Laboratory of Food Biotechnology, Institute of Food, Nutrition and Health, ETH Zürich, Schmelzbergstrasse 7, 8092 Zürich, Switzerland.
BMC Microbiol. 2011 Dec 15;11:264. doi: 10.1186/1471-2180-11-264.
Accurate assessment of probiotics with targeted anti-Salmonella activity requires suitable models accounting for both, microbe-microbe and host-microbe interactions in gut environments. Here we report the combination of two original in vitro intestinal models closely mimicking the complex in vivo conditions of the large intestine. Effluents from continuous in vitro three-stage fermentation colonic models of Salmonella Typhimurium infection inoculated with immobilized child microbiota and Salmonella were directly applied to confluent mucus-secreting HT29-MTX cell layers. The effects of Salmonella, addition of two bacteriocinogenic strains, Bifidobacterium thermophilum RBL67 (thermophilicin B67) and Escherichia coli L1000 (microcin B17), and inulin were tested on Salmonella growth and interactions with epithelial cell layers. Salmonella adhesion and invasion were investigated and epithelial integrity assessed by transepithelial electrical resistance (TER) measurements and confocal microscopy observation. Data from complex effluents were compared with pure Salmonella cultures.
Salmonella in effluents of all reactors of the colonic fermentation model stabilized at mean values of 5.3 ± 0.8 log10 cfu/ml effluent. Invasion of cell-associated Salmonella was up to 50-fold lower in complex reactor samples compared to pure Salmonella cultures. It further depended on environmental factors, with 0.2 ± 0.1% being measured with proximal, 0.6 ± 0.2% with transverse and 1.3 ± 0.7% with distal reactor effluents, accompanied by a similar high decrease of TER across cell monolayers (minus 45%) and disruption of tight junctions. Subsequent addition of E. coli L1000 stimulated Salmonella growth (6.4 ± 0.6 log10 cfu/ml effluent of all 3 reactors) and further decreased TER, but led to 10-fold decreased invasion efficiency when tested with distal reactor samples. In contrast, presence of B. thermophilum RBL67 revealed a protective effect on epithelial integrity compared to previous E. coli L1000 periods, as reflected by a significant mean increase of TER by 58% in all reactors. Inulin addition enhanced Salmonella growth and invasion when tested with distal and proximal reactor samples, respectively, but induced a limited decrease of TER (minus 18%) in all reactors.
Our results highlight the benefits of combining suitable cellular and colonic fermentation models to assess strain-specific first-level host protection properties of probiotics during Salmonella infection, providing an efficient system biology tool for preclinical development of new antimicrobials.
准确评估具有靶向抗沙门氏菌活性的益生菌需要合适的模型，这些模型既要考虑肠道环境中的微生物-微生物相互作用，也要考虑宿主-微生物相互作用。本研究报告了两种原始的体外肠道模型的结合，这些模型紧密模拟了沙门氏菌感染的大肠复杂的体内条件。将固定化儿童微生物群和沙门氏菌接种到连续体外三阶段发酵结肠模型的流出物直接应用于汇合的粘液分泌 HT29-MTX 细胞层。测试了沙门氏菌、添加两种细菌素产生菌株双歧杆菌 RBL67（热稳定素 B67）和大肠杆菌 L1000（微菌素 B17）以及菊粉对沙门氏菌生长和与上皮细胞层相互作用的影响。通过跨上皮电阻（TER）测量和共聚焦显微镜观察研究了沙门氏菌的粘附和侵袭，并评估了上皮完整性。将复杂流出物的数据与纯沙门氏菌培养物进行了比较。
沙门氏菌在结肠发酵模型所有反应器的流出物中稳定在 5.3 ± 0.8 log10 cfu/ml 流出物的平均值。与纯沙门氏菌培养物相比，复杂反应器样品中细胞相关沙门氏菌的侵袭率降低了多达 50 倍。它进一步取决于环境因素，近端测量值为 0.2 ± 0.1%，横向为 0.6 ± 0.2%，远端为 1.3 ± 0.7%，同时穿过细胞单层的 TER 也有类似的大幅下降（减去 45%）和紧密连接的破坏。随后添加大肠杆菌 L1000 刺激沙门氏菌生长（所有 3 个反应器的流出物中为 6.4 ± 0.6 log10 cfu/ml），并进一步降低 TER，但当用远端反应器样品进行测试时，侵袭效率降低了 10 倍。相比之下，与之前的大肠杆菌 L1000 时期相比，双歧杆菌 RBL67 的存在对上皮完整性表现出保护作用，这反映在所有反应器中 TER 平均增加了 58%。菊粉添加分别增强了远端和近端反应器样品中的沙门氏菌生长和侵袭，但在所有反应器中仅引起 TER 减少（减去 18%）。
我们的结果强调了结合合适的细胞和结肠发酵模型来评估沙门氏菌感染期间益生菌的特定一级宿主保护特性的优势，为新抗菌药物的临床前开发提供了一种有效的系统生物学工具。
