Direct analysis of genes encoding 16S rRNA from complex communities reveals many novel molecular species within the human gut

Abstract

The human intestinal tract harbors a complex microbial ecosystem which plays a key role in nutrition and health. Although this microbiota has been studied in great detail by culture techniques, microscopic counts on human feces suggest that 60 to 80% of the observable bacteria cannot be cultivated. Using comparative analysis of cloned 16S rRNA gene (rDNA) sequences, we have investigated the bacterial diversity (both cultivated and noncultivated bacteria) within an adult-male fecal sample. The 284 clones obtained from 10-cycle PCR were classified into 82 molecular species (at least 98% similarity). Three phylogenetic groups contained 95% of the clones: the Bacteroides group, the Clostridium coccoides group, and the Clostridium leptum subgroup. The remaining clones were distributed among a variety of phylogenetic clusters. Only 24% of the molecular species recovered corresponded to described organisms (those whose sequences were available in public databases), and all of these were established members of the dominant human fecal flora (e.g., Bacteroides thetaiotaomicron, Fusobacterium prausnitzii, and Eubacterium rectale). However, the majority of generated rDNA sequences (76%) did not correspond to known organisms and clearly derived from hitherto unknown species within this human gut microflora.

FIG. 1

Phylogenetic tree derived from partial 16S rDNA sequence data for members of the Bacteroides group. Bar represents 2% sequence divergence. Designations of clones and key organisms used to name the subgroups are in boldface type. The tree was constructed with the SIMILARITY and NEIGHBOR programs. Bootstrap values are based on 500 replications. Vertical bars correspond to the Anaeroflexus assemblage (a) Bacteroides fragilis subgroup (b), Prevotella subgroup (c), and Bacteroides distasonis subgroup (d).

FIG. 2

Phylogenetic tree derived from partial 16S rDNA sequence data for members of the Clostridium coccoides group. Bar represents 2% sequence divergence. Designations of clones and the key organism used to name the group are in boldface type. The tree was constructed with the SIMILARITY and NEIGHBOR programs. Bootstrap values are based on 500 replications.

FIG. 3

Phylogenetic tree derived from partial 16S rDNA sequence data for members of the Clostridium leptum subgroup. Bar represents 2% sequence divergence. Designations of clones and the key organism used to name the group are in boldface type. The tree was constructed with the SIMILARITY and NEIGHBOR programs. Bootstrap values are based on 500 replications.

FIG. 4

Estimation of the biodiversity which was obtained by direct community analysis of a fecal sample. The cumulative number of OTUs is given as a function of the number of clones sequenced. Clones were randomly used.

FIG. 5

Phylogenetic tree derived from partial 16S rDNA sequence data for members of the Verrucomicrobium subdivision. The bar represents 2% sequence divergence. Designations of a clone and the key organism used to name the cluster are in boldface type. The tree was constructed with the SIMILARITY and NEIGHBOR programs. Bootstrap values are given for 500 replications.