Stable Isotope Biochemistry Laboratory, Scottish Universities Environmental Research Centre, Rankine Avenue, Scottish Enterprise Technology Park, East Kilbride, Glasgow G750QF, United Kingdom.
Appl Environ Microbiol. 2011 Nov;77(22):8009-14. doi: 10.1128/AEM.05573-11. Epub 2011 Sep 23.
The human intestinal microbiota is a complex biological system comprising a vast repertoire of microbes with considerable metabolic activity relevant to both bacterial growth and host health. Greater strides have been made in the analysis of microbial diversity than in the measurement of functional activity, particularly in vivo. Stable isotope probing offers a new approach by coupling measurements of metabolic activity with microbial identification. Using a low-enrichment labeling strategy in vitro, this study has identified metabolically active bacterial groups via magnetic-bead capture methodology and stable isotope ratio analysis. Using five probes (EUB338, Bac303, Bif164, EREC482, and Clep866), changes in the activities of key intestinal microbial groups were successfully measured by exploiting tracers of de novo RNA synthesis. Perturbation of the nutrient source with oligofructose generated changes in the activity of bifidobacteria as expected, but also in the Bacteroides-Prevotella group, the Eubacterium rectale-Clostridium coccoides group, and the Clostridium leptum subgroup. Changes in activity were also observed in response to the medium type. This study suggests that changes in the functional activity of the gut microbiota can be assessed using tracers of de novo nucleic acid synthesis combined with measurement of low isotopic enrichment in 16S rRNA. Such tracers potentially limit substrate bias because they are universally available to bacteria. This low-enrichment labeling approach does not depend on the commercial availability of specific labeled substrates and can be easily translated to in vivo probing experiments of the functional activity of the microbiota in the human gut.
人类肠道微生物群是一个复杂的生物系统，由大量具有相当代谢活性的微生物组成，这些微生物与细菌生长和宿主健康都有密切关系。与功能活性的测量相比，人们在微生物多样性的分析方面已经取得了更大的进展，尤其是在体内。稳定同位素示踪为我们提供了一种新的方法，它将代谢活性的测量与微生物鉴定相结合。本研究通过体外低富集标记策略，利用磁珠捕获方法和稳定同位素比分析，通过代谢活性细菌群体的识别，鉴定了代谢活跃的细菌群体。使用五种探针（EUB338、Bac303、Bif164、EREC482 和 Clep866），通过利用从头合成 RNA 的示踪剂，成功地测量了关键肠道微生物群体活性的变化。用低聚果糖作为营养源进行的扰动实验，如预期的那样，双歧杆菌的活性发生了变化，但拟杆菌-普雷沃氏菌组、直肠真杆菌-梭菌组和丁酸弧菌亚组的活性也发生了变化。对培养基类型的改变也观察到了活性的变化。本研究表明，使用从头合成核酸的示踪剂结合 16S rRNA 的低同位素富集测量，可以评估肠道微生物群的功能活性变化。这种示踪剂潜在地限制了底物的偏向性，因为它们普遍存在于细菌中。这种低富集标记方法不依赖于特定标记底物的商业可用性，并且可以很容易地转化为人类肠道微生物群功能活性的体内探测实验。
