The Fecal Microbiome in Quiescent Crohn’s Disease with Persistent Gastrointestinal Symptoms Show Enrichment of Oral Microbes But Depletion of Butyrate and Indole Producers

  1. Jonathan Golob
  2. Krishna Rao
  3. Jeffrey Berinstein
  4. William Chey
  5. Chung Owyang
  6. Nobuhiko Kamada
  7. Peter Higgins
  8. Vincent Young
  9. Shrinivas Bishu
  10. Allen Lee

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Abstract

Background and Aims

Even in the absence of inflammation, persistent symptoms in Crohn’s disease (CD) are prevalent and negatively impact quality of life. We aimed to determine whether quiescent CD patients with persistent symptoms ( qCD+symptoms ) have changes in microbial structure and functional potential compared to those without symptoms ( qCD-symptoms ).

Methods

We performed a prospective multi-center observational study nested within the SPARC IBD study. CD patients were included if they had evidence of quiescent disease as defined by fecal calprotectin level < 150 mcg/g. Persistent symptoms were defined by the CD-PRO2 questionnaire. Active CD ( aCD ), diarrhea-predominant irritable bowel syndrome ( IBS-D ), and healthy controls ( HC ) were included as controls. Stool samples underwent whole genome shotgun metagenomic sequencing.

Results

A total of 424 patients were analyzed, including 39 qCD+symptoms, 274 qCD-symptoms, 21 aCD, 40 IBS-D, and 50 HC. Patients with qCD+symptoms had a less diverse microbiome, including significant reductions in Shannon diversity ( P <.001) and significant differences in microbial community structure ( P <.0001), compared with qCD-symptoms, IBS-D, and HC. Further, patients with qCD+symptoms showed significant enrichment of bacterial species that are normal inhabitants of the oral microbiome, including Klebsiella pneumoniae (q=.003) as well as depletion of important butyrate and indole producers, such as Eubacterium rectale (q=.001), Lachnospiraceae spp . (q<.0001), and Faecalibacterium prausnitzii (q<.0001), compared with qCD-symptoms. Finally, qCD+symptoms showed significant reductions in bacterial tnaA genes, which mediate tryptophan metabolism, as well as significant tnaA allelic variation, compared with qCD-symptoms.

Conclusion

The microbiome in patients with qCD+symptoms show significant changes in diversity, community profile, and composition compared with qCD-symptoms. Future studies will focus on the functional significance of these changes.

What You Need to Know

Background

Persistent symptoms in quiescent Crohn’s disease (CD) are prevalent and lead to worse outcomes. While changes in the microbial community have been implicated, the mechanisms by which altered microbiota may lead to qCD+symptoms remain unclear.

Findings

Quiescent CD patients with persistent symptoms demonstrated significant differences in microbial diversity and composition compared to those without persistent symptoms. Specifically, quiescent CD patients with persistent symptoms were enriched in bacterial species that are normal inhabitants of the oral microbiome but depleted in important butyrate and indole producers compared to those without persistent symptoms.

Implications for Patient Care

Alterations in the gut microbiome may be a potential mediator of persistent symptoms in quiescent CD. Future studies will determine whether targeting these microbial changes may improve symptoms in quiescent CD.

Article activity feed

  1. PREreview

    This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/8200142.

    This full PREreview is the result of a live-streamed preprint journal club organized and hosted by PREreview & the Crohn's & Colitis Foundation as the final module of the Open Reviewers workshop. We thank all participants who contributed to the discussion and made it possible for us to provide feedback to this preprint. 

    Summary

    In their study, Golob et. al., explore the mechanisms underlying persistent gastrointestinal symptoms in patients with quiescent Crohn's disease (qCD) by investigating alterations in the fecal microbiota, specifically focusing on microbial genes related to tryptophan metabolism. In a prospective multi-center observational study, they analyzed stool samples from individuals with qCD+ symptoms, qCD- symptoms, active Crohn's disease, diarrhea-predominant irritable bowel syndrome (IBS-D), and healthy controls (HC) for for microbial diversity, functional profile, and community structure via metagenomic sequencing. 

    The results of the study revealed significant differences in microbial diversity and community structure in individuals with qCD+ symptoms compared to those with qCD- symptoms, IBS-D patients, and healthy controls. Specifically, qCD+ symptom patients exhibited decreased Shannon diversity, indicating a less diverse microbiota composition. Furthermore, the microbial community structure of qCD+ symptom patients differed significantly from that of qCD- symptom patients, IBS-D patients, and healthy controls. These findings suggest that altered microbial communities, including changes in microbial genes involved in tryptophan metabolism, may play a role in the persistence of gastrointestinal symptoms in patients with qCD. Tryptophan is a key regulator of host-microbial interactions and is known to influence intestinal barrier function, visceral sensation, and brain-gut interactions. Understanding these mechanisms could pave the way for the development of targeted therapies and interventions to alleviate symptoms and improve the quality of life for patients with qCD. 

    Overall, the reviewers appreciated that the manuscript was very clearly written, with results presented in a way that the reader could easily understand. The method section and the supplementary information provide enough information for a thorough assessment of the research. As a result of the collaborative review discussion we identified a few concerns that we list below accompanied, when appropriate, by suggested ways to address them. 

    List of major issues and feedback

    • Several reviewers participating in the discussion noted that the sample sizes of qCD+ and qCD- symptoms patient groups were very different and wondered if the statistical methods used were robust to having such discrepancies. 

    • The conclusion that altered microbiota may contribute to symptoms in qCD could be strengthened by a more detailed discussion on the association between the two with regards to mechanisms. Although depletion of butyrate and indole producing microbes are well known to influence inflammation, there is not a clear link between these findings. It would be helpful to discuss potential reasons why oral microbiota shift to the gut, and why this would lead to symptoms in qCD patients.

    • "Racially and ethnically diverse SPARC IBD cohort" could be considered misleading since >90% of the patients are white and >97% are non-hispanic/latino in qCD +/- symptoms. Consider revising this statement and possibly provide an explanation for this lack of diversity (if any) or mention that further studies are needed to enhance diversity.

    List of minor issues and feedback

    • Alcohol abuse is related to infections of oral microbes in the gut. If these data are available in the cohort history, consider adding to the study and if a correlation is found it could influence the strength of the conclusions.

    • Figure 1C and D could be combined for clarity.

    • Figure 2 in general should include y-axis labels as qCD+symptoms/qCD-symptoms.

    • In functional metagenomic-related alterations in qCD+ symptoms subsection of results, the different pathways mentioned should be referenced.

    • The three different tryptophan metabolism pathways should be referenced. Ref 13 relates only to the metabolism of Tryptophan to indole.

    • Consider increasing font size in figures for clarity.

    We thank the authors for sharing their work as a preprint. We hope that our feedback above is helpful as the authors consider any revisions to the manuscript or future lines of work.

    Competing interests

    The author declares that they have no competing interests.

  2. Version published to 10.1101/2023.05.16.23290065v1 on medRxiv