1917 (EcN) on the stabilization of the intestinal barrier under healthy conditions, germfree mice were colonized with EcN or K12 E. coli strain MG1655. IECs were isolated and analyzed for gene and protein expression of the tight junction molecules ZO-1 and ZO-2. Then, in order to analyze beneficial effects of EcN under inflammatory conditions, the probiotic was orally administered to BALB/c mice with acute dextran sodium sulfate (DSS) induced colitis. Colonization of gnotobiotic mice with EcN resulted in an up-regulation of ZO-1 in IECs at both mRNA and protein levels. EcN administration to DSS-treated mice reduced the loss of body weight and colon shortening. In addition, infiltration of the colon with leukocytes was ameliorated in EcN inoculated mice. Acute DSS colitis did not result in an anion secretory defect, but abrogated the sodium absorptive function of the mucosa. Additionally, intestinal barrier function was severely affected as evidenced by a strong increase in the mucosal uptake of Evans blue in vivo. Concomitant administration of EcN to DSS treated animals resulted in a significant protection against intestinal barrier dysfunction and IECs isolated from these mice exhibited a more pronounced expression of ZO-1. Conclusion and Significance This study convincingly demonstrates that probiotic EcN is able to mediate up-regulation of ZO-1 expression in murine IECs and confer protection from the DSS colitis-associated increase in mucosal permeability to luminal substances. Citation: Ukena SN, Singh A, Dringenberg U, Engelhardt R, Seidler U, Hansen W, et al. (2007) Probiotic Escherichia coli Nissle 1917 Inhibits Leaky Gut by Enhancing Mucosal Integrity. PLoS ONE 2(12): e1308. Editor: Debbie Fox, The Research Institute for Children, United States of AmericaReceived: September 17, 2007; Accepted: November 21, 2007; Published: December 12, 2007Copyright: © 2007 Ukena 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 Supported by grants from the Deutsche Forschungsgemeinschaft (SFB 621).Competing interests: The authors have declared that no competing interests exist. IntroductionThe epithelial layer of the gastrointestinal tract serves as one of the primary interfaces with the outside world. The mucosal surface of the intestinal epithelium is in constant contact with abundant populations of microbes and their metabolites. The intestinal barrier formed by the epithelial cells and the junctional complex, consisting of tight junctions (TJ), adherens junctions, gap junctions and desmosomes, excludes the majority of these microbes and their metabolites from access to the subepithelial cells. Its effectiveness and stability are ensured by the junctional complex [1]. Compromising the integrity of this barrier can promote manifestation of enteric infections and is a key feature of IBDs like Crohn's disease (CD) and ulcerative colitis (UC). Intestinal permeability was also found to be increased in HIV infection [2], and diarrhea is one of the most predominant symptoms of HIV-infected patients. The diarrhea of these patients is mainly due to infections with enteropathogens. However, in a number of HIV patients with gastrointestinal complaints no enteropathogen can be identified [3], [4]. The role of a ‘leaky gut’ in the pathogenesis of gastrointestinal diseases is increasingly recognized. Consequently, reduction of the increased permeability is an interesting target for improvement of the clinical status of gastrointestinal diseases. Tight junctions are intricate macromolecular protein structures located at the most apical regions of the junctional complex, sealing the spaces between the IECs. The first junction-associated protein identified was zonula occludens 1 (ZO-1), with a molecular mass between ∼210–225 kDa [5]. This molecule constitutes the structural link between the cytoskeleton and the tight junction by binding to both actin filaments and the TJ protein occludin [6]. Reorganization of TJ proteins like ZO-1, triggered by cytokines produced secondary to the inflammatory processes in IBDs, results in increased intestinal permeability [7], [8]. ZO-2 is another TJ associated protein that forms a complex together with ZO-1 [9] and has recently been shown to be up-regulated by EcN in vitro [10]. Probiotics are defined as live microorganisms which, when administered in adequate amounts, confer health benefits to the host [11]. The use of such microorganisms as novel therapeutic agents and as an alternative to standard medication in gastrointestinal diseases is promising, although their mechanism of action is still under investigation. EcN has evolved into one of the best characterized probiotics, and its therapeutic efficacy and safety have convincingly been proven [12]–[16]. A potential mechanism by which probiotics may exhibit their beneficial activities is modulation of the epithelial barrier function [17], [18]. This hypothesis is also supported by a recent study demonstrating that probiotic Streptococcus thermophilus and Lactobacillus acidophilus can prevent invasion of enteroinvasive E. coli and enhance intestinal epithelial barrier function by amplifying phosphorylation of occludin and ZO-1 in vitro [19]. Driven by mounting evidence affirming the beneficial effects of probiotics on the intestinal epithelial barrier and the already well-documented therapeutic efficacy of EcN, we set out to investigate the impact of EcN on the intestinal epithelial barrier function in vivo. Materials and Methods Mice All mice used in this study were 6–8 weeks old females. Conventional BALB/c mice were obtained from Harlan (Borchen, Germany). Gnotobiotic BALB/c mice were obtained from colonies maintained germfree at the Central Animal Facility of Hannover Medical School, as described previously [20]. The animal experiments reported here were conducted in accordance with the German Animal Welfare Law and with the European Communities Council Directive 86/609/EEC for the protection of animals used for experimental purposes. All experiments were approved by the local institutional animal care and