Comparison of microbial signatures between paired faecal and rectal biopsy samples from healthy volunteers using next-generation sequencing and culturomics

Indrani Mukhopadhya* (Corresponding Author), Jennifer C. Martin, Sophie Shaw, Aileen J. McKinley, Silvia W. Gratz, Karen P. Scott* (Corresponding Author)

*Corresponding author for this work

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Faecal samples are frequently used to characterise the gut microbiota in health and disease, yet there is considerable debate about how representative faecal bacterial profiles are of the overall gut community. A particular concern is whether bacterial populations associated with the gut mucosa are properly represented in faecal samples, since these communities are considered critical in the aetiology of gastrointestinal diseases. In this study we compared the profiles of the faecal and mucosal microbiota from ten healthy volunteers using bacterial culturing (culturomics) and next-generation sequencing targeting the 16S ribosomal nucleic acid (rRNA) gene. Paired fresh rectal biopsies and faecal samples were processed under stringent anaerobic conditions to maintain the viability of the bacteria. Four different sample types were analysed: faecal (F), faecal homogenised (FHg), biopsy tissue (B) and biopsy wash (BW) samples.

There were no significant statistical differences in either bacterial richness or diversity between biopsy washes (BW) and faecal (F) or faecal homogenised (FHg) samples. Principal coordinates analysis of a Bray–Curtis distance matrix generated from sequence variant tables did not show distinct clustering between these samples (PERMANOVA; p = 0.972) but showed strong clustering of samples from individual donors. However, the rectal biopsy tissue (B) samples had a significantly altered bacterial signature with greater abundance of Proteobacteria and Acidobacteria compared to faecal (F) and faecal homogenised (FHg) samples. A total of 528 bacteria encompassing 92 distinct bacterial species were isolated and cultured from a subset of six volunteer samples (biopsy washes and faeces). This included isolation of 22 novel bacterial species. There was significant similarity between the bacterial species grown in anaerobic culture and those identified by 16S rRNA gene sequencing (Spearman correlation; rho = 0.548, p = 0.001).

This study showed that the bacterial profiles of paired faecal and rectal biopsy wash samples were very similar, validating the use of faecal samples as a convenient surrogate for rectal biopsy-associated microbiota. Anaerobic bacterial culture results showed similar taxonomic patterns to the amplicon sequence analysis disproving the dogma that culture-based methods do not reflect findings of molecular assessments of gut bacterial composition.

Original languageEnglish
Article number171
Number of pages17
Issue number1
Early online date14 Oct 2022
Publication statusPublished - 14 Oct 2022

Bibliographical note

We are indebted to our volunteers for providing the faecal and biopsy samples without which this study would not have been possible. We thank the members of the Rowett Gut Health research team for discussions and advice. The authors thank the Centre for Genome Enabled Biology and Medicine for Illumina sequencing and useful discussions.

This work was supported by funding from Probi AB (Grant Ref: RG14104). The Rowett Institute (University of Aberdeen) receives financial support from the Scottish Government Rural and Environmental Sciences and Analytical Services (RESAS).


  • Faecal microbiota
  • Mucosa-associated microbiota
  • Microbiome analysis
  • Next-generation sequencing
  • Anaerobic bacteria
  • Culture
  • SCFA


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