Fusobacterium prausnitzii and Related Species Represent a Dominant Group Within the Human Fecal Flora ANTONIA S UAU, VIO LAIN E ROCHET, ABDELGHANI S GHIR, GEN EVIÈVE GRAMET, S TÉPHANIE BREWAEYS, M ALÈN E S UTREN , LIONEL RIGOTTIER-GOIS and JOËL DORÉ Unité d’écologie et de physiologie du système digestif, Institut National de la Recherche Agronomique, Jouy-en-Josas, France Received: February 15, 2001 Summary The human gut microflora plays a key role in nutrition and health. It has been extensively studied by conventional culture techniques. However these methods are difficult, time consuming and their results not always consistent. Furthermore microscopic counts indicate that only 20 to 40% of the total flora can be cultivated. Among the predominant species of the human gut, Fusobacterium prausnitzii was re- ported either as one of the most frequent and numerous species or was seldom retrieved. We designed and validated a specific rRNA-targeted oligonucleotide probe, called S-*-Fprau-0645-a-A-23, to accu- rately detect and quantify F. prausnitzii and relatives within the human fecal microflora. The target group accounted for 5.3 ± 3% of total bacterial 16S rRNA using dot blot hybridization (10 human fecal samples) and 16.5 ± 7% of cells stained with Dapi using in situ hybridization (10 other human fecal sam- ples). A specific morphology seemed to be typical and dominant: two cells forming an asymmetrical dou- ble droplet. This work showed that F. prausnitzii and phylogenetically related species represent a domi- nant group within the human fecal flora. Key words: rRNA – oligonucleotide probe – in situ hybridization – dot blot hybridization – Fusobacte- rium prausnitzii – human fecal flora Introduction There are up to 10 14 total bacteria in the human in- testinal tract, which is ten to twenty times the total num- ber of tissue cells in the entire body. This flora is complex in term of species diversity: according to statistical analy- ses of data from culture-based studies, it could be esti- mated that “the total number of different kind of bacteria probably exceeds 400 or 500 species but many could be represented by less than 10 8 cells per g of faeces”. The en- dogenous flora contributes to human health through its fermentation products, protection against pathogens and stimulation of the immune system (DUCLUZEAU, 1988; GIBSO N and ROBERFROID, 1995). According to fecal flora analysis based on phenotypic identification of isolates (FINEGOLD et al., 1983; M OORE and H OLDEMAN , 1974), to direct analysis of 16S rRNA genes (SUAU et al., 1999; WILSO N and BLITCHINGTON , 1996; Z OETENDAL et al., 1998) and to oligonucleotide probe hybridization (FRANKS et al., 1998; SGHIR et al., 1998), the vast majority of human fecal bacteria belong to four distinct phylogenetic groups defined in the RDP classification (M AIDAK et al., 2001): Bifidobacterium, Bacteroides, Clostridium coccoides and Clostridium lep- tum . The Bifidobacterium subgroup gathers all the Bifi- dobacterium species and Gardnerella vaginalis (M IYAKE et al., 1998). The Bacteroides group encloses Bacteroides, Porphyromonas and Prevotella genera (PASTER et al., 1994). Named after a species, the Clostridium coccoides group is a large group gathering species from Clostridium , Eubacterium , Ruminococcus and Butyrivib- rio genera. It corresponds to Clostridium rRNA subclus- ter XIVa (COLLINS et al., 1994). The fourth cluster has also been named after a species: the Clostridium leptum subgroup encompasses species belonging to genera Clostridium , Eubacterium , Ruminococcus and Anaero- filum . It corresponds to Clostridium rRNA cluster IV (COLLINS et al., 1994). Obligatory anaerobic, Gram-negative, nonsporeform- ing, chemoorganotrophic rods belong to the family Bac- teroidaceae. Among this family, those which produce butyrate as a major product, smaller amounts of acetate and sometimes propionate, formate or lactate and are nonmotile belong to the genus Fusobacterium (M OORE 0723-2020/01/24/01-139 $ 15.00/0 System. Appl. Microbiol. 24, 139–145 (2001) © Urban & Fischer Verlag http://www.urbanfischer.de/journals/sam