research advisory committee and authorized by the district authority of Braunschweig and Hannover. Bacterial colonization of gnotobiotic BALB/c mice EcN or E. coli MG1655 was freshly grown to an OD600 = 1 (∼ CFU/ml) from an overnight culture diluted 1:500 in LB media [21]. Bacteria were collected by centrifugation (1 ml, 3 min at 1000×g). The resulting pellet was redissolved in 200 µl sterile PBS and administered by oral gavage. Application was repeated two days later. After 6 days of colonization, fecal CFU were determined by plate count from pooled stool samples and were calculated per gram feces. The animals grew comparable numbers of both bacterial strains in all experiments averaging CFU/g feces with EcN and CFU/g feces with E. coli MG1655. Feces of mice that had received PBS remained sterile. Induction of colitis Acute colitis was induced in BALB/c mice by addition of 4–6% dextran sodium sulfate (DSS) (MP Biomedicals, Eschwege, Germany) to drinking water for a period of 8 days, according to a protocol recently described by Grabig et al. [22]. Animals were separated into the following groups: Group I was treated orally with PBS two times a day. Group II received drinking water with 4–6% DSS and was treated orally with PBS twice daily. Group III also received drinking water with 4–6% DSS and was given CFU (Mutaflor mite, Ardeypharm, Germany) EcN twice daily by oral application (DSS+EcN) (Figure 1). Colitis induction indicated by weight loss of the mice was monitored by comparing the body weight upon DSS treatment to the initial body weight of the respective animals. Figure 1. Experimental design of the DSS colitis colitis was induced by administration of 4–6% DSS to drinking water (DSS). (A) Group I was orally treated with PBS twice daily. Group II was given 4–6% DSS in drinking water and orally treated with PBS twice daily. Group III received CFU EcN two times a day by oral application in combination with 4–6% DSS in drinking water (DSS+EcN). Isolation of IECs IECs were isolated as described elsewhere [23]. Briefly, the small and/or large intestine were isolated, rinsed with PBS and opened longitudinally. Mucus was removed by treatment with DTT for 15 min at 37°C on a shaker. Having washed the mucosa in PBS, it was placed in HBSS/ mM EDTA and tumbled for 10 min at 37°C. The supernatant was collected and the remaining mucosa was vortexed in PBS. This supernatant was also collected. Pooled IECs were centrifuged with HBSS/PBS; the pellet was resuspended in FACS buffer (PBS+2% FCS+2 mM EDTA) and stained with anti-CD45 APC antibody (BD Biosciences, Heidelberg, Germany) to deplete hematopoietic cells [24]. IECs were sorted with a MoFlow cell sorter (Cytomation, Fort Collins, CO, USA). RNA isolation and expression analysis To analyze ZO-1 and ZO-2 mRNA expression in murine IECs, total RNA was isolated using the RNeasy Minikit (Qiagen, Hilden, Germany) with on-column DNase digestion using the RNase-Free DNase set (Qiagen). Isolated mRNA was reverse transcribed with 200 U Superscript II® (Invitrogen, Karlsruhe, Germany), oligo dT- and random hexamer primers (Invitrogen). PCR was performed using the following primers: ribosomal protein 9 (RPS9) mouse (mm) sense primer CTG GAC GAG GGC AAG ATG AAG C, RPS9 mm anti-sense primer TGA CGT TGG CGG ATG AGC ACA; ZO-1 mm sense primer TTT TTG ACA GGG GGA GTG G, ZO-1 mm anti-sense primer TGC TGC AGA GGT CAA AGT TCA AG; ZO-2 mm sense primer CTA GAC CCC CAG AGC CCC AGA AA, ZO-2 mm anti-sense primer TCG CAG GAG TCC ACG CAT ACA AG. Quantitative real-time RT-PCR was done with the GeneAmp 5700 Sequence Detection System (Perkin Elmer, Rodgau-Jügesheim, Germany) using Brilliant SYBR Green QPCR Core Reagent Kit (Stratagene, Heidelberg, Germany). RPS9 served as control. Western blot analysis To examine the ZO-1 protein expression in IECs, lysates of sorted cells from colonized gnotobiotic mice were homogenized and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE), followed by blotting the proteins on a PVDF membrane. After the blocking of unsaturated protein binding sites, the membrane was incubated with the primary antibody rabbit anti-ZO-1 (Zymed, South San Francisco, CA, USA) or rabbit anti-β actin (Sigma-Aldrich, Taufkirchen, Germany) and the secondary antibody goat anti-rabbit IgG (Dianova, Hamburg, Germany), respectively. Electricphysiologic measurements The colonic mucosa was mounted between two chambers with an exposed area of cm2 and placed in an Ussing chamber. Parafilm “O” rings were used to minimize edge damage to the tissue where it was secured between the chamber halves. Tissues were bathed with HCO3− containing solutions on both sides which were gassed with 95% O2/5% CO2. The composition (in mM) was 108 NaCl, 22 NaHCO3, 3 KCl, MgSO4, 2 CaCl2, KH2PO4, at pH The serosal bath contained (in mM) glucose, 10 sodium pyruvate, 10−3 indomethacin, and 10−3 tetrodotoxin; the luminal bath contained mannitol and 10−2 amiloride (to block potential amiloride-sensitive Na+ channels). Short-circuit current (Isc), potential difference (PD) and tissue resistance (R) were recorded using the Mussler 6-channel voltage clamp system (Mussler, Aachen, Germany). 22Na+ studies were performed during voltage clamp to zero PD. 74 kBq/ml 22Na+ was added to either the serosal or the mucosal solution after reaching stable electrical parameters. After stabilization (approximately 20–30 minutes after mounting), a 45-minute period of equilibration followed, then aliquots were taken in 15-minute intervals (two intervals for basal flux, two after forskolin, and two after luminal glucose). For the presented results, we used the values from the second basal flux period, the first period after forskolin, and the first period after glucose. There were no statistically significant differences between the values obtained in the first and second flux period after forskolin and after glucose. Radioactivity was determined in a liquid scintillation counter, and bidirectional flux rates for the respective substance were calculated. The values for Isc represent the average value of the 