Green fluorescent protein for detection of the probiotic microorganism Escherichia coli strain Nissle 1917 (EcN) in vivo

Department of Internal Medicine I, University of Regensburg, Regensburg, Germany.
Journal of Microbiological Methods (Impact Factor: 2.03). 07/2005; 61(3):389-98. DOI: 10.1016/j.mimet.2005.01.007
Source: PubMed


Probiotic microorganisms are defined as viable nutritional agents conferring benefit to the health of the human host. Especially, Escherichia coli strain Nissle 1917 (EcN) was shown to be equally effective as mesalazine in the maintenance of remission in ulcerative colitis (UC). Presumably, the therapeutic effect of EcN is linked to the presence of the strain in the region of interest; however, it remains difficult to follow the orally administered strain on its passage through the complex microbial environment of the intestine in vivo, inhabited dominantly by various E. coli strains, using traditional culturing methods. In this study we transformed EcN and a wild-type E. coli from a laboratory rat (EcR) with a plasmid carrying a gfp gene (pUC-gfp) to obtain EcN- and EcR-GFP to allow in vivo detection without alteration of strain-specific characteristics. Analysis of different strain-specific characteristics included the measurement of stimulation of IL-8 secretion and adhesion in vitro using the epithelial cell line HT-29. The kinetics of intestinal distribution in mice and colonization properties in rats following oral administration was studied in vivo. Detectability of the strain in histologic specimens was analysed using fluorescence microscopy and immunohistochemistry. The identity of fluorescent E. coli strains isolated from stool samples, Peyer's patches (PP) and mesenteric lymph nodes (MLN) was determined by REP-PCR. We were able to demonstrate that EcN and EcN-GFP do not differ in stimulation of IL-8 secretion or adhesion to HT-29 cells. In vivo, EcN-GFP colonies were readily detectable by fluorescence microscopy in luminal samples and also by immunohistochemistry in histological sections allowing analysis of the kinetics of the intestinal passage following oral administration. Translocation of fluorescent and non-fluorescent bacteria into PP and MLN was noted at 6 h post oral administration. EcN-GFP was detectable initially for 14 days in faecal samples of rats, while EcR-GFP was detectable throughout the whole experiment (45 days). Challenge with ampicillin at day 45 demonstrated continuing presence of EcN-GFP in small numbers by reappearing fluorescent colonies. The plasmid was not stable in vivo since non-fluorescent EcN colonies were detected also in faecal samples by REP-PCR. In summary, transformation of EcN to obtain EcN-GFP in our study had no detectable influence on the probiotic microorganism regarding adhesion on and induction of IL-8 secretion of HT-29 cells and allows the detection in mixed microbial environments in vivo but the stability of EcN-GFP in vivo is limited.

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    • "After 24 h, the plates were inspected under UV light for the fluorescence. As soon as the reduction of fluorescent colonies of fecal samples was noted, rats were given ampicillin (50 mg/kg body weight) (days 23–25 and days 48–51) in drinking water [16, 26]. "
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    ABSTRACT: The present study describes the beneficial effects of potential probiotic E. coli 16 (pUC8:16gfp) expressing Vitreoscilla hemoglobin (vgb) gene, associated with bacterial respiration under microaerobic condition, on gastrointestinal (GI) colonization and its antioxidant activity on carbon tetrachloride (CCl4) induced toxicity in Charles Foster rats. In vitro, catalase activity in E. coli 16 (pUC8:16gfp) was 1.8 times higher compared to E. coli 16 (pUC-gfp) control. In vivo, E. coli 16 (pUC8:16gfp) not only was recovered in the fecal matter after 70 days of oral administration but also retained antibacterial activities, whereas E. coli 16 (pUC-gfp) was not detected. Oral administration of 200 and 500 μL/kg body weight of CCl4 to rats at weekly interval resulted in elevated serum glutamyl pyruvate transaminase (SGPT) and serum glutamyl oxalacetate transaminase (SGOT) levels compared to controls. Rats prefed with E. coli 16 (pUC8:16gfp) demonstrated near to normal levels for SGPT and SGOT, whereas the liver homogenate catalase activity was significantly increased compared to CCl4 treated rats. Thus, pUC8:16gfp plasmid encoding vgb improved the growth and GI tract colonization of E. coli 16. In addition, it also enhanced catalase activity in rats harboring E. coli 16 (pUC8:16gfp), thereby preventing the absorption of CCl4 to GI tract.
    Full-text · Article · Jun 2014 · BioMed Research International
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    • "It can be applied as a useful vehicle in expression of molecular therapy in intestinal epithelium [7]. EcN can survive in the gastrointestinal system after orally use and it is propagated in the gut and can be colonized in the intestinal environment for long-term periods [8]. The genetic "
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    ABSTRACT: Regarding to the high prevalence and comorbidities of chronic high blood glucose in diabetic patients and the limited efficacy and current painful treatments. It is necessary to improve new treatments that are non-invasive and long-term for controlling blood glucose. Recent studies have shown that the healthy microflora in different body organs can perform as the gene vectors for expressing different types of gene therapies in situ. We have proposed that by constructing a recombinant Escherichia coli Nissle 1917 that expresses CTB-IGF-1 hybrid gene under control of ompC glucose sensitive promoter, the intestinal glucose level can be regulated. This method in comparison with other methods is a non-invasive way to control the blood glucose orally and it can be used for all types of diabetes.
    Full-text · Article · Sep 2013 · Medical Hypotheses
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    • "PP and MLN after oral administration to mice [26]. Lactobacillus casei strain Shirota was also incorporated to M cells of PP and digested by innate immune cells to form bioactive components [27]. "
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    ABSTRACT: The intestinal tract mucosa is exposed to a vast number of environmental antigens and a large community of commensal bacteria. The mucosal immune system has to provide both protection against pathogens and tolerance to harmless bacteria. Immune homeostasis depends on the interaction of indigenous commensal and transient bacteria (probiotics) with various components of the epithelium and the gut-associated lymphoid tissue. Herein, an update is given of the mechanisms by which the gut microbiota and probiotics are translocated through the epithelium, sensed via pattern-recognition receptors, and activate innate and adaptive immune responses.
    Preview · Article · Dec 2009 · International Reviews Of Immunology
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