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Stool frequency is associated with gut microbiota composition
  1. Fatemeh Hadizadeh1,2,
  2. Susanna Walter3,
  3. Meriem Belheouane4,5,
  4. Ferdinando Bonfiglio1,
  5. Femke-Anouska Heinsen6,
  6. Anna Andreasson7,8,
  7. Lars Agreus7,
  8. Lars Engstrand9,10,
  9. John F Baines4,5,
  10. Joseph Rafter1,
  11. Andre Franke6,
  12. Mauro D'Amato1,11
  1. 1Department of Biosciences and Nutrition, Karolinska Institutet, Stockholm, Sweden
  2. 2School of Nutrition, Isfahan University of Medical Sciences, Isfahan, Iran
  3. 3Division of Gastroenterology, Institution of Clinical and Experimental Medicine, Linköping University, Linköping, Sweden
  4. 4Max Planck Institute for Evolutionary Biology, Plön, Germany
  5. 5Institute for Experimental Medicine, Christian-Albrechts-University of Kiel, Kiel, Germany
  6. 6Institute of Clinical Molecular Biology, Kiel University, Kiel, Germany
  7. 7Division for Family Medicine, Department of Neurobiology, Care Sciences and Society, Karolinska Institutet, Stockholm, Sweden
  8. 8Stress Research Institute, Stockholm University, Stockholm, Sweden
  9. 9Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden
  10. 10Clinical Genomics Facility, Science for Life Laboratory, Stockholm, Sweden
  11. 11BioCruces Health Research Institute and IKERBASQUE, Basque Foundation for Science, Bilbao, Spain
  1. Correspondence to Professor Mauro D'Amato, BioCruces Health Research Institute, Cruces University Hospital, Barakaldo 48903, Spain; mauro.damato{at}, mauro.damato{at}

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We read with great interest the two recent communications by Vandeputte et al1 and Tigchelaar et al,2 which reported an association between stool consistency and gut microbiota composition. Both studies focused on stool consistency, based on the Bristol Stool Scale (BSS) as the main trait, due to its reported correlation with colonic transit time (CTT) that is of clinical relevance for several GI conditions. However, stool frequency, another feature of human bowel function that is easy to measure and record, has also been shown to correlate with CTT,3 ,4 although to a lesser extent than stool form, and has not yet been investigated in relation to microbiota composition.

We examined the association between gut microbiota and stool frequency in the Population-based Colonoscopy study, a general population-based cohort from Stockholm, Sweden, previously described in detail.5 Sixty-nine individuals (21 males and 48 females, aged 55.6±10.33) with available frozen faecal samples and daily recordings of defaecation patterns (number of bowel movement and associated BSS scores from 1-week diaries) over the same period (12.2±16.8 days) were included. The mean daily stool frequency and BSS score of the participants were 1.38 (range 0.57–3) and 3.91 (range 1.5–6), respectively, and these two measures correlated significantly (Spearman p=0.007, r=0.32). Stool bacterial DNA was extracted using easyMAG NucliSens kits (Biomerieux), and the V1–V2 hypervariable region of the 16S rRNA gene was amplified using the 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 338R (5′-TGCTGCCTCCCGTAGGAGT-3′) primers prior to sequencing on the Illumina MiSeq platform. Data processing was performed using Mothur ( on a normalised data set of 10 000 reads per sample, which was used for taxonomic classification and operational taxonomic unit assignment. α-diversity was calculated using the vegan and phyloseq R packages.

Similar to Vandeputte et al, we detected a negative correlation between stool consistency and Observed Species and Chao1 α-diversity indices. Faith's phylogenetic diversity and Shannon–Weaver entropy also displayed similar negative correlations (figure 1A). However, stronger and more significant correlations were observed for daily stool frequency (figure 1B). Similar results were obtained upon exclusion of individuals with IBS (n=9), identified based on consensus Rome criteria from questionnaire data.

Figure 1

Correlation between α-diversity indices, stool consistency and stool frequency. Observed species (Observed sp.), Chao1, Faith's phylogenetic diversity (PD) and Shannon–Weaver (Shannon) indices are plotted on the same graph, omitting relative scale units for simplicity, together with their respective statistics. (A) Mean daily stool consistency scores based on the Bristol Stool Scale. (B) Stool frequency expressed as mean nr of bowel movements per day.

At individual taxonomic levels, the ratio of Bacteroides:Firmicutes and Bacteroides:unclassified_Ruminococcaceae displayed significant correlations with stool frequency (Spearman; p=0.017; r=0.29 and p=0.0001; r=0.45, respectively), but not with consistency (p>0.05). Similar to Tigchelaar et al, we could not replicate Vandeputte et al1 BSS associations with specific genera, although some of these associations correlated with stool frequency in our samples (q=0.015; r=0.35 for Bacteroides and q=0.021; r=−032 for Oxalobacter). When testing the 10 most abundant genera in our dataset, the highest (negative) correlation with mean stool frequency was observed for unclassified_Ruminococcaceae (q=0.002, r=−0.44). Alistipes and Oscillobacter were also negatively associated (respective q=0.049; r=−0.27 and q=0.0021; r=−0.42). Similar results were observed for stool consistency (data not shown). Finally, in line with Tigchelaar's findings for BSS, we did not detect gender-specific correlations between stool frequency and microbiota composition. Our results add to accumulating evidence linking intestinal transit time to human gut microbiota composition, which warrants larger scale surveys of the potential effect that more frequent colonic content renewal may have on the intestinal ecosystem, ultimately determining bowel faecal content and associated microbiota composition.

In summary, despite inherent differences in individual population properties, stool collection methods and analysis protocols, this and previous reports provide compelling evidence of a correlation between human defaecation patterns (stool consistency and frequency) and several indicators of faecal microbiota composition. Although (eventual) causal relationships are still poorly understood, these findings are important because they contribute to describe gut microbiota and investigate its role in health and disease.


We would like to thank the DFG Research Training Group RTG 1743/1—Genes, Environment and Inflammation (Kiel University).



  • Contributors MD had the original idea, and designed the study together with AF; SW, AA, LE and LA collected material, characterised the study subjects and acquired data; F-AH, AF and JFB performed microbiota sequencing; data analysis and interpretation were done by FH, MB, FB, JR and MD. FH, MD and JR drafted the manuscript with critical revision from all authors.

  • Funding Supported by funds from the Swedish Research Council (Vetenskapsradet) to MD; European Union Seventh Framework Programme (FP7/2007-2013, grant number 262055, ESGI) to MD and AF; Iranian ministry of health and medical education to FH.

  • Competing interests None declared.

  • Ethics approval Karolinska Institutet, Stockholm, Sweden.

  • Provenance and peer review Not commissioned; internally peer reviewed.