15-minute period. Measurement of colonic epithelial permeability Animals were maintained with free access to food and water. Induction of anesthesia was achieved by the administration of 10 µl/g intraperitoneal haloperidol/midazolam/fentanyl cocktail (haloperidol mg/kg, fentanyl mg/kg and midazolam 5 mg/kg body weight). The lower abdomen was opened by one small central incision, and a small polyethylene tube (PE100) with a distal flange was advanced to the proximal colon (immediately after the cecum), and secured by a ligature that served as inlet tube. A PE200 flanged tubing was inserted through the rectum and secured by ligature to allow for drainage through the rectum. The isolated colon segment with an intact blood supply was gently flushed and then perfused (Perfusor compact, BRAUN, Melsungen, Germany) at a rate of 30 ml/h with 150 mmol/l NaCl for 5 min, followed by perfusion with 1% Evans Blue in NaCl for 10 min. To wash-out the sticking dye in the mucus, the lumen was perfused with 6 mM acetylcysteine for 5 min followed by NaCl for 10 min. The animals were then sacrificed by cervical dislocation and the ligated colon was removed. The colon was rinsed once more with saline, its length was recorded and it was then placed in 5 ml N,N- dimethyl-formamide overnight to extract the Evans Blue. The dye concentration was measured spectrophotometrically at 620 nm (Hitachi U-2000 UV/VIS, Hitachi, Japan). Immunofluorescence Tissue sections were fixed with 4% paraformaldehyde, washed extensively and blocked with porcine serum. Subsequently, the sections were incubated with the primary antibody rabbit anti-ZO-1 (Zymed) followed by incubation with a Cy3 labeled secondary goat anti-rabbit IgG antibody (Jackson Immunoresearch, Cambridgeshire, UK). The sections were then dried, covered with gelatine and visualized by fluorescence microscopy. Statistical analysis Statistical analysis was performed with Origin software (OriginLab, Northampton, MA, USA). For analysis of numeric values, the one-tailed analysis of variance and the Student's t-test were used. A p-value of < was considered significant. Error bars represent the standard error of the mean. Results ZO-1 mRNA and protein expression are elevated in gnotobiotic mice colonized with EcN In order to investigate the impact of a single bacterial species on host IEC gene expression, we established a model for colonization of gnotobiotic mice with EcN and E. coli MG1655. To further investigate the in vivo impact of EcN on the epithelial barrier, primary IECs from the intestine of gnotobiotic mice were isolated and sorted by FACS resulting in an IEC population with greater than 94% purity (Figure 2A). ZO-1 is a TJ protein which has been described to play an important role in the prevention of intestinal barrier disruption by probiotics. Therefore, we investigated whether EcN differentially regulates ZO-1 mRNA expression in vivo. As depicted in figure 2B colonization of gnotobiotic mice with EcN resulted in a specific up-regulation of ZO-1 mRNA in IECs (p< To confirm these data at protein level, sections of ileum and colon from gnotobiotic control mice and animals colonized with EcN were stained with anti-ZO-1 antibody followed by a fluorescence-labeled secondary antibody (Figure 3A). Comparison of ZO-1 staining along the surface of the crypts revealed only a slight increase of ZO-1 protein in IECs of mice colonized with EcN. To further analyze the up-regulated ZO-1 expression at protein level, isolated IECs were investigated by Western blotting. In comparison to IECs isolated from control mice and those colonized with E. coli MG1655, IECs from EcN treated animals showed a markedly elevated expression of ZO-1 protein (Figure 3B). Thus, we could clearly demonstrate that colonization of gnotobiotic mice with EcN specifically up-regulates ZO-1 expression at the mRNA as well as at the protein level. Due to the important functional role of ZO-1 in the junctional complex, these findings suggest that enhancement of the intestinal epithelial barrier function by EcN could at least in part be attributed to up-regulation of ZO-1. It has also been previously demonstrated that treatment of T84 cells with EcN leads to an up-regulation of ZO-2 in vitro [10]. In contrast to the data generated with the T84 cell line, analysis of ZO-2 mRNA expression in IECs isolated from gnotobiotic mice colonized with EcN or E. coli MG1655 did not result in an increase of ZO-2 mRNA levels (fold change <2) in vivo (Figure 2C). Figure 2. Isolation of IECs from gnotobiotic mice colonized with EcN or E. coli MG1655 and ZO-1 expression BALB/c mice were colonized with E. coli MG1655 (K12) or EcN for 6 days, respectively. Application of PBS was used as control. (A) Whole intestinal cell populations were isolated from gnotobiotic mice treated either with PBS, K12 or EcN. For sorting of a pure intestinal epithelial cell population, cells were labeled with anti-CD45 antibody to exclude hematopoietic cells and further distinguished by cell granularity and size (SSC) (Pre sorting). IECs were FACS-sorted by negative selection. Re-analysis was performed to determine the purity of sorted IECs (Post sorting). (B) Quantitative ZO-1 mRNA expression in IECs. Relative mRNA amounts were normalized with respect to expression levels of IECs from control mice (fold change = 1). Data are presented as mean of three independent experiments (n = 3/group). (C) Quantitative ZO-2 mRNA expression in IECs. Relative mRNA amounts were normalized with respect to expression levels of IECs from control mice (fold change = 1). Data are presented as mean of three independent experiments (n = 3/group). *p< EcN vs. Ctrl (relative expression values). 3. ZO-1 protein expression in ileum and colon of gnotobiotic mice.