In Vivo Assessment of Resistant Starch Degradation by the Caecal Microbiota of Mice Using RNA-Based Stable Isotope Probing-A Proof-of-Principle Study.

Resistant starch (RS) is the digestion resistant fraction of complex polysaccharide starch. By reaching the large bowel, RS can function as a prebiotic carbohydrate, i.e., it can shape the structure and activity of bowel bacterial communities towards a profile that confers health benefits. However, knowledge about the fate of RS in complex intestinal communities and the microbial members involved in its degradation is limited. In this study, 16S ribosomal RNA (rRNA)-based stable isotope probing (RNA-SIP) was used to identify mouse bowel bacteria involved in the assimilation of RS or its derivatives directly in their natural gut habitat. Stable-isotope [U13C]-labeled native potato starch was administrated to mice, and caecal contents were collected before 0 h and 2 h and 4 h after administration. 'Heavy', isotope-labeled [13C]RNA species, presumably derived from bacteria that have metabolized the labeled starch, were separated from 'light', unlabeled [12C]RNA species by fractionation of isolated total RNA in isopycnic-density gradients. Inspection of different density gradients showed a continuous increase in 'heavy' 16S rRNA in caecal samples over the course of the experiment. Sequencing analyses of unlabeled and labeled 16S amplicons particularly suggested a group of unclassified Clostridiales, Dorea, and a few other taxa (Bacteroides, Turicibacter) to be most actively involved in starch assimilation in vivo. In addition, metabolic product analyses revealed that the predominant 13C-labeled short chain fatty acid (SCFA) in caecal contents produced from the [U13C] starch was butyrate. For the first time, this study provides insights into the metabolic transformation of RS by intestinal bacterial communities directly within a gut ecosystem, which will finally help to better understand its prebiotic potential and possible applications in human health.

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

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.

RNA-Based Stable Isotope Probing Suggests Allobaculum spp. as Particularly Active Glucose Assimilators in a Complex Murine Microbiota Cultured In Vitro.

RNA-based stable isotope probing (RNA-SIP) and metabolic profiling were used to detect actively glucose-consuming bacteria in a complex microbial community obtained from a murine model system. A faeces-derived microbiota was incubated under anaerobic conditions for 0, 2, and 4 h with 40 mM [U13C]glucose. Isopycnic density gradient ultracentrifugation and fractionation of isolated RNA into labeled and unlabeled fractions followed by 16S rRNA sequencing showed a quick adaptation of the bacterial community in response to the added sugar, which was dominated by unclassified Lachnospiraceae species. Inspection of distinct fractions of isotope-labeled RNA revealed Allobaculum spp. as particularly active glucose utilizers in the system, as the corresponding RNA showed significantly higher proportions among the labeled RNA. With time, the labeled sugar was used by a wider spectrum of faecal bacteria. Metabolic profiling indicated rapid fermentation of [U13C]glucose, with lactate, acetate, and propionate being the principal 13C-labeled fermentation products, and suggested that "cross-feeding" occurred in the system. RNA-SIP combined with metabolic profiling of 13C-labeled products allowed insights into the microbial assimilation of a general model substrate, demonstrating the appropriateness of this technology to study assimilation processes of nutritionally more relevant substrates, for example, prebiotic carbohydrates, in the gut microbiota of mice as a model system.

Effect of rotor type on the separation of isotope-labeled and unlabeled Escherichia coli RNA by isopycnic density ultracentrifugation.

Separation of differentially isotope-labeled bacterial RNA by isopycnic density gradient centrifugation is a critical step in RNA-based stable isotope probing analyses, which help to link the structure and function of complex microbial communities. Using isotope-labeled Escherichia coli RNA, we showed that an 8 mL near-vertical rotor performed better than a 2 mL fixed-angle rotor, thereby corroborating current recommendations. Neither increased concentrations of formamide nor urea in the medium improved the separation results using the fixed-angle rotor.

Impaired ß-defensin expression in human skin links DEFB1 promoter polymorphisms with persistent Staphylococcus aureus nasal carriage.

BACKGROUND: Genetically determined variation in the expression of innate defense molecules may explain differences in the propensity to be colonized with Staphylococcus aureus. METHODS: We determined S. aureus nasal carriage in 603 volunteers; analyzed polymorphisms in the DEFB1 promoter at positions -52 G>A (rs1799946), -44 C>G (rs1800972), and -20 G>A (rs11362); and measured the content of human ß-defensin 1 (hBD-1) and hBD-3 messenger RNA (mRNA) in 192 samples of healthy and experimentally wounded human skin. RESULTS: Compared with GGG at the positions -52/-44/-20, the ACG haplotype was more common among persistent S. aureus nasal carriers (odds ratio, 1.93; 95% confidence interval [CI], 1.2-3.1; P = .006) and was associated with reduced expression of hBD-1 (GGG>ACG>GCA; P < .001) and hBD-3 (GGG>GCA>ACG; P = .04) in skin when measured 72 hours after wounding. Furthermore, a 50% decrease in hBD-1 and hBD-3 mRNA expression in wounded skin increased the odds of persistent carriage by 1.45 (95% CI, .93-2.26; P = .1) and 1.48 (95% CI, 1.01-2.17; P = .04), respectively. Adjustment for known risk factors of persistent S. aureus carriage did not substantially change the associations of both DEFB1 haplotypes and ß-defensin expression with S. aureus colonization. CONCLUSIONS: DEFB1 polymorphisms may promote persistent S. aureus colonization by altering ß-defensin expression in keratinocytes of human skin.