Adjusting microbiome profiles for differences in microbial load by spike-in bacteria

Frank Stämmler,Joachim Gläsner,Rainer Spang,André Gessner,Peter J Oefner,Daniela Weber,Ernst Holler,Andreas Hiergeist

doi:10.1186/s40168-016-0175-0

Frank Stämmler, Joachim Gläsner + Show 6 more

Open Access

https://doi.org/10.1186/s40168-016-0175-0

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Abstract

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.

Highlights

Next-generation 16S ribosomal RNA gene sequencing is widely used to determine the relative composition of the mammalian gut microbiomes
Choice of spike - in bacteria We used Salinibacter ruber (S. ruber, GenBank ID: CP000159), an extreme halophilic bacterium found in hypersaline environments [21], Rhizobium radiobacter (R. radiobacter, GenBank ID: ASXY01000000), a nonphytopathogenic member of the Biovar I group of Agrobacterium found in the soil and the plant rhizosphere [22], as well as the thermo-acidophilic, endospore forming soil bacterium Alicyclobacillus acidiphilus (A. acidiphilus, GenBank ID: PRJDB697) [23]
They do not exist in the gut microbiome under physiological conditions and are well distinguishable from the bacteria in the gut using 16S rRNA gene sequencing

Summary

Introduction

Next-generation 16S ribosomal RNA gene sequencing is widely used to determine the relative composition of the mammalian gut microbiomes. Generation sequencing of the 16S rRNA gene allows profiling of the bacterial and archaeal components of the gut microbiome at unprecedented precision and depth [8,9,10]. Computational tools such as QIIME [11] and mothur [12] cluster reads into operational taxonomic units (OTUs) [13], which may be jointed into taxonomic groups at the genus, family, order, class, and phylum level. If we further assume that the total number of bacteria or microbial load of in A is four times larger than in B, the 5 % in A account for twice as many bacteria as the 10 % in B, bringing the actual ratio to 2:1

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