(A) Immunofluorescence staining of tissue sections from gnotobiotic control mice and mice colonized with EcN for 6 days with a fluorescent anti-ZO-1 antibody (orange). Original magnification×20. (B) Protein expression of ZO-1 in IECs. Western blot analysis of FACS sorted IECs from colonized mice was performed using anti-ZO-1 antibody. Anti-β actin antibody was used as internal control, binding to the corresponding protein with a molecular weight of 42 kDa. The ZO-1 antibody detects endogenous ZO-1 protein, displayed as a band at ∼210 kDa. Elevated ZO-1 expression after EcN treatment in experimental colitis It has been shown that the impaired barrier function in IBD is associated with an altered TJ structure [7], [8], [25]–[28]. Recently it was demonstrated that EcN, used as a therapeutic for the treatment of ulcerative colitis, ameliorates acute colitis in mice [29]. These observations raised the question whether an alleviated acute colitis-as a consequence of EcN treatment-may be due to its effect on the epithelial barrier. To investigate this aspect acute colitis was induced in BALB/c mice by administration of 4–6% DSS in drinking water for a period of 8 days [22]. In addition, mice were given CFU EcN or PBS orally two times a day. In contrast to untreated control mice of group I, mice exposed to DSS (group II) developed symptoms of acute colitis with diarrhea, rectal bleeding and wasting, loosing 10% of their initial body weight within 8 days (Figure 4A). Concomitant oral administration of EcN (group III) significantly ameliorated the severity of DSS-induced colitis and the loss of body weight was reduced (6%) (p< Healthy control mice (group I) exhibited an average colon length of (± cm, whereas, as a consequence of severe intestinal inflammation, the colon length of DSS treated diseased mice (group II) was reduced to (± cm (p< In contrast, in DSS and EcN treated mice (group III), this colon shortening was significantly attenuated with (± cm (p< (Figure 4B). Colonic inflammation is correlated with strong infiltration of hematopoietic cells into the intestine. To further elaborate the beneficial effect of EcN in DSS treated mice, FACS analysis of hematopoietic cells in the colon was performed. Consistent with the reduction in loss of body weight and colon shortening, mice treated with DSS and EcN (group III) exhibited significantly lower leukocyte infiltrates in the colon in comparison to DSS treated mice (group II) (Figure 4C). To analyze whether the improved state of health after EcN treatment is accompanied with an increased ZO-1 expression, IECs were isolated from the colon of treated mice and analyzed for ZO-1 gene expression. IECs of mice treated with DSS and EcN showed elevated ZO-1 mRNA levels in comparison to DSS treated animals (p< (Figure 5). Although ZO-2 mRNA was not up-regulated by EcN under healthy conditions, a slight increase of ZO-2 mRNA could be detected in DSS mice treated with EcN (data not shown). These results further underline the beneficial effects of EcN on the intestinal barrier even under inflammatory conditions. Figure 4. Administration of EcN in DSS induced colitis.(A) Disease severity was measured daily and is expressed in terms of body weight loss. (group II: n = 23, group III: n = 25, group I: n = 17) *p< group III vs. group II. (B) Reduction of colon length. Measurement of colon length [cm] after preparation. (group II: n = 24, group III: n = 25, group I: n = 13) *p< group III vs. group II or group III vs. group I **p< group II vs. group I. (C) Infiltration of hematopoietic cells into the colon. Whole intestinal cell populations were labeled with anti-CD45 APC antibody and measured by FACS analysis (n = 6/group). 5. Increased ZO-1 mRNA expression in mice treated with DSS and were isolated from indicated mice and relative levels of ZO-1 mRNA were normalized with respect to the expression level of IECs from DSS treated mice (fold change = 1). Data are presented as mean of four independent experiments (n = 3/group). *p< group III vs. group II (relative expression values). Electrolyte transport capacity and tissue resistance after EcN treatment in experimental colitis In order to assess the electrolyte transport capacity and the tissue resistance (R) in acute DSS colitis, and the influence of EcN treatment on these parameters, the basal and forskolin-stimulated Isc, basal and forskolin-inhibited Na+ absorption, and the tissue-resistance R in isolated colonic mucosa of inflamed DSS treated mice (group II), DSS and EcN treated mice (group III), and healthy controls (group I) were studied. To ensure that any potential inflammation-related changes would be detected, the mucosa was neither stripped nor were the prostaglandin production or neural transmission inhibited. Interestingly, no difference was found in either the basal and forskolin-stimulated Isc (Figure 6A), or in the transmucosal electrical resistance (R) between the different groups (data not shown). This demonstrates that in acute DSS colitis, the anion secretory capacity, which originates from the cryptal region of the colonic epithelium, is not perturbed. On the other hand, net Na+ absorption was significantly decreased in colonic mucosa of DSS and EcN treated mice (group III), and reversed to Na+ leakage into the luminal fluid in the mucosa of DSS treated mice (group II) (Figure 6B). Since the transporters for Na+ absorption are expressed in the surface colonic enterocytes, this demonstrates severe alterations in surface cell electrolyte transport following treatment with EcN. Figure 6. Secretory and absorptive function of isolated colonic mucosa from DSS-treated mice, with and without EcN administration, and healthy controls.