Determination of Resistant Starch Assimilating Bacteria in Fecal Samples of Mice by In vitro RNA-Based Stable Isotope Probing.

Elena Herrmann,Ralf Conrad,Markus Egert,Christian U Riedel,Douglas Rosendale,Wayne Young

doi:10.3389/fmicb.2017.01331

Abstract

The impact of the intestinal microbiota on human health is becoming increasingly appreciated in recent years. In consequence, and fueled by major technological advances, the composition of the intestinal microbiota in health and disease has been intensively studied by high throughput sequencing approaches. Observations linking dysbiosis of the intestinal microbiota with a number of serious medical conditions including chronic inflammatory disorders and allergic diseases suggest that restoration of the composition and activity of the intestinal microbiota may be a treatment option at least for some of these diseases. One possibility to shape the intestinal microbiota is the administration of prebiotic carbohydrates such as resistant starch (RS). In the present study, we aim at establishing RNA-based stable isotope probing (RNA-SIP) to identify bacterial populations that are involved in the assimilation of RS using anaerobic in vitro fermentation of murine fecal material with stable [U13C] isotope-labeled potato starch. Total RNA from these incubations was extracted, processed by gradient ultracentrifugation and fractionated by density. 16S rRNA gene sequences were amplified from reverse transcribed RNA of high and low density fractions suspected to contain labeled and unlabeled RNA, respectively. Phylogenetic analysis of the obtained sequences revealed a distinct subset of the intestinal microbiota involved in starch metabolism. The results suggest Bacteroidetes, in particular genera affiliated with Prevotellaceae, as well as members of the Ruminococcacea family to be primary assimilators of resistant starch due to a significantly higher relative abundance in higher density fractions in RNA samples isolated after 2 h of incubation. Using high performance liquid chromatography coupled to isotope ratio mass spectrometry (HPLC-IRMS) analysis, some stable isotope label was recovered from acetate, propionate and butyrate. Here, we demonstrate the suitability of RNA-SIP to link specific groups of microorganisms with fermentation of a specific substrate. The application of RNA-SIP in future in vivo studies will help to better understand the mechanisms behind functionality of a prebiotic carbohydrate and its impact on an intestinal ecosystem with potential implications for human health.

Highlights

The human large bowel is a highly complex and dynamic ecosystem harboring an immense number of microorganisms (Riedel et al, 2014) and representing one of the most metabolically active sites in our body (Slavin, 2013)
resistant starch (RS) is defined as the fraction of food-derived starch that is resistant to digestion by host amylases in the upper digestive tract and transits intact to the large bowel, where it serves as a substrate for microbial growth and is transformed to short-chain fatty acids (SCFA; butyrate, propionate and acetate; Asp, 1992; Englyst et al, 1996)
In gradients containing only unlabeled [12C]RNA species originating from fresh fecal slurry (0 h; Figure 1B) and from slurries incubated with [12C]starch for 2 h (Figure 1C) and 4 h (Figure 1D), an almost identical RNA distribution pattern was observed: The bulk of the unlabeled RNA species was present in low density fractions (≤1.796 g mL−1; ≥fraction 8) with peak amounts detected in fraction 9 (1.792 g mL−1) after 0 h and 2 h of incubation and in fraction 10 (1.787 g mL−1) after 4 h of incubation

Summary

Introduction

The human large bowel is a highly complex and dynamic ecosystem harboring an immense number of microorganisms (Riedel et al, 2014) and representing one of the most metabolically active sites in our body (Slavin, 2013). An essential function is the metabolic capacity to ferment complex dietary carbohydrates such resistant starch (RS) (Guarner and Malagelada, 2003). RS is defined as the fraction of food-derived starch that is resistant to digestion by host amylases in the upper digestive tract and transits intact to the large bowel, where it serves as a substrate for microbial growth and is transformed to short-chain fatty acids (SCFA; butyrate, propionate and acetate; Asp, 1992; Englyst et al, 1996). SCFAs are known to play a crucial role in host physiology (Koh et al, 2016; Morrison and Preston, 2016)

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