Direct Analysis of Genes Encoding 16S rRNA from Complex Communities Reveals Many Novel Molecular Species within the Human Gut

TLDR

The human intestinal tract harbors a complex microbial ecosystem essential for nutrition and health, yet 60–80 % of its bacteria remain uncultivated. This study used comparative analysis of cloned 16S rRNA gene sequences to investigate the bacterial diversity of an adult male’s fecal sample. A total of 284 clones amplified by 10‑cycle PCR were grouped into 82 molecular species based on ≥98 % sequence similarity. Three phylogenetic groups (Bacteroides, Clostridium coccoides, and Clostridium leptum) comprised 95 % of the clones, but only 24 % of the 82 species matched known organisms, indicating that 76 % represent novel gut species.

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.