(A) Basal and forskolin-stimulated Isc in DSS treated (group II n = 7), DSS+EcN (group III n = 12) and control animals (group I n = 10). The different numbers resulted from the fact that considerably more colonic segments from the DSS treated mice were so friable that they ruptured before measurements could be taken. (B) Basal and post-forskolin net Na+ flux rates in the three different groups. Whereas Na+ absorption was completely abolished in group II, active Na+ absorption was only partially inhibited in group III. Only in the control group I could an inhibition of Na+ absorption by forskolin be observed, indicating normal regulation (n = 6/group). *p< Reduction of colonic epithelial permeability after EcN treatment in experimental colitis In order to address the question whether the pronounced ZO-1 expression in EcN treated mice (group III) impacts the permeability of the colonic epithelium for transport of luminal substances, the uptake of Evans Blue into the mucosa in anesthetized mice after a short-term luminal perfusion with the dye was measured. In comparison to untreated mice (group I), a strong increase of Evans Blue uptake into the colonic mucosa of DSS treated mice (group II), and a much lesser increase into the colonic mucosa of DSS and EcN treated mice (group III) was detected (Figure 7). This demonstrates that the concomitant application of EcN during colitis induction with DSS markedly ameliorates the leakiness of the colonic epithelium. Figure 7. Permeability to Evans Blue in the colon of DSS treated (group II), DSS+EcN (group III) treated mice and healthy controls (group I).The graph shows a significant increase in Evans Blue uptake into the colonic mucosa of group II mice and a strong reduction in Evans Blue uptake in group III mice to almost normal values (n = 6/group). ***p< DiscussionThe current study establishes that E. coli Nissle 1917 positively impacts the intestinal epithelial barrier in vivo in three different ways. First, EcN is capable of producing a specific up-regulation of ZO-1 expression in IECs of healthy gnotobiotic mice. When treated concomitantly with EcN, IECs of mice with DSS-induced colitis also exhibit a pronounced expression of ZO-1 mRNA. Finally, EcN provides protection against the DSS-mediated leakiness of the gut in our mouse model. Our data strongly suggest that one of the protective effects of EcN treatment on colitis prevention could be a modulation of tight junctional integrity which in turn leads to preserved intestinal barrier function against noxious or infectious agents. Critical for the development of IBDs are imbalances in mucosal immunity as well as a disturbed function of the epithelial barrier, which leads to a marked infiltration of luminal microflora [30]. Moreover, in the majority of cases, gastrointestinal diseases develop from the disruption of the intestinal epithelial barrier by enteropathogenic bacteria that alter the cellular cytoskeleton [31], [32] or affect specific tight junction proteins like ZO-1 [33]. In the past years, probiotics have been shown to be effective in the treatment of mild to moderately active IBD [18] and to reduce inflammation in animal models of colitis [29], [34]–[36]. Three large clinical trials have investigated the therapeutic efficacy of EcN in maintaining remission of UC. EcN was reported to be as efficacious as standard medication in preventing the relapse of UC [12]–[14]. Although the mechanisms leading to relapses in the pathogenesis of IBDs have not yet been clarified, there is growing evidence that increased intestinal permeability plays a key role. This report is the first to demonstrate a direct influence of the therapeutic EcN on expression of the TJ associated molecule ZO-1 in vivo under healthy and inflammatory conditions. We demonstrate that EcN specifically up-regulates ZO-1 expression both at the mRNA and at the protein level in IECs of gnotobiotic mice. Very recently, Zyrek et al. described the up-regulation of ZO-2 after EcN exposure to the T84 cell line in vitro [10]. However, in this study analysis of IECs from gnotobiotic mice colonized with EcN did not reveal a differential ZO-2 mRNA expression in vivo following EcN treatment. In order to study functional consequences of DSS-mediated colitis and EcN treatment and also the potential significance of ZO-1 up-regulation for an enhancement of intestinal barrier function, we performed experiments aimed to assess transport and barrier function of the colonic epithelium of DSS-treated mice with and without application of EcN. To evaluate secretory and absorptive function of the colonic epithelium, we measured the basal and forskolin-stimulated Isc (which is an assessment of the electrogenic anion secretion, and its stimulation by an increase in intracellular cAMP levels), as well as net Na+ absorption before and after an increase in cAMP levels. Interestingly, after one week of DSS treatment, the acute colitis did not compromise the secretory function of the epithelium at all, but abolished the Na+ absorptive function completely. Na+ absorption is mediated by electroneutral (apical Na+/H+ exchangers NHE3 and possibly NHE2) and electrogenic (apical Na+ channel ENaC) pathways in the colon [37]. Na+ absorptive transporters are compromised during acute colitis, and this is one major reason for diarrhea during colonic inflammation [38]. Active fluid absorption is also dependent on intact tight junctions, otherwise a phenomenon called “leak flux diarrhea” occurs [39]–[41]. A cytokine-induced intestinal barrier dysfunction via a leak flux mechanism has recently been proposed by Schmitz et al. as potential cause for non-infectious diarrhea in HIV-infected patients, in addition to mucosal transformation with a consecutive malabsorptive mechanism [42]. When cholera toxin deletion mutants of Vibrio cholerae were given to healthy volunteers they still developed mild diarrhea [43]. Searching for the causative agent led to the detection of the ZOT, a toxin which causes a disruption of the tight junctions in isolated intestine [44]. Later studies revealed yet another agent from Vibrio cholerae, the HA/protease, that specifically interferes with the tight junction proteins occludin and ZO-1 resulting in barrier disruption [45], [46]. Thus, it became clear that the integrity of the tight junctions was necessary for the maintenance of an absorptive state of the gut epithelium. In addition, it was found that a disruption of the tight junctional complex allowed easier permeation of substances from the lumen [47]. Our experiments demonstrated a strongly elevated influx of Evans Blue into the colonic mucosa in the live mouse with DSS colitis which is indicative of increased gut permeability. Much less dye was bound to IECs