Kyle Bittinger

Optimizing methods and dodging pitfalls in microbiome research

Research on the human microbiome has yielded numerous insights into health and disease, but also has resulted in a wealth of experimental artifacts. Here, we present suggestions for optimizing experimental design and avoiding known pitfalls, organized in the typical order in which studies are carried out. We first review best practices in experimental design and introduce common confounders such as age, diet, antibiotic use, pet ownership, longitudinal instability, and microbial sharing during cohousing in animal studies. Typically, samples will need to be stored, so we provide data on best practices for several sample types. We then discuss design and analysis of positive and negative controls, which should always be run with experimental samples. We introduce a convenient set of non-biological DNA sequences that can be useful as positive controls for high-volume analysis. Careful analysis of negative and positive controls is particularly important in studies of samples with low microbial biomass, where contamination can comprise most or all of a sample. Lastly, we summarize approaches to enhancing experimental robustness by careful control of multiple comparisons and to comparing discovery and validation cohorts. We hope the experimental tactics summarized here will help researchers in this exciting field advance their studies efficiently while avoiding errors.

Diabetes Enhances IL-17 Expression and Alters the Oral Microbiome to Increase Its Pathogenicity

Diabetes is a risk factor for periodontitis, an inflammatory bone disorder and the greatest cause of tooth loss in adults. Diabetes has a significant impact on the gut microbiota; however, studies in the oral cavity have been inconclusive. By 16S rRNA sequencing, we show here that diabetes causes a shift in oral bacterial composition and, by transfer to germ-free mice, that the oral microbiota of diabetic mice is more pathogenic. Furthermore, treatment with IL-17 antibody decreases the pathogenicity of the oral microbiota in diabetic mice; when transferred to recipient germ-free mice, oral microbiota from IL-17-treated donors induced reduced neutrophil recruitment, reduced IL-6 and RANKL, and less bone resorption. Thus, diabetes-enhanced IL-17 alters the oral microbiota and renders it more pathogenic. Our findings provide a mechanistic basis to better understand how diabetes can increase the risk and severity of tooth loss.

Protective major histocompatibility complex allele prevents type 1 diabetes by shaping the intestinal microbiota early in ontogeny

Significance This report brings a new perspective on the decades-old question of how MHC and HLA complexes can potently protect against a variety of autoimmune diseases, including type 1 diabetes. We demonstrated that protection by the MHC-II Eα:Eβ complex operated via modulation of the composition of the intestinal microbiota during a critical early window of ontogeny, associated with modification of the local immune system. These findings prompt a model of HLA/MHC-mediated protection from autoimmunity, and raise the question of whether disease-protective alleles in other human autoimmune diseases or models thereof might operate by a similar mechanism. They also argue that treating infants and pregnant mothers with antibiotics should be minimized. Certain MHC-II or HLA-D alleles dominantly protect from particular autoimmune diseases. For example, expression of the MHC-II Eα:Eβ complex potently protects nonobese diabetic (NOD) mice, which normally lack this isotype, from spontaneous development of type 1 diabetes. However, the underlying mechanisms remain debated. We investigated MHC-II–mediated protection from type 1 diabetes using a previously reported NOD mouse line expressing an Eα transgene and, thereby, the Eα:Eβ complex. Eα16/NOD females vertically protected their NOD offspring from diabetes and insulitis, an effect that was dependent on the intestinal microbiota; moreover, they developed autoimmunity when treated with certain antibiotics or raised in a germ-free environment. Genomic and proteomic analyses revealed NOD and Eα16/NOD mice to host mild but significant differences in the intestinal microbiotas during a critical early window of ontogeny, and transfer of cecal contents from the latter to the former suppressed insulitis. Thus, protection from autoimmunity afforded by particular MHC/HLA alleles can operate via intestinal microbes, highlighting potentially important societal implications of treating infants, or even just their pregnant mothers, with antibiotics.

A role for bacterial urease in gut dysbiosis and Crohn’s disease

Bacterial urease activity of the gut microbiota alters nitrogen flux, leading to gut dysbiosis and worsening of colitis in mice. Nitrogen flux and gut dysbiosis Ni et al. used shotgun metagenomic and metabolomic analysis of fecal samples from pediatric patients with Crohn’s disease. They demonstrated an association between disease severity, gut dysbiosis, and free amino acids. A heavy isotope–labeled nitrogen flux analysis showed that bacterial urease activity led to the transfer of host-derived nitrogen to the gut microbiota, boosting amino acid synthesis. Inoculation of a murine host with Escherichia coli engineered to express urease led to dysbiosis associated with worsened immune-mediated colitis and increased amino acid production. A potential role for nitrogen flux in the development of gut dysbiosis suggests that urease may be a potential target for developing treatments for inflammatory bowel diseases. Gut dysbiosis during inflammatory bowel disease involves alterations in the gut microbiota associated with inflammation of the host gut. We used a combination of shotgun metagenomic sequencing and metabolomics to analyze fecal samples from pediatric patients with Crohn’s disease and found an association between disease severity, gut dysbiosis, and bacterial production of free amino acids. Nitrogen flux studies using 15N in mice showed that activity of bacterial urease, an enzyme that releases ammonia by hydrolysis of host urea, led to the transfer of murine host-derived nitrogen to the gut microbiota where it was used for amino acid synthesis. Inoculation of a conventional murine host (pretreated with antibiotics and polyethylene glycol) with commensal Escherichia coli engineered to express urease led to dysbiosis of the gut microbiota, resulting in a predominance of Proteobacteria species. This was associated with a worsening of immune-mediated colitis in these animals. A potential role for altered urease expression and nitrogen flux in the development of gut dysbiosis suggests that bacterial urease may be a potential therapeutic target for inflammatory bowel diseases.

