Colonic Microbiota Signatures across Five Northern European Countries

The composition of the colonic microbiota of 91 northern Europeanswas characterized by fluorescent in situ hybridization using18 phylogenetic probes. On average 75% of the bacteria wereidentified, and large interindividual variations were observed.Clostridium coccoides and Clostridium leptum were the dominantgroups (28.0% and 25.2%), followed by the Bacteroides (8.5%).According to principal component analysis, no significant groupingwith respect to geographic origin, age, or gender was observed.

INTRODUCTION

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Introduction

For the past 10 years, the progress made in molecular technologieshas given rise to new ways to explore the human colonic microbiota.Investigations based on 16S rRNA sequences have revealed thepresence of hundreds of molecular species, the majority uncultivatedand/or not yet cultivated, unique to their host and with fewspecies shared between two individuals (5, 10, 13). Fluorescentin situ hybridization (FISH) (1) of the 16S rRNA has shown thatthe species diversity comprised less than 20 dominant phylogeneticgroups (2-4, 7). However, the molecular analysis has so farbeen restricted to limited cohorts of individuals, recruitedwithin a single geographic region or country (3, 4, 7, 8, 14).In this study, we characterized the fecal microbiota of 91 individualsfrom five northern European countries to provide a large-scalemolecular analysis of the normal colonic microbiota in healthyhumans. Multivariate data analysis was performed in order toseek a possible link between the composition of the fecal microbiotaand age, gender, or geographic origin parameters.

Composition of the fecal microbiota assessed by FISH combined with flow cytometry.

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Composition of the fecal...

Fecal samples were collected from 91 healthy humans aged between7 and 52 years. These donors were from France (n = 21), Denmark(n = 20), Germany (n = 20), The Netherlands (n = 20), and theUnited Kingdom (n = 10) and had a nonrestricted Western Europeandiet. None had a history of digestive pathology nor had receivedantibiotic treatment within 6 months before the study. The fixationprocedure was adapted from the study of Schwiertz et al. (9).A set of 18 group- and species-specific probes defined previously(4) was applied to describe the fecal microbiota composition.Fluorescent in situ hybridization combined with flow cytometrywas performed as described previously (4, 7, 8). The proportionsof cells that hybridized with the phylogenetic probes relativeto the total bacteria are presented in Table 1. For the fiveEuropean countries, the highest percentages were detected withthe Clostridium coccoides-Eubacterium rectale probe (Erec 482)and the Clostridium leptum probe (Clep 866), representing 28.0%and 25.2% of total bacteria, respectively. These groups codominatedin France, the United Kingdom, and Germany, compared to Denmarkand The Netherlands, where the Clostridium coccoides-Eubacteriumrectale group predominated. Within the Clostridium leptum subgroupthe Faecalibacterium prausnitzii cluster (Fprau 645) was themost abundant in all five countries, followed by the Ruminococcusbromii and related species (Rbro 730). The Bacteroides group(Bac 303) came third and accounted for 8.5% of bacterial cells.Members of the Atopobium (Ato 291) and the Bifidobacterium (Bif164) groups represented 3.1% and 4.4% of the cells, respectively.The composition of the fecal microbiota obtained in this studycorrelated well with the composition observed in previous studiesusing FISH in combination with either microscopy and image analysisor flow cytometry (2-4, 7, 8, 11, 14). When the relative proportionsof bacterial cells obtained with the panel of 12 nonoverlappingphylogenetic probes were added, a mean of 75.7% (ranging from66.4% in France to 82.3% in The Netherlands) was calculated.Consequently, more than 24% of the human fecal microbiota stillremained unidentified. Compared to the study of Rigottier-Goiset al. (7), the number of phylogenetic probes in the set wasincreased from 6 to 18, and the proportion of untargeted bacteriawas thereby reduced from 49 to 24%. Nevertheless, efforts tocharacterize this phylogenetic gap must be continued. One approachwill be to use the potential of flow cytometric cell sortingto select bacterial cells of interest (12), combined with molecularinventory to characterize the molecular species and therebyto design new probes.

