Fluorescent hybridisation combined with flow cytometry and hybridisation of total RNA to analyse the composition of microbial communities in human faeces using 16S rRNA probes

Authors

  • Lionel Rigottier-Gois,

    Corresponding author
    1. Institut National de la Recherche Agronomique, Unité d'Ecologie et de Physiologie du Système Digestif, bât. 405, Domaine de Vilvert, 78352 Jouy en Josas Cedex, France
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  • Anne-Gaëlle Le Bourhis,

    1. Institut National de la Recherche Agronomique, Unité d'Ecologie et de Physiologie du Système Digestif, bât. 405, Domaine de Vilvert, 78352 Jouy en Josas Cedex, France
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  • Geneviève Gramet,

    1. Institut National de la Recherche Agronomique, Unité d'Ecologie et de Physiologie du Système Digestif, bât. 405, Domaine de Vilvert, 78352 Jouy en Josas Cedex, France
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  • Violaine Rochet,

    1. Institut National de la Recherche Agronomique, Unité d'Ecologie et de Physiologie du Système Digestif, bât. 405, Domaine de Vilvert, 78352 Jouy en Josas Cedex, France
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  • Joël Doré

    1. Institut National de la Recherche Agronomique, Unité d'Ecologie et de Physiologie du Système Digestif, bât. 405, Domaine de Vilvert, 78352 Jouy en Josas Cedex, France
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*Corresponding author. Tel.: +33 (1) 34 65 23 08; Fax: +33 (1) 34 65 24 92. gois@diamant.jouy.inra.fr

Abstract

To determine the structure of human faecal microbiota, faecal samples from 23 healthy individuals were analysed with a similar set of probes targeting six phylogenetic groups using rRNA dot-blot hybridisation and whole cell fluorescent in situ hybridisation (FISH) combined with flow cytometry. When microbiota compositions derived by each method were compared, the results were not statistically different for Clostridium coccoides, Fusobacterium prausnitzii, Bifidobacterium spp. and Enterobacteria. Conversely, the proportions were significantly different for Bacteroides and Atopobium (P<0.05). The metabolic state of these bacteria within the colon could explain the discrepancy observed between the rRNA level and the actual cell proportion. However, both approaches supplied consistent and complementary information on the structure of the faecal microbiota. FISH combined with flow cytometry appears best suited to future high throughput analysis.

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