of mice treated concomitantly with EcN. Combined with the absorptive Na+ flux in these animals, this is likely to explain the nearly normal appearance of the feces in the DSS plus EcN treated mice compared to the liquid stools of the mice with DSS colitis. It is feasible that the reduction of DSS-mediated downregulation of ZO-1 expression by EcN treatment is one reason for the enhanced barrier stability. Several lines of evidence suggest that increased intestinal permeability has a central role in the pathogenesis of IBDs. For example, between 10–20% of presymptomatic CD patients have been shown to exhibit increased gut permeability [48], [49]. Alteration of TJ structure in UC for instance results in impaired barrier function [40]. Localization studies in mucosal biopsies of IBD patients have revealed disappearance of key TJ proteins from intercellular junctions [26], [50]. Probiotics have been shown to reduce the increased intestinal permeability in vitro [51] as well as in clinical trials [52]. Here we demonstrate for the first time that the substantially increased intestinal permeability of mice treated with DSS is significantly alleviated by simultaneous oral application of EcN. In addition, we observed an altered ZO-1 expression profile in IECs of mice with DSS-induced colitis. Recently, a study described the translocation of ZO-1 from the apical to the basolateral side in CD patients [53] indicating an alteration of ZO-1 under pathological conditions. Moreover, using a mouse colitis model, Resta-Lenert et al. have shown that the increase in intestinal permeability is associated with a decrease of occludin and ZO-1 phosphorylation [54]. However, administration of EcN in our mouse models not only diminished the clinical signs of colitis like colon shortening and weight loss, but also prevented an increase in intestinal permeability while concurrently minimizing the down-regulation of IEC ZO-1 expression. This indicates a considerable association between the severity of colitis, as evidenced by increased gut permeability, and altered ZO-1 mRNA expression levels. It can be speculated that the rise of ZO-1 expression results in a reduced intestinal permeability by an enhanced junctional complex or a reinforced interaction of the junctional complex with actin. This hypothesis is consistent with recently published data regarding the antrum mucosal protein (AMP)-18 that ameliorates DSS colitis in mice and also enhances accumulation of occludin and ZO-1 in TJ domains in vitro [55]. Since AMP peptide also prevented a fall in transepithelial resistance during disruption of actin filaments and stabilized the perijunctional actin during oxidant injury, it has been suggested that AMP-18 could protect the intestinal mucosal barrier by acting on specific TJ proteins and stabilizing perijunctional actin [55]. Using another murine colitis model, administration of n-3 polyunsaturated fatty acids resulted not only in reduced pathological scores but also an increase of ZO-1 protein expression [56]. The present study clearly demonstrates that EcN specifically up-regulates ZO-1 mRNA expression in IECs in vivo. Together with the influence of EcN on intestinal permeability and an enhanced ZO-1 expression under pathological conditions, it can be speculated that EcN augments mucosal barrier function. These in vivo results corroborate the in vitro findings from other groups by demonstrating that probiotic EcN plays an important role in the maintenance of intestinal barrier function. An improved barrier integrity elicited by EcN is an appealing explanation for the success of this probiotic in the therapy of UC and could be an important aspect in treating further human intestinal disorders, including HIV-associated diarrhea. Acknowledgments We thank further Lothar Gröbe for FACS sorting, Marco Metzger for performing immunohistochemistry and Silvia Prettin for technical support as well as Anna Smoczek and Ina Köhn for animal care. The authors also gratefully acknowledge Michael J. Schubert for critically reading the manuscript. Author ContributionsConceived and designed the experiments: SU US JB FG AW. Performed the experiments: SU AS UD RE AB. Analyzed the data: SU AW. Contributed reagents/materials/analysis tools: SS AF SU WH DB GR. Wrote the paper: SU FG Farquhar MG, Palade GE (1963) Junctional complexes in various epithelia. J Cell Biol 17: 375–412. View Article Google Scholar 2. Kapembwa MS, Fleming SC, Sewankambo N, Serwadda D, Lucas S, et al. (1991) Altered small-intestinal permeability associated with diarrhoea in human-immunodeficiency-virus-infected Caucasian and African subjects. Clin Sci (Lond) 81: 327–334. View Article Google Scholar 3. Rene E, Marche C, Regnier B, Saimot AG, Vilde JL, et al. (1989) Intestinal infections in patients with acquired immunodeficiency syndrome. A prospective study in 132 patients. Dig Dis Sci 34: 773–780. View Article Google Scholar 4. Smith PD, Lane HC, Gill VJ, Manischewitz JF, Quinnan GV, et al. (1988) Intestinal infections in patients with the acquired immunodeficiency syndrome (AIDS). Etiology and response to therapy. Ann Intern Med 108: 328–333. View Article Google Scholar 5. Stevenson BR, Siliciano JD, Mooseker MS, Goodenough DA (1986) Identification of ZO-1: a high molecular weight polypeptide associated with the tight junction (zonula occludens) in a variety of epithelia. J Cell Biol 103: 755–766. View Article Google Scholar 6. Wittchen ES, Haskins J, Stevenson BR (1999) Protein interactions at the tight junction. Actin has multiple binding partners, and ZO-1 forms independent complexes with ZO-2 and ZO-3. J Biol Chem 274: 35179–35185. View Article Google Scholar 7. Ma TY, Boivin MA, Ye DM, Pedram A, Said HM (2005) Mechanism of tumor necrosis factor-alpha modulation of Caco-2 intestinal epithelial tight junction barrier: Role of myosin light chain kinase protein expression. Am J Physiol Gastrointest Liver Physiol 288: G422–G430. View Article Google Scholar 8. Wang F, Graham WV, Wang Y, Witkowski ED, Schwarz BT, et al. (2005) Interferon-gamma and tumor necrosis factor-alpha synergize to induce intestinal epithelial barrier dysfunction by up-regulating myosin light chain kinase expression. Am J Pathol 166: 409–419. View Article Google Scholar 9. Gumbiner B, Lowenkopf T, Apatira D (1991) Identification of a 160-kDa polypeptide that binds to the tight junction protein ZO-1. Proc Natl Acad Sci U S A 88: 3460–3464. View Article Google Scholar 10. Zyrek AA, Cichon C, Helms S, Enders C, Sonnenborn U, et al. (2007) Molecular mechanisms underlying the probiotic effects of Escherichia coli Nissle 1917 involve ZO-2 and PKCzeta redistribution resulting in tight junction and epithelial barrier repair. Cell Microbiol 9: 804–816. View Article Google Scholar 11. Havenaar R, Huis in 't Veld JHJ (1992) Probiotics: a general view. In: Wood BJB, editor. The Lactic Acid Bacteria. New York: Chapman & Hall. pp. 209–224. 