BackgroundNext-generation 16S ribosomal RNA gene sequencing is widely used to determine the relative composition of the mammalian gut microbiomes. However, in the absence of a reference, this does not reveal alterations in absolute abundance of specific operational taxonomic units if microbial loads vary across specimens.ResultsHere we suggest the spiking of exogenous bacteria into crude specimens to quantify ratios of absolute bacterial abundances. We use the 16S rDNA read counts of the spike-in bacteria to adjust the read counts of endogenous bacteria for changes in total microbial loads. Using a series of dilutions of pooled faecal samples from mice containing defined amounts of the spike-in bacteria Salinibacter ruber, Rhizobium radiobacter and Alicyclobacillus acidiphilus, we demonstrate that spike-in-based calibration to microbial loads allows accurate estimation of ratios of absolute endogenous bacteria abundances. Applied to stool specimens of patients undergoing allogeneic stem cell transplantation, we were able to determine changes in both relative and absolute abundances of various phyla, especially the genus Enterococcus, in response to antibiotic treatment and radio-chemotherapeutic conditioning.ConclusionExogenous spike-in bacteria in gut microbiome studies enable estimation of ratios of absolute OTU abundances, providing novel insights into the structure and the dynamics of intestinal microbiomes.Electronic supplementary materialThe online version of this article (doi:10.1186/s40168-016-0175-0) contains supplementary material, which is available to authorized users.

Key Points• Urinary 3-IS levels predict outcome after ASCT and are associated with antibiotics and NOD2/CARD15 variants.Indole, which is produced from L-tryptophan by commensal bacteria expressing tryptophanase, not only is an important intercellular signal in microbial communities, but also modulates mucosal barrier function and expression of pro-and anti-inflammatory genes by intestinal epithelial cells. Here, we hypothesized that decreased urinary excretion of 3-indoxyl sulfate (3-IS), the major conjugate of indole found in humans, may be a marker of gut microbiota disruption and increased risk of developing gastrointestinal (GI) graftversus-host-disease. Using liquid chromatography/tandem mass spectrometry, 3-IS was determined in urine specimens collected weekly within the first 28 days after allogeneic stem cell transplantation (ASCT) in 131 patients. Low 3-IS levels within the first 10 days after ASCT were associated with significantly higher transplant-related mortality (P 5 .017) and worse overall survival (P 5 .05) 1 year after ASCT. Least absolute shrinkage and selection operator regression models trained on lognormalized counts of 763 operational taxonomic units derived from next-generation sequencing of the hypervariable V3 region of the 16S ribosomal RNA gene showed members of the families of Lachnospiraceae and Ruminococcaceae of the class of Clostridia to be associated with high urinary 3-IS levels, whereas members of the class of Bacilli were associated with low 3-IS levels. Risk factors of early suppression of 3-IS levels were the type of GI decontamination (P 5 .01), early onset of antibiotic treatment (P 5 .001), and recipient NOD2/ CARD15 genotype (P 5 .04). In conclusion, our findings underscore the relevance of microbiota-derived indole and metabolites thereof in mucosal integrity and protection from inflammation. (Blood. 2015;126(14):1723-1728) IntroductionAllogeneic stem cell transplantation (ASCT) constitutes a potential curative therapy for various hematologic malignancies, bone marrow failure, and immune deficiency syndromes. However, this treatment is still associated with a high risk of mortality because of infectious complications and acute graft-versus-host disease (GVHD). A significant part of these severe complications originates from the gastrointestinal (GI) tract. 1The introduction of 16S ribosomal RNA (rRNA) sequencing has provided novel insights into the diversity and complexity of the gut ecosystem.2 Loss of a diverse composition of the microbiome has been associated with a variety of diseases including inflammatory bowel and autoimmune diseases. At least in part this may be because of the increasingly recognized role that commensal bacteria play in maintaining immunologic homeostasis and epithelial integrity and in exerting anti-inflammatory effects and intestinal tolerance by inducing regulatory T cells. 3,4 Recent studies have demonstrated an association between intestinal bacterial diversity in both mouse models and humans and outcome of ASCT.5-7 A significantly higher ...

Intestinal dysbiosis has been associated with acute gastrointestinal GvHD and poor outcome following allogeneic stem cell transplantation (ASCT). To assess the effect of a switch in 2012 from ciprofloxacin/metronidazole to rifaximin for gut decontamination on intestinal microbiota composition and ASCT outcome, we retrospectively analyzed 394 patients receiving ASCT from September 2008 through June 2015. In 131 and 90 patients, respectively, urinary 3-indoxyl sulfate levels and intestinal enterococcal load were measured before conditioning and weekly within the first 28 days after ASCT. The use of rifaximin correlated with lower enterococcal positivity (6.9 vs 21.9%, P = 0.05) and higher urinary 3-indoxyl sulfate concentrations (10.5 vs 4.6 μmoL/mmoL crea, P o0.001) after ASCT. Patients on rifaximin showed lower 1-year transplant-related mortality (P = 0.04) and higher overall survival (P = 0.008). Treatment of infectious complications with systemic antibiotics did not abrogate the beneficial effects of rifaximin on intestinal microbiota composition in the early course of ASCT and outcome. The data underscore the importance of maintaining a diverse population of symbiotic and mutualistic bacteria in the gut on ASCT outcome.

Motivation: In biomedicine, every molecular measurement is relative to a reference point, like a fixed aliquot of RNA extracted from a tissue, a defined number of blood cells, or a defined volume of biofluid. Reference points are often chosen for practical reasons. For example, we might want to assess the metabolome of a diseased organ but can only measure metabolites in blood or urine. In this case the observable data only indirectly reflects the disease state. The statistical implications of these discrepancies in reference points have not yet been discussed. Results: Here we show that reference point discrepancies compromise the performance of regression models like the LASSO. As an alternative, we suggest zero-sum regression for a reference point insensitive analysis. We show that zero-sum regression is superior to the LASSO in case of a poor choice of reference point both in simulations and in an application that integrates intestinal microbiome analysis with metabolomics. Moreover, we describe a novel coordinate descent based algorithm to fit zero-sum elastic nets. Availability: The R-package "zeroSum" can be downloaded at https://github.com/rehbergT/zeroSum. Moreover, we provide all R-scripts and data used to produce the results of this manuscript as supplementary material.

Hyperhomocysteinaemia occurs frequently in patients with TAO. It may play an important and nicotine-independent role in its pathogenesis.

Colour-coded sonography with a high-frequency transducer head probably provides reliable diagnosis of TA in patients with RP, even in the absence of clinical signs of vascular inflammation. It remains to be proven whether sonography without biopsy is reliable enough for the diagnosis and treatment of asymptomatic TA.