E-Poster with pre-recorded video presentation Lorne Infection and Immunity 2021

Identification of novel bacterial species associated with paediatric inflammatory bowel disease through direct mucosal sampling (#281)

Gemma L DAdamo 1 , Michelle Chonwerawong 1 , Jodee Gould 1 , Jamie Gearing 1 , Vanessa Marcelino 1 , Tamblyn Thomason 1 , Emily Rutten 1 , Paul Hertzog 1 2 , Edward Giles 1 3 , Samuel Forster 1 2
  1. Centre for Innate Immunity and Infectious Diseases, Hudson Institute , Clayton, Melbourne , Victoria, Australia
  2. Wellcome Trust Sanger Institute, Cambridge, UK
  3. Department of Paediatrics, Monash University, Melbourne , Victoria, Australia

Inflammatory bowel disease (IBD) is a chronic, incurable condition, comprised of Crohn’s Disease and Ulcerative Colitis. Over 20% of IBD is diagnosed in childhood; however, there are no universally effective treatments and clinical management is complicated by a plethora of side-effects. Until recently, investigation of the microbiota has been limited by an inability to culture the majority of intestinal bacteria. This has led to reliance on microbial sequencing to understand bacterial community structure; however, sequencing is limited by an inability to unambiguously identify causative bacteria.

Applying recently developed culturing techniques1, we investigated the microbiota in Paediatric Inflammatory Bowel Disease (PIBD) patients presenting for colonoscopy at Monash Children’s Hospital. Mucosal biopsy samples were collected across three intestinal regions (terminal Ileum, caecum and rectum) in order to characterise microbial signatures among a PIBD cohort. Concurrent bacterial culturing, shotgun metagenomic sequencing and molecular profiling was performed on 242 samples from this paediatric cohort. Bacterial culturing allowed 8347 purified isolates to be archived and identified using 16S rRNA sequencing, with whole genome sequencing performed on 192 of these isolates. Microbial analysis was complemented with examination of the molecular responses initiated at sample sites.
RNA-sequencing, combined with qPCR validation of 12 genes of interest (IL6, IL8, IL12, IL17A, IL17F, IL23, CXCL10, TNF-α, STAT3, TREM1, EPCAM, IFN-γ), provided a detailed understanding of the transcriptional responses initiated.   

Reference based metagenomic analysis2 allowed for correlation of the bacterial isolates present with disease states and molecular inflammatory profiles. The therapeutic candidates identified through the unification of these multi-omics approaches were functionally validated in-vitro via Caco2 cell culture models, to begin testing their causative potential. This work develops and applies a novel workflow for the identification, classification and functional validation of microbiome-based disease biomarkers and may be significant for a huge multitude of diseases, unrestricted to the gastrointestinal microbiome.

 

1          Browne, H. P. et al. Culturing of ‘unculturable’ human microbiota reveals novel taxa and extensive sporulation. Nature 533, 543-546 (2016).

2          Forster, S. C. et al. A human gut bacterial genome and culture collection for improved metagenomic analyses.  37, 186 (2019).