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TABLE 1. Proportions of the C. coccoides-E. rectale (Erec 482) group, the C. leptum (Clep 866, Cvir 1414, Edes 635) subgroup, and the Bacteroides (Bac 303), Atopobium (Ato 291), Bifidobacterium (Bif 164), Lactobacillus-Enterococcus (Lab 158), Eubacterium cylindroides (Ecyl 387), Veillonella (Veil 223), Streptococcus (Strc 493), and Enterobacteriaceae (Enter 1432) groups in healthy adults assessed with a combination of FISH and flow cytometry^c

PCA of bacterial composition with biometric and geographic data.

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Pca of bacterial composition...

All proportions of a given group or species as a function ofgeographic origin, age and gender were organized and subjectedto principal component analysis (PCA) using the Unscramblerprogram version 7.6 (Cano AS, Trondheim, Norway). Altogether91 individual fecal samples were analyzed in duplicate, givinga total of 182 data points in the PCA model. The generated outputof the PCA revealed that not very much of the variation couldbe described by individual principal components. These valuessuggested that no single or smaller groups of variables couldassist in simplifying the interpretation of the data set. Consequently,results were evaluated for all six principal components, PC1to PC6. The principal component analysis revealed large interindividualvariations, showing that each individual harbors a specificcolonic microbiota or signature in terms of bacterial composition.In our study, samples from one Danish family with two childrenwere analyzed, and results showed that all members of the familyare different. According to the PCA, no significant groupingof samples with respect to age or gender was observed, no matterwhether the analysis was done for the entire cohort of samplesor in a country-specific manner. No grouping of samples withrespect to geographic origin was observed when the five countrieswere considered. However, as shown in Fig. 1 there is a tendencyfor the Dutch samples to accumulate in the right sector, whilethe French samples pull more in the left direction of the coordinatesystem, suggesting a weak difference between the French andDutch samples. On the basis of the distribution of major dominantgroups, the colonic microbiota of healthy humans thus appearscomparable throughout the northern European countries investigated.

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FIG. 1. Score plot of the PCA analysis with focus on groupings of samples with respect to geographic origin using principal components PC1 and PC2. The score plot shows the clustering of different samples, and samples that lie close to each other are similar with respect to the measured variables. Conversely, samples that lie diametrically opposite to each other will be very different from each other. Crosses, French; open squares, German; open circles, Danish; filled squares, English; filled triangles, Dutch.

Nutrigenomics and nutrigenetics are two emerging fields of nutritionresearch, which seek to study the effects of nutrition on healthand disease by examining the influence of dietary signatures(6). Microbial ecology of the gastrointestinal tract plays akey role in nutrition research into the relation between colonicmicrobiota signatures and age, diet, dietary allergies, or diseases.Molecular approaches based on 16S rRNA sequences provide toolsfor understanding this complex microbiota in relation to dietand human health. In this study, we showed that the colonicmicrobiota of healthy humans is comparable throughout the northof Europe. Targeting other ethnic populations will enable thecomposition of the human colonic microbiota around the worldto be established. Performing such a study on infants and theelderly will facilitate the assessment of how the human digestivemicrobiota evolves with age. This baseline study on healthyhumans will also contribute to eventually identifying changesin the composition of the intestinal microbiota in patientssuffering from inflammatory bowel diseases.

ACKNOWLEDGMENTS

We thank Erwin Zoetendal, Sylvie Rabot, Gérard Corthier,and Maria J. Flores for critically reading the manuscript.

This study was carried out with financial support from the Commissionof the European Communities, specifically the RTD programs Qualityof Life and Management of Living Resources, QLK1-2000-108, MicrobeDiagnostics, coordinated by Michaël Blaut (Dife, Germany).The study does not necessarily reflect the Commission's viewsand in no way anticipates its future policy in this area.

FOOTNOTES

* Corresponding author. Mailing address: INRA, UEPSD, bât 405, Domaine de Vilvert, 78352 Jouy en Josas cedex, France. Phone: 33 (1) 34 65 23 08. Fax: 33 (1) 34 65 24 92. E-mail: lionel.rigottier-gois{at}jouy.inra.fr.

REFERENCES

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