12. Kruis W, Schutz E, Fric P, Fixa B, Judmaier G, et al. (1997) Double-blind comparison of an oral Escherichia coli preparation and mesalazine in maintaining remission of ulcerative colitis. Aliment Pharmacol Ther 11: 853–858. View Article 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. View Article Google Scholar 14. Rembacken BJ, Snelling AM, Hawkey PM, Chalmers DM, Axon ATR (1999) Non-pathogenic Escherichia coli versus mesalazine for the treatment of ulcerative colitis: a randomised trial. Lancet 354: 635–639. View Article Google Scholar 15. Westendorf AM, Gunzer F, Deppenmeier S, Tapadar D, Hunger JK, et al. (2005) Intestinal immunity of Escherichia coli NISSLE 1917: a safe carrier for therapeutic molecules. FEMS Immunol Med Microbiol 43: 373–384. View Article Google Scholar 16. Henker J, Laass M, Blokhin BM, Bolbot YK, Maydannik VG, et al. (2007) The probiotic Escherichia coli strain Nissle 1917 (EcN) stops acute diarrhoea in infants and toddlers. Eur J Pediatr 166: 311–318. View Article Google Scholar 17. Dotan I, Rachmilewitz D (2005) Probiotics in inflammatory bowel disease: possible mechanisms of action. Curr Opin Gastroenterol 21: 426–430. View Article Google Scholar 18. Sartor RB (2004) Therapeutic manipulation of the enteric microflora in inflammatory bowel diseases: Antibiotics, probiotics, and prebiotics. Gastroenterology 126: 1620–1633. View Article Google Scholar 19. Resta-Lenert S, Barrett KE (2003) Live probiotics protect intestinal epithelial cells from the effects of infection with enteroinvasive Escherichia coli (EIEC). Gut 52: 988–997. View Article Google Scholar 20. Bleich A, Sundberg JP, Smoczek A, von Wasielewski R, de Buhr MF, et al. (2007) Sensitivity to Escherichia coli Nissle 1917 in mice is dependent on environment and genetic background. Int J Exp Pathol Nov 13: Epub ahead of print. View Article Google Scholar 21. Gunzer F, Hennig-Pauka I, Waldmann KH, Mengel M (2003) Gnotobiotic piglets as an animal model for oral infection with O157 and non-O157 serotypes of STEC. Methods Mol Med 73: 307–327. View Article Google Scholar 22. Grabig A, Paclik D, Guzy C, Dankof A, Baumgart DC, et al. (2006) Escherichia coli strain Nissle 1917 ameliorates experimental colitis via toll-like receptor 2- and toll-like receptor 4-dependent pathways. Infect Immun 74: 4075–4082. View Article Google Scholar 23. Rogler G, Daig R, Aschenbrenner E, Vogl D, Schlottmann K, et al. (1998) Establishment of long-term primary cultures of human small and large intestinal epithelial cells. Lab Invest 78: 889–890. View Article Google Scholar 24. Spyridonidis A, Schmitt-Graff A, Tomann T, Dwenger A, Follo M, et al. (2004) Epithelial tissue chimerism after human hematopoietic cell transplantation is a real phenomenon. Am J Pathol 164: 1147–1155. View Article Google Scholar 25. Schürmann G, Brüwer M, Klotz A, Schmid KW, Senninger N, et al. (1999) Transepithelial transport processes at the intestinal mucosa in inflammatory bowel disease. Int J Colorectal Dis 14: 41–46. View Article Google Scholar 26. Ivanov AI, Nusrat A, Parkos CA (2004) The epithelium in inflammatory bowel disease: potential role of endocytosis of junctional proteins in barrier disruption. Novartis Found Symp 263: 115–124. View Article Google Scholar 27. Nejdfors P, Wang Q, Ekelund M, Westrom BR, Jansson O, et al. (1998) Increased colonic permeability in patients with ulcerative colitis: An in vitro study. Scand J Gastroenterol 33: 749–753. View Article Google Scholar 28. Wyatt J, Vogelsang H, Hubl W, Waldhoer T, Lochs H (1993) Intestinal permeability and the prediction of relapse in Crohn's disease. Lancet 341: 1437–1439. View Article Google Scholar 29. Schultz M, Strauch UG, Linde HJ, Watzl S, Obermeier F, et al. (2004) Preventive effects of Escherichia coli strain Nissle 1917 on acute and chronic intestinal inflammation in two different murine models of colitis. Clin Diagn Lab Immunol 11: 372–378. View Article Google Scholar 30. Podolsky DK (2002) Inflammatory bowel disease. N Engl J Med 347: 417–429. View Article Google Scholar 31. Jepson MA, Collares-Buzato CB, Clark MA, Hirst BH, Simmons NL (1995) Rapid disruption of epithelial barrier function by Salmonella typhimurium is associated with structural modification of intercellular junctions. Infect Immun 63: 356–359. View Article Google Scholar 32. Jepson MA, Schlecht HB, Collares-Buzato CB (2000) Localization of dysfunctional tight junctions in Salmonella enterica serovar typhimurium-infected epithelial layers. Infect Immun 68: 7202–7208. View Article Google Scholar 33. Philpott DJ, McKay DM, Mak W, Perdue MH, Sherman PM (1998) Signal transduction pathways involved in enterohemorrhagic Escherichia coli-induced alterations in T84 epithelial permeability. Infect Immun 66: 1680–1687. View Article Google Scholar 34. McCarthy J, O'Mahony L, O'Callaghan L, Sheil B, Vaughan EE, et al. (2003) Double blind, placebo controlled trial of two probiotic strains in interleukin 10 knockout mice and mechanistic link with cytokine balance. Gut 52: 975–980. View Article Google Scholar 35. Rachmilewitz D, Katakura K, Karmeli F, Hayashi T, Reinus C, et al. (2004) Toll-like receptor 9 signaling mediates the anti-inflammatory effects of probiotics