Microbes vs. chemistry in the origin of the anaerobic gut lumen

Significance It is generally thought that the gut microbes consume oxygen and maintain the lumen in a deeply anaerobic state. However, we found that the gut of germ-free mice is also deeply anaerobic, suggesting that there exist other mechanisms responsible for oxygen consumption in addition to the microbes’ respiration. These mechanisms comprise oxidative reactions, such as the oxidation of lipids and other organic substrates. Both the microbiota and the oxidative chemistry regulate luminal oxygen levels that in turn influence the composition of the microbial communities throughout the intestinal tract. The succession from aerobic and facultative anaerobic bacteria to obligate anaerobes in the infant gut along with the differences between the compositions of the mucosally adherent vs. luminal microbiota suggests that the gut microbes consume oxygen, which diffuses into the lumen from the intestinal tissue, maintaining the lumen in a deeply anaerobic state. Remarkably, measurements of luminal oxygen levels show nearly identical pO2 (partial pressure of oxygen) profiles in conventional and germ-free mice, pointing to the existence of oxygen consumption mechanisms other than microbial respiration. In vitro experiments confirmed that the luminal contents of germ-free mice are able to chemically consume oxygen (e.g., via lipid oxidation reactions), although at rates significantly lower than those observed in the case of conventionally housed mice. For conventional mice, we also show that the taxonomic composition of the gut microbiota adherent to the gut mucosa and in the lumen throughout the length of the gut correlates with oxygen levels. At the same time, an increase in the biomass of the gut microbiota provides an explanation for the reduction of luminal oxygen in the distal vs. proximal gut. These results demonstrate how oxygen from the mammalian host is used by the gut microbiota, while both the microbes and the oxidative chemical reactions regulate luminal oxygen levels, shaping the composition of the microbial community throughout different regions of the gut.

Allometry and Ecology of the Bilaterian Gut Microbiome.

ABSTRACT Classical ecology provides principles for construction and function of biological communities, but to what extent these apply to the animal-associated microbiota is just beginning to be assessed. Here, we investigated the influence of several well-known ecological principles on animal-associated microbiota by characterizing gut microbial specimens from bilaterally symmetrical animals (Bilateria) ranging from flies to whales. A rigorously vetted sample set containing 265 specimens from 64 species was assembled. Bacterial lineages were characterized by 16S rRNA gene sequencing. Previously published samples were also compared, allowing analysis of over 1,098 samples in total. A restricted number of bacterial phyla was found to account for the great majority of gut colonists. Gut microbial composition was associated with host phylogeny and diet. We identified numerous gut bacterial 16S rRNA gene sequences that diverged deeply from previously studied taxa, identifying opportunities to discover new bacterial types. The number of bacterial lineages per gut sample was positively associated with animal mass, paralleling known species-area relationships from island biogeography and implicating body size as a determinant of community stability and niche complexity. Samples from larger animals harbored greater numbers of anaerobic communities, specifying a mechanism for generating more-complex microbial environments. Predictions for species/abundance relationships from models of neutral colonization did not match the data set, pointing to alternative mechanisms such as selection of specific colonists by environmental niche. Taken together, the data suggest that niche complexity increases with gut size and that niche selection forces dominate gut community construction. IMPORTANCE The intestinal microbiome of animals is essential for health, contributing to digestion of foods, proper immune development, inhibition of pathogen colonization, and catabolism of xenobiotic compounds. How these communities assemble and persist is just beginning to be investigated. Here we interrogated a set of gut samples from a wide range of animals to investigate the roles of selection and random processes in microbial community construction. We show that the numbers of bacterial species increased with the weight of host organisms, paralleling findings from studies of island biogeography. Communities in larger organisms tended to be more anaerobic, suggesting one mechanism for niche diversification. Nonselective processes enable specific predictions for community structure, but our samples did not match the predictions of the neutral model. Thus, these findings highlight the importance of niche selection in community construction and suggest mechanisms of niche diversification. The intestinal microbiome of animals is essential for health, contributing to digestion of foods, proper immune development, inhibition of pathogen colonization, and catabolism of xenobiotic compounds. How these communities assemble and persist is just beginning to be investigated. Here we interrogated a set of gut samples from a wide range of animals to investigate the roles of selection and random processes in microbial community construction. We show that the numbers of bacterial species increased with the weight of host organisms, paralleling findings from studies of island biogeography. Communities in larger organisms tended to be more anaerobic, suggesting one mechanism for niche diversification. Nonselective processes enable specific predictions for community structure, but our samples did not match the predictions of the neutral model. Thus, these findings highlight the importance of niche selection in community construction and suggest mechanisms of niche diversification.

