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P852 Characterisation of Crohn’s disease mucosa-associated microbiota by a novel combination of microbe culture and metagenomic sequencing (MC-MGS): the ENIGMA study

E. M. Berendsen*1,2, E. C. Hoedt1,2,3, J-J. Teh1,2, J. Zhang4,5, F. Zhang4,5, Q. Liu4,5, A. L. Hamilton6, A. Wilson-O'Brien6, J. Ching4,5, J. J. Sung4,5, J. Yu4,5, S. C. Ng4,5,7, M. A. Kamm6, M. Morrison1,2

1The University of Queensland Diamantina Institute, Faculty of Medicine, Brisbane, Australia, 2Translational Research Institute, Brisbane, Australia, 3University College Cork, APC Microbiome Ireland, Cork, Ireland, 4The Chinese University of Hong Kong, Department of Medicine and Therapeutics, Hong Kong, Hong Kong, 5The Chinese University of Hong Kong, LKS Institute of Health Sciences, Institute of Digestive Disease and State Key Laboratory of Digestive Diseases, Hong Kong, Hong Kong, 6The University of Melbourne and St Vincent's Hospital, Melbourne, Department of Medicine and Department of Gastroenterology, Melbourne, Australia, 7The Chinese University of Hong Kong, Centre for Gut Microbiota Research, Hong Kong, Hong Kong

Background

Mucosal microbiota characterisation by shotgun metagenome sequencing is challenging due to limited microbial density and predominant host DNA. We have developed and evaluated microbe-culture metagenome sequencing (MC-MGS) to better characterise the mucosa-associated microbiota, in this case from 5 Crohn’s disease (CD) patients in the post-operative POCER study.1

Methods

Total DNA was extracted from biopsies stored in RNA later, and microbial DNA enrichment done with the NEBNext® protocol (New England Biolabs). In parallel, matched biopsies stored in anaerobically prepared glycerol buffer were used to produce microbial consortia with a habitat-simulating medium (37°C, 24 h). Total, enriched, and microbe culture DNA was sequenced using the Illumina NextSeq500 platform to produce 3 gbp of data per sample (as 150bp paired-end reads). The microbiota profiles for the respective samples was evaluated using GraftM2, and metagenome-assembled genomes produced using MetaBAT3.

Results

DNA sequence data directly isolated from biopsies, was >90% human and not microbial. Subtractive enrichment of microbial DNA resulted in a 2–8 fold increase in microbial read counts as assessed by GraftM and qPCR. In contrast, the MC-MGS datasets were exclusively microbial and represented 56–84% of the biodiversity captured from total biopsy DNA, and 75–92% of the biodiversity recovered after microbial DNA enrichment. MC-MGS samples also produced 16 metagenome assembled genomes representing diverse facultative and fastidious anaerobes (Table 1).

Table 1. Metagenome-assembled genomes produced from biopsy samples of CD patients by MC-MGS

Conclusion

The novel MC-MGS approach recovers much of the mucosa-associated microbiota, enabling production of both metagenome-assembled genomes and isolation of ‘new’ bacterial strains. MC-MGS provides a valuable new approach to provide a holistic, functional characterisation of the mucosa-associated microbiota in health and disease. This work is supported by The Leona M. and Harry B. Helmsley Charitable Trust.

Reference

1. De Cruz P, Kamm MA, Hamilton AL, et al. Crohn's disease management after intestinal resection: a randomised trial. Lancet. 2015;385:1406–17.

2. Boyd JA, Woodcroft J, Tyson GW. GraftM: a tool for scalable, phylogenetically informed classification of genes within metagenomes. Nucleic Acids Res 2018;46:e59-e59.

3. Kang DD, Froula J, Egan R, et al. MetaBAT, an efficient tool for accurately reconstructing single genomes from complex microbial communities. PeerJ 2015;3:e1165.