in murine experimental colitis. Gastroenterology 126: 520–528. View Article Google Scholar 36. Schultz M, Veltkamp C, Dieleman LA, Grenther WB, Wyrick PB, et al. (2002) Lactobacillus plantarum 299V in the treatment and prevention of spontaneous colitis in interleukin-10-deficient mice. Inflamm Bowel Dis 8: 71–80. View Article Google Scholar 37. Kunzelmann K, Mall M (2002) Electrolyte transport in the mammalian colon: Mechanisms and implications for disease. Physiol Rev 82: 245–289. View Article Google Scholar 38. Seidler U, Lenzen H, Cinar A, Tessema T, Bleich A et al (2006) Molecular mechanisms of disturbed electrolyte transport in intestinal inflammation. Ann N Y Acad Sci 1072: 262–275. View Article Google Scholar 39. Clayburgh DR, Barrett TA, Tang YM, Meddings JB, Van Eldik LJ, et al. (2005) Epithelial myosin light chain kinase-dependent barrier dysfunction mediates T cell activation-induced diarrhea in vivo. J Clin Invest 115: 2702–2715. View Article Google Scholar 40. Schmitz H, Barmeyer C, Fromm M, Runkel N, Foss HD, et al. (1999) Altered tight junction structure contributes to the impaired epithelial barrier function in ulcerative colitis. Gastroenterology 116: 301–309. View Article Google Scholar 41. Stockmann M, Fromm M, Schmitz H, Schmidt W, Riecken EO, et al. (1998) Duodenal biopsies of HIV-infected patients with diarrhoea exhibit epithelial barrier defects but no active secretion. AIDS 12: 43–51. View Article Google Scholar 42. Schmitz H, Rokos K, Florian P, Gitter AH, Fromm M, et al. (2002) Supernatants of HIV-infected immune cells affect the barrier function of human HT-29/B6 intestinal epithelial cells. AIDS 16: 983–991. View Article Google Scholar 43. Levine MM, Kaper JB, Herrington D, Losonsky G, Morris JG, et al. (1988) Volunteer studies of deletion mutants of Vibrio cholerae O1 prepared by recombinant techniques. Infect Immun 56: 161–167. View Article Google Scholar 44. Fasano A, Baudry B, Pumplin DW, Wasserman SS, Tall BD, et al. (1991) Vibrio cholerae produces a second enterotoxin, which affects intestinal tight junctions. Proc Natl Acad Sci U S A 88: 5242–5246. View Article Google Scholar 45. Wu Z, Milton D, Nybom P, Sjo A, Magnusson KE (1996) Vibrio cholerae hemagglutinin/protease (HA/protease) causes morphological changes in cultured epithelial cells and perturbs their paracellular barrier function. Microb Pathog 21: 111–123. View Article Google Scholar 46. Wu Z, Nybom P, Magnusson KE (2000) Distinct effects of Vibrio cholerae haemagglutinin/protease on the structure and localization of the tight junction-associated proteins occludin and ZO-1. Cell Microbiol 2: 11–17. View Article Google Scholar 47. Fasano A, Uzzau S (1997) Modulation of intestinal tight junctions by Zonula occludens toxin permits enteral administration of insulin and other macromolecules in an animal model. J Clin Invest 99: 1158–1164. View Article Google Scholar 48. Hollander D (1999) Intestinal permeability, leaky gut, and intestinal disorders. Curr Gastroenterol Rep 1: 410–416. View Article Google Scholar 49. Meddings JB (1997) Intestinal permeability in Crohn's disease. Aliment Pharmacol Ther 11: 45–53. View Article Google Scholar 50. Barmeyer C, Harren M, Schmitz H, Heinzel-Pleines U, Mankertz J, et al. (2004) Mechanisms of diarrhea in the interleukin-2-deficient mouse model of colonic inflammation. Am J Physiol Gastrointest Liver Physiol 286: G244–G252. View Article Google Scholar 51. Parassol N, Freitas M, Thoreux K, Dalmasso G, Bourdet-Sicard R, et al. (2005) Lactobacillus casei DN-114 001 inhibits the increase in paracellular permeability of enteropathogenic Escherichia coli-infected T84 cells. Res Microbiol 156: 256–262. View Article Google Scholar 52. Alberda C, Gramlich L, Meddings J, Field C, McCargar L, et al. (2007) Effects of probiotic therapy in critically ill patients: a randomized, double-blind, placebo-controlled trial. Am J Clin Nutr 85: 816–823. View Article Google Scholar 53. Oshitani N, Watanabe K, Nakamura S, Fujiwara Y, Higuchi K, et al. (2005) Dislocation of tight junction proteins without F-actin disruption in inactive Crohn's disease. Int J Mol Med 15: 407–410. View Article Google Scholar 54. Resta-Lenert S, Smitham J, Barrett KE (2005) Epithelial dysfunction associated with the development of colitis in conventionally housed mdr1a(-/-) mice. Am J Physiol Gastrointest Liver Physiol 289: G153–G162. View Article Google Scholar 55. Walsh-Reitz MM, Huang EF, Musch MW, Chang EB, Martin TE, et al. (2005) AMP-18 protects barrier function of colonic epithelial cells: role of tight junction proteins. Am J Physiol Gastrointest Liver Physiol 289: G163–G171. View Article Google Scholar 56. Whiting CV, Bland PW, Tarlton JE (2005) Dietary n-3 polyunsaturated fatty acids reduce disease and colonic proinflammatory cytokines in a mouse model of colitis. Inflamm Bowel Dis 11: 340–349. View Article Google Scholar

E. coli Nissle 1917. Several investigations have been conducted during the past years to examine the correlation between dysbiosis and both intestinal and extra-intestinal diseases such as inflammatory bowel disease (IBD) and ulcerative colitis (UC). E. coli Nissle 1917 (EcN) is a nonpathogenic gram-negati ….

Nonpathogenic Escherichia coli strain Nissle 1917 (O6:K5:H1) is used as a probiotic agent in medicine, mainly for the treatment of various gastroenterological diseases. To gain insight on the genetic level into its properties of colonization and commensalism, this strain's genome structure has been …

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

The probiotic Escherichia coli Nissle 1917 (EcN) was engineered to synthesize the ketone body (R)-3-hydroxybutyrate (3HB) for sustainable production in the gut lumen of mice suffering from colitis.

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

SHib.

bc04p2mbkf.pages.dev/45

bc04p2mbkf.pages.dev/293

bc04p2mbkf.pages.dev/358

bc04p2mbkf.pages.dev/273

bc04p2mbkf.pages.dev/3

bc04p2mbkf.pages.dev/301

bc04p2mbkf.pages.dev/166

bc04p2mbkf.pages.dev/64

bc04p2mbkf.pages.dev/372

escherichia coli nissle 1917