Characterisation of the nasal microbiota in granulomatosis with polyangiitis

Objectives Prior studies have suggested a potential link between nasal microbes and granulomatosis with polyangiitis (GPA; Wegener’s), but these studies relied on culture-dependent methods. This study comprehensively examined the entire community of nasal microbiota (bacteria and fungi) in participants with GPA compared with healthy controls using deep sequencing methods. Methods 16S rRNA and internal transcribed spacer gene sequencing were performed on nasal microbial DNA isolated from nasal swabs of 60 participants with GPA and 41 healthy controls. Alpha and beta diversity were assessed as well as the relative abundance of the most abundant bacterial and fungal taxa. The effects of covariates including disease activity and immunosuppressive therapies on microbial composition were evaluated. Results Compared with controls, participants with GPA had a significantly different microbial composition (weighted UniFrac p=0.04) and lower relative abundance of Propionibacterium acnes and Staphylococcus epidermidis (for both, false discovery rate-corrected p=0.02). Disease activity in GPA was associated with a lower abundance of fungal order Malasseziales compared with participants with GPA in remission (p=0.04) and controls (p=0.01). Use of non-glucocorticoid immunosuppressive therapy was associated with ‘healthy’ nasal microbiota while participants with GPA who were off immunosuppressive therapy had more dysbiosis (weighted UniFrac p=0.01). No difference in the relative abundance of Staphylococcus aureus was observed between GPA and controls. Conclusions GPA is associated with an altered nasal microbial composition, at both the bacterial and fungal levels. Use of immunosuppressive therapies and disease remission are associated with healthy microbial communities.

Sunbeam: a pipeline for next-generation metagenomic sequencing experiments

Background: Metagenomic sequencing experiments require a number of preprocessing and analytical steps to interpret the microbial and genetic composition of biological samples. Such steps include quality control, adapter trimming, host decontamination, metagenomic classification, read assembly, and alignment to reference genomes. Results: We present here an extensible and modular pipeline called Sunbeam that performs these steps in a consistent and reproducible way. It features a one-step installation and novel tools for eliminating artifactual sequences that may interfere with downstream analysis including Komplexity, a novel software tool to eliminate potentially problematic, low-complexity nucleotide sequences from metagenomic data. Another unique component of the Sunbeam pipeline is an easy-to-use extension framework that enables users to add custom processing or analysis steps directly to the Sunbeam workflow. Conclusions: Sunbeam provides a foundation to build more in-depth analyses and to enable comparisons between disparate sequencing experiments by standardizing routine pre-processing and analytical steps. Sunbeam is written in Python using the Snakemake workflow management software and is freely available at github.com/sunbeam-labs/sunbeam under the GPLv3.

FXR-Dependent Modulation of the Human Small Intestinal Microbiome by the Bile Acid Derivative Obeticholic Acid

BACKGROUND & AIMSnIntestinal bacteria can modify the composition of bile acids and bile acids, which are regulated by the farnesoid X receptor, affect the survival and growth of gut bacteria. We studied the effects of obeticholic acid (OCA), a bile acid analogue and farnesoid X receptor agonist, on the intestinal microbiomes of humans and mice.nnnMETHODSnWe performed a phase I study in 24 healthy volunteers given OCA (5, 10, or 25 mg/d for 17 days). Fecal and plasma specimens were collected at baseline (day 0) and on days 17 (end of dosing) and 37 (end of study). The fecal specimens were analyzed by shotgun meta-genomic sequencing. A Uniref90 high-stringency genomic analysis was used to assign specific genes to the taxonomic signature of bacteria whose abundance was associated with OCA. Male C57BL/6 mice were gavage fed daily with water, vehicle, or OCA (10 mg/kg) for 2 weeks. Small intestine luminal contents were collected by flushing with saline and fecal pellets were collected at baseline and day 14. Mouse samples were analyzed by 16S-tagged sequencing. Culture experiments were performed to determine the taxonomic-specific effects of bile acids and OCA on bacterial growth.nnnRESULTSnSuppression of endogenous bile acid synthesis by OCA in subjects led to a reversible induction of gram-positive bacteria that are found in the small intestine and are components of the diet and oral microbiota. We found that bile acids decreased proliferation of these bacteria in minimum inhibitory concentration assays. In these organisms, there was an increase in the representation of microbial genomic pathways involved in DNA synthesis and amino acid metabolism with OCA treatment of subjects. Consistent with these findings, mice fed OCA had lower endogenous bile acid levels and an increased proportion of Firmicutes, specifically in the small intestine, compared with mice fed water or vehicle.nnnCONCLUSIONSnIn studying the effects of OCA in humans and mice, we found evidence for interactions between bile acids and features of the small intestinal microbiome. These findings indicate that farnesoid X receptor activation alters the intestinal microbiota and could provide opportunities for microbiome biomarker discovery or new approaches to engineering the human microbiome. ClinicalTrials.gov, NCT01933503.