Green fluorescent protein for detection of the probiotic microorganism Escherichia coli strain Nissle 1917 (EcN) in vivo.
ABSTRACT 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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ABSTRACT: A recombinant DNA, pMG36e::luc+ (5.3 kb), was constructed and transferred to Escherichia coli MC1061 and Lactococcus lactis MG1363 for in vivo imaging of the bacteria in murine intestines. E. coli MC1061 (pMG36e::luc+) and L. lactis MG1363 (pMG36e::luc+) displayed luciferase activities in vitro, where the bioluminescent signal of the E. coli was much stronger than that of the L. lactis by approximately 100-fold. These 2 recombinant bacteria were orally administered into rats. The bioluminescent signals of the E. coli and L. lactis in the gastrointestinal (GI) tracts of rats were detected and maintained up to 2 h via whole body imaging, indicating that the luciferase gene expression system for bacteria could be applied for in vivo imaging.Food science and biotechnology 06/2012; 21(3). · 0.66 Impact Factor
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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.BioMed Research International 06/2014; · 2.71 Impact Factor
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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.Medical Hypotheses 09/2013; · 1.15 Impact Factor