Novel strain-level resolution of Crohn\u2019s disease mucosa-associated microbiota via an ex vivo combination of microbe culture and metagenomic sequencing

J J Teh,J Yu,F Zhang,A L Hamilton,S C Ng,J Zhang,Q Liu,M Morrison,E M Berendsen,S Kang,E C Hoedt,A Wilson-O’Brien,J Ching,J J Y Sung,M A Kamm

doi:10.1038/s41396-021-00991-1

Abstract

The mucosa-associated microbiota is widely recognized as a potential trigger for Crohn’s disease pathophysiology but remains largely uncharacterised beyond its taxonomic composition. Unlike stool microbiota, the functional characterisation of these communities using current DNA/RNA sequencing approaches remains constrained by the relatively small microbial density on tissue, and the overwhelming amount of human DNA recovered during sample preparation. Here, we have used a novel ex vivo approach that combines microbe culture from anaerobically preserved tissue with metagenome sequencing (MC-MGS) to reveal patient-specific and strain-level differences among these communities in post-operative Crohn’s disease patients. The 16 S rRNA gene amplicon profiles showed these cultures provide a representative and holistic representation of the mucosa-associated microbiota, and MC-MGS produced both high quality metagenome-assembled genomes of recovered novel bacterial lineages. The MC-MGS approach also produced a strain-level resolution of key Enterobacteriacea and their associated virulence factors and revealed that urease activity underpins a key and diverse metabolic guild in these communities, which was confirmed by culture-based studies with axenic cultures. Collectively, these findings using MC-MGS show that the Crohn’s disease mucosa-associated microbiota possesses taxonomic and functional attributes that are highly individualistic, borne at least in part by novel bacterial lineages not readily isolated or characterised from stool samples using current sequencing approaches.

Highlights

A step advance in human microbiome research was catalysed by 16 S rRNA gene amplicon sequencing
These differences had limited effects on the 16 S rRNA gene amplicon profiles produced from the DNA preparations of matched tissue samples, showing that the patient-specific community structure was at least retained, and even augmented by the MC-metagenomic sequencing (MGS) approach
In their study of treatment naïve pediatric Crohn’s disease (CD) subjects, Gevers et al [1] reported that the 16 S rRNA gene amplicon profiles from the mucosal biopsies was not greatly different between collection sites from the same individual, which is consistent with our findings here, using either DNA extracted directly from biopsy tissue or via microbial culture using tissue from 3 different sites

Summary

Introduction

A step advance in human microbiome research was catalysed by 16 S rRNA gene amplicon sequencing. For the last two decades, this approach has produced a characterisation of the taxonomic profiles of stool and more recently, the. Mucosa-associated microbiota (MAM) in health and disease [1] This is exemplified by the large number of case-control and observational studies of the stool and MAM from newonset and chronic Crohn’s disease (CD) patients, which invariably show a decrease in bacterial diversity coincident with disease and in comparison to non-CD ‘healthy’ subjects. Recent ‘multi-omics’ approaches have provided further insights beyond 16 S rRNA gene-based profiling [5, 6], much still needs to be learned about the microbial biology and functional implications of the MAM and in particular, its involvement with respect to disease location, severity, therapeutic modality (e.g., exclusive enteral nutrition or biologic drugs) and patient immune status [7]

Methods

Results

Conclusion