Schultz Michael, Watzl Sonja, Oelschlaeger Tobias A, Rath Heiko C, Göttl Claudia, Lehn Norbert, Schölmerich Jürgen, Linde Hans-Jörg
Department of Internal Medicine I, University of Regensburg, Regensburg, Germany.
J Microbiol Methods. 2005 Jun;61(3):389-98. doi: 10.1016/j.mimet.2005.01.007.
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.
益生菌被定义为对人类宿主健康有益的活营养剂。特别是，大肠杆菌菌株Nissle 1917（EcN）在维持溃疡性结肠炎（UC）缓解方面显示出与美沙拉嗪同样有效。据推测，EcN的治疗效果与其在感兴趣区域的存在有关；然而，使用传统培养方法，很难在体内追踪口服给药的菌株在通过由各种大肠杆菌菌株主导的复杂肠道微生物环境时的情况。在本研究中，我们用携带绿色荧光蛋白基因（pUC - gfp）的质粒转化EcN和来自实验室大鼠的野生型大肠杆菌（EcR），以获得EcN - GFP和EcR - GFP，以便在不改变菌株特异性特征的情况下进行体内检测。对不同菌株特异性特征的分析包括使用上皮细胞系HT - 29测量体外IL - 8分泌刺激和黏附情况。在体内研究了口服给药后小鼠肠道分布动力学和大鼠的定植特性。使用荧光显微镜和免疫组织化学分析组织学标本中菌株的可检测性。通过REP - PCR确定从粪便样本、派尔集合淋巴结（PP）和肠系膜淋巴结（MLN）分离的荧光大肠杆菌菌株的身份。我们能够证明EcN和EcN - GFP在刺激IL - 8分泌或黏附于HT - 29细胞方面没有差异。在体内，通过荧光显微镜在管腔样本中很容易检测到EcN - GFP菌落，在组织学切片中也可通过免疫组织化学检测到，从而可以分析口服给药后肠道通过的动力学情况。口服给药后6小时，观察到荧光和非荧光细菌向PP和MLN的易位。EcN - GFP最初在大鼠粪便样本中可检测14天，而EcR - GFP在整个实验期间（45天）均可检测到。在第45天用氨苄青霉素进行挑战，通过再次出现荧光菌落证明仍有少量EcN - GFP持续存在。由于通过REP - PCR在粪便样本中也检测到了非荧光EcN菌落，所以该质粒在体内不稳定。总之，在我们的研究中，将EcN转化为EcN - GFP对益生菌在黏附于HT - 29细胞和诱导其IL - 8分泌方面没有可检测到的影响，并允许在体内混合微生物环境中进行检测，但EcN - GFP在体